David Craik

Evaluation of Efficient Non-reducing Enzymatic and Chemical Ligation Strategies for Complex Disulfide-Rich Peptides.

Publisher: American Chemical Society (ACS)

Date: 09-11-2021

DOI: 10.1021/ACS.BIOCONJCHEM.1C00452

Abstract: Double-knotted peptides identified in venoms and synthetic bivalent peptide constructs targeting ion channels are emerging tools for the study of ion channel pharmacology and physiology. These highly complex and disulfide-rich peptides contain two in idual cystine knots, each comprising six cysteines and three disulfide bonds. Until now, native double-knotted peptides, such as Hi1a and DkTx, have only been isolated from venom or produced recombinantly, whereas engineered double-knotted peptides have successfully been produced through enzymatic ligation using sortase A to form a seamless amide bond at the ligation site between two knotted toxins, and by alkyne/azide click chemistry, joining two peptide knots via a triazole linkage. To further pursue these double-knotted peptides as pharmacological tools or probes for therapeutically relevant ion channels, we sought to identify a robust methodology resulting in a high yield product that lends itself to rapid production and facile mutational studies. In this study, we evaluated the ligation efficiency of enzymatic (sortase A5°, butelase 1, wild-type OaAEP 1, C247A-OaAEP 1, and peptiligase) and mild chemical approaches (α-ketoacid-hydroxylamine, KAHA) for forming a native amide bond linking the toxins while maintaining the native disulfide connectivity of each pre-folded peptide. We used two Na

Quantitative analysis of backbone-cyclised peptides in plants

Publisher: Elsevier BV

Date: 09-2008

DOI: 10.1016/J.JCHROMB.2008.07.023

Abstract: Progress in understanding the biosynthetic pathway of the cyclotides has been h ered as this unique family of cyclic plant peptides are notoriously difficult to analyse by standard proteomic approaches such as gel electrophoresis. We have developed a simple, rapid and robust strategy for the quantification of cyclotides in crude plant extracts using MALDI-TOF MS making use of generic peptides similar in mass to the analyte as internal standards for calibration. Linearity (r(2)>0.99) over two orders of magnitude (down to femtomole levels) was achieved in plant extracts, allowing quantitative analysis of transgenic and endogenous peptide expression.

The Cyclotide Fingerprint inOldenlandia affinis: Elucidation of Chemically Modified, Linear and Novel Macrocyclic Peptides

Publisher: Wiley

Date: 06-2007

DOI: 10.1002/CBIC.200700097

Abstract: The complete suite of cyclotides present in Oldenlandia affinis (Rubiaceae), the plant that was originally found to contain this unique family of circular proteins, has been characterised. This study expands the number of known cyclotides in this plant to 17, of which nine new sequences (kalata B9-B17) were characterised in this work. In addition, five derivatives that contain oxidation products of the conserved tryptophan were identified, and it was shown that the formation of these derivatives is catalysed by exposure to sunlight. Furthermore, we describe two "linear" cyclotide analogues. These acyclic peptides have three intact disulfide bonds, and their N and C termini coincide with the hypothesised cleavage sites from the precursor protein. This work increases our knowledge about the sequence variation that is accommodated by the cyclic cystine knot scaffold, confirms its applicability as a template for drug design, and also shows the first natural degradation pathways for cyclotides. These pathways have important implications for the persistence and environmental fate of the cyclotides if used as crop-protection agents.

Characterization of synthetic Tf2 as a NaV1.3 selective pharmacological probe

Publisher: MDPI AG

Date: 11-06-2020

DOI: 10.3390/BIOMEDICINES8060155

Abstract: NaV1.3 is a subtype of the voltage-gated sodium channel family. It has been implicated in the pathogenesis of neuropathic pain, although the contribution of this channel to neuronal excitability is not well understood. Tf2, a β-scorpion toxin previously identified from the venom of Tityus fasciolatus, has been reported to selectively activate NaV1.3. Here, we describe the activity of synthetic Tf2 and assess its suitability as a pharmacological probe for NaV1.3. As described for the native toxin, synthetic Tf2 (1 µM) caused early channel opening, decreased the peak current, and shifted the voltage dependence of NaV1.3 activation in the hyperpolarizing direction by −11.3 mV, with no activity at NaV1.1, NaV1.2, and NaV1.4-NaV1.8. Additional activity was found at NaV1.9, tested using the hNav1.9_C4 chimera, where Tf2 (1 µM) shifted the voltage dependence of activation by −6.3 mV. In an attempt to convert Tf2 into an NaV1.3 inhibitor, we synthetized the analogue Tf2[S14R], a mutation previously described to remove the excitatory activity of related β-scorpion toxins. Indeed, Tf2[S14R](10 µM) had reduced excitatory activity at NaV1.3, although it still caused a small −5.8 mV shift in the voltage dependence of activation. Intraplantar injection of Tf2 (1 µM) in mice caused spontaneous flinching and swelling, which was not reduced by the NaV1.1/1.3 inhibitor ICA-121431 nor in NaV1.9-/- mice, suggesting off-target activity. In addition, despite a loss of excitatory activity, intraplantar injection of Tf2[S14R](10 µM) still caused swelling, providing strong evidence that Tf2 has additional off-target activity at one or more non-neuronal targets. Therefore, due to activity at NaV1.9 and other yet to be identified target(s), the use of Tf2 as a selective pharmacological probe may be limited.

Cyclotides: macrocyclic peptides with applications in drug design and agriculture

Publisher: Springer Science and Business Media LLC

Date: 10-2009

DOI: 10.1007/S00018-009-0159-3

Abstract: Cyclotides are disulfide-rich peptides from plants that are exceptionally stable as a result of their unique cyclic cystine knot structural motif. Their natural role is thought to be as plant defence agents, most notably against insect pests, but they also have potential applications in drug design and agriculture. This article identifies gaps in current knowledge on cyclotides and suggests future directions for research into this fascinating family of ultra-stable mini-proteins.

Kalata B8, a novel antiviral circular protein, exhibits conformational flexibility in the cystine knot motif

Publisher: Portland Press Ltd.

Date: 13-01-2006

DOI: 10.1042/BJ20051371

Abstract: The cyclotides are a family of circular proteins with a range of biological activities and potential pharmaceutical and agricultural applications. The biosynthetic mechanism of cyclization is unknown and the discovery of novel sequences may assist in achieving this goal. In the present study, we have isolated a new cyclotide from Oldenlandia affinis, kalata B8, which appears to be a hybrid of the two major subfamilies (Möbius and bracelet) of currently known cyclotides. We have determined the three-dimensional structure of kalata B8 and observed broadening of resonances directly involved in the cystine knot motif, suggesting flexibility in this region despite it being the core structural element of the cyclotides. The cystine knot motif is widespread throughout Nature and inherently stable, making this apparent flexibility a surprising result. Furthermore, there appears to be isomerization of the peptide backbone at an Asp-Gly sequence in the region involved in the cyclization process. Interestingly, such isomerization has been previously characterized in related cyclic knottins from Momordica cochinchinensis that have no sequence similarity to kalata B8 apart from the six conserved cysteine residues and may result from a common mechanism of cyclization. Kalata B8 also provides insight into the structure–activity relationships of cyclotides as it displays anti-HIV activity but lacks haemolytic activity. The ‘uncoupling’ of these two activities has not previously been observed for the cyclotides and may be related to the unusual hydrophilic nature of the peptide.

Formation of cyclotides and variations in cyclotide expression in Oldenlandia affinis suspension cultures

Publisher: Springer Science and Business Media LLC

Date: 05-09-2007

DOI: 10.1007/S00253-007-1159-6

Abstract: Cyclotides, a family of disulfide-rich mini-proteins, show a wide range of biological activities, making them interesting targets for pharmaceutical and agrochemical applications, but little is known about their natural function and the events that trigger their expression. An investigation of nutritional variations and irradiation during a batch process involving plant cell cultures has been performed, using the native African medical herb, Oldenlandia affinis, as a model plant. The results demonstrated the biosynthesis of kalata B1, the main cyclotide in O. affinis, in a combined growth/nongrowth-associated pattern. The highest concentration, 0.37 mg g(-1) dry weight, was accumulated in irradiated cells at 35 mumol m(-2) s(-1). Furthermore, 12 novel cyclotides were identified and the expression of various cyclotides compared in irradiated vs non-irradiated cultures. The results indicate that cyclotide expression varies greatly depending on physiological conditions and environmental stress. Kalata B1 is the most abundant cyclotide in plant suspension cultures, which underlies its importance as a natural defense molecule. The identification of novel cyclotides in suspension cultures, compared to whole plants, indicates that there may be more novel cyclotides to be discovered and that the genetic network regulating cyclotide expression is a very sensitive system, ready to adapt to the current environmental growth condition.

CyBase: A database of cyclic protein sequences and structures, with applications in protein discovery and engineering

Publisher: Oxford University Press (OUP)

Date: 23-12-2008

DOI: 10.1093/NAR/GKM953

Isolation and characterization of cytotoxic cyclotides from Viola philippica

Publisher: Elsevier BV

Date: 08-2011

DOI: 10.1016/J.PEPTIDES.2011.06.016

Abstract: Cyclotides are a large family of plant peptides characterized by a macrocyclic backbone and knotted arrangement of three disulfide bonds. This unique structure renders cyclotides exceptionally stable to thermal, chemical and enzymatic treatments. They exhibit a variety of bioactivities, including uterotonic, anti-HIV, cytotoxic and hemolytic activity and it is these properties that make cyclotides an interesting peptide scaffold for drug design. In this study, eight new cyclotides (Viphi A-H), along with eight known cyclotides, were isolated from Viola philippica, a plant from the Violaceae family. In addition, Viba 17 and Mram 8 were isolated for the first time as peptides. The sequences of these cyclotides were elucidated primarily by using a strategy involving reduction, enzymatic digestion and tandem mass spectroscopy sequencing. Several of the cyclotides showed cytotoxic activities against the cancer cell lines MM96L, HeLa and BGC-823. The novel cyclotides reported here: (1) enhance the known sequence variation observed for cyclotides (2) extend the number of species known to contain cyclotides (3) provide interesting structure-activity relationships that delineate residues important for cytotoxic activity. In addition, this study provides insights into the potential active ingredients of traditional Chinese medicines.

Structure and Activity Studies of Disulfide-Deficient Analogues of αO-Conotoxin GeXIVA.

Publisher: American Chemical Society (ACS)

Date: 27-01-2020

DOI: 10.1021/ACS.JMEDCHEM.9B01409

Abstract: αO-conotoxin GeXIVA from

Cyclotide Structure and Function: The Role of Membrane Binding and Permeation

Publisher: American Chemical Society (ACS)

Date: 27-01-2017

DOI: 10.1021/ACS.BIOCHEM.6B01212

Abstract: There is growing interest in the use of peptides as therapeutic drugs and, in particular, in the potential of cyclotides, a family of cyclic peptides with remarkable stability and amenability to sequence engineering, as scaffolds in drug design. As well as having an ultrastable structure, many natural cyclotides have intrinsic biological activities with potential pharmaceutical or agricultural applications. Some cyclotides also have the ability to cross membrane barriers and to enter into cells in particular, cyclotides that belong to the Möbius and bracelet subfamilies have been found to harbor lipid-binding domains, which allow for the specific recognition of phosphatidylethanolamine phospholipids in biological membranes. This lipid selectivity is intimately correlated with the highly conserved three-dimensional structures of cyclotides and is important for their reported biological properties and cell penetration ability. The membrane binding features of Möbius and bracelet cyclotides contrast with the lack of membrane binding of trypsin inhibitor cyclotides, which have physicochemical properties and bioactivities different from those of the other two subfamilies of cyclotides but are also able to enter cells. This review discusses the structures of cyclotides with regard to their myriad of biological activities and describes the role of membrane binding in their functions and ability to enter cells.

Mutagenesis of bracelet cyclotide hyen D reveals functionally and structurally critical residues for membrane binding and cytotoxicity

Publisher: Elsevier BV

Date: 04-2022

DOI: 10.1016/J.JBC.2022.101822

Molecular dynamics simulations of dihydro‐β‐erythroidine bound to the human α4β2 nicotinic acetylcholine receptor

Publisher: Wiley

Date: 07-06-2019

DOI: 10.1111/BPH.14698

STRUCTURE AND FUNCTION OF PLANT TOXINS (WITH EMPHASIS ON CYSTINE KNOT TOXINS)

Publisher: Informa UK Limited

Date: 2002

DOI: 10.1081/TXR-120014405

Conotoxins and Other Conopeptides

Publisher: Wiley

Date: 07-03-2014

DOI: 10.1002/9783527681501.CH14

Chemical Re-engineering of Chlorotoxin Improves Bioconjugation Properties for Tumor Imaging and Targeted Therapy

Publisher: American Chemical Society (ACS)

Date: 06-01-2011

DOI: 10.1021/JM101018R

Microcin J25 Has a Threaded Sidechain-to-Backbone Ring Structure and Not a Head-to-Tail Cyclized Backbone

Publisher: American Chemical Society (ACS)

Date: 20-09-2003

DOI: 10.1021/JA0367703

Abstract: Microcin J25 is a 21 amino acid bacterial peptide that has potent antibacterial activity against Gram-negative bacteria, resulting from its interaction with RNA polymerase. The peptide was previously proposed to have a head-to-tail cyclized peptide backbone and a tight globular structure (Blond, A., Péduzzi, J., Goulard, C., Chiuchiolo, M. J., Barthélémy, M., Prigent, Y., Salomón, R. A., Farías, R. N., Moreno, F. & Rebuffat, S. Eur. J. Biochem. 1999, 259, 747-755). It exhibits remarkable thermal stability for a peptide of its size lacking disulfide bonds and in part this was previously proposed to derive from its macrocyclic structure. We show here that in fact the peptide does not have a head-to-tail cyclic structure but rather a side chain to backbone cyclization between Glu8 and the N-terminus. This creates an embedded ring that is threaded by the C-terminal tail of the molecule, forming a noose-like feature. The three-dimensional structure deduced from NMR data suggests that slippage of the noose is prevented by two aromatic residues flanking the embedded ring. Unthreading does not occur even when the molecule is enzymatically digested with thermolysin. The new structural interpretation fully accounts for previously reported NMR and biophysical data and is consistent with the remarkable stability of this potent antimicrobial peptide.

Histidine-Rich Defensins from the Solanaceae and Brasicaceae Are Antifungal and Metal Binding Proteins

Publisher: MDPI AG

Date: 24-08-2020

DOI: 10.3390/JOF6030145

Abstract: Plant defensins are best known for their antifungal activity and contribution to the plant immune system. The defining feature of plant defensins is their three-dimensional structure known as the cysteine stabilized alpha-beta motif. This protein fold is remarkably tolerant to sequence variation with only the eight cysteines that contribute to the stabilizing disulfide bonds absolutely conserved across the family. Mature defensins are typically 46–50 amino acids in length and are enriched in lysine and/or arginine residues. Examination of a database of approximately 1200 defensin sequences revealed a subset of defensin sequences that were extended in length and were enriched in histidine residues leading to their classification as histidine-rich defensins (HRDs). Using these initial HRD sequences as a query, a search of the available sequence databases identified over 750 HRDs in solanaceous plants and 20 in brassicas. Histidine residues are known to contribute to metal binding functions in proteins leading to the hypothesis that HRDs would have metal binding properties. A selection of the HRD sequences were recombinantly expressed and purified and their antifungal and metal binding activity was characterized. Of the four HRDs that were successfully expressed all displayed some level of metal binding and two of four had antifungal activity. Structural characterization of the other HRDs identified a novel pattern of disulfide linkages in one of the HRDs that is predicted to also occur in HRDs with similar cysteine spacing. Metal binding by HRDs represents a specialization of the plant defensin fold outside of antifungal activity.

Cyclic thrombospondin-1 mimetics: grafting of a thrombospondin sequence into circular disulfide-rich frameworks to inhibit endothelial cell migration

Publisher: Portland Press Ltd.

Date: 26-11-2015

DOI: 10.1042/BSR20150210

Abstract: Tumour formation is dependent on nutrient and oxygen supply from adjacent blood vessels. Angiogenesis inhibitors can play a vital role in controlling blood vessel formation and consequently tumour progression by inhibiting endothelial cell proliferation, sprouting and migration. The primary aim of the present study was to design cyclic thrombospondin-1 (TSP-1) mimetics using disulfide-rich frameworks for anti-angiogenesis therapies and to determine whether these peptides have better potency than the linear parent peptide. A short anti-angiogenic heptapeptide fragment from TSP-1 (GVITRIR) was incorporated into two cyclic disulfide-rich frameworks, namely MCoTI-II (Momordica cochinchinensis trypsin inhibitor-II) and SFTI-1 (sunflower trypsin inhibitor-1). The cyclic peptides were chemically synthesized and folded in oxidation buffers, before being tested in a series of in vitro evaluations. Incorporation of the bioactive heptapeptide fragment into the cyclic frameworks resulted in peptides that inhibited microvascular endothelial cell migration, and had no toxicity against normal primary human endothelial cells or cancer cells. Importantly, all of the designed cyclic TSP-1 mimetics were far more stable than the linear heptapeptide in human serum. The present study has demonstrated a novel approach to stabilize the active region of TSP-1. The anti-angiogenic activity of the native TSP-1 active fragment was maintained in the new TSP-1 mimetics and the results provide a new chemical approach for the design of TSP-1 mimetics.

Solution structure and proposed binding mechanism of a novel potassium channel toxin κ-conotoxin PVIIA

Publisher: Elsevier BV

Date: 12-1997

DOI: 10.1016/S0969-2126(97)00307-9

Abstract: kappa-PVIIA is a 27-residue polypeptide isolated from the venom of Conus purpurascens and is the first member of a new class of conotoxins that block potassium channels. By comparison to other ion channels of eukaryotic cell membranes, voltage-sensitive potassium channels are relatively simple and methodology has been developed for mapping their interactions with small-peptide toxins. PVIIA, therefore, is a valuable new probe of potassium channel structure. This study of the solution structure and mode of channel binding of PVIIA forms the basis for mapping the interacting residues at the conotoxin-ion channel interface. The three-dimensional structure of PVIIA resembles the triple-stranded beta sheet/cystine-knot motif formed by a number of toxic and inhibitory peptides. Subtle structural differences, predominantly in loops 2 and 4, are observed between PVIIA and other conotoxins with similar structural frameworks, however. Electrophysiological binding data suggest that PVIIA blocks channel currents by binding in a voltage-sensitive manner to the external vestibule and occluding the pore. Comparison of the electrostatic surface of PVIIA with that of the well-characterised potassium channel blocker charybdotoxin suggests a likely binding orientation for PVIIA. Although the structure of PVIIA is considerably different to that of the alphaK scorpion toxins, it has a similar mechanism of channel blockade. On the basis of a comparison of the structures of PVIIA and charybdotoxin, we suggest that Lys19 of PVIIA is the residue which is responsible for physically occluding the pore of the potassium channel.

Solution structure and novel insights into the determinants of the receptor specificity of human relaxin-3

Publisher: Elsevier BV

Date: 03-2006

DOI: 10.1074/JBC.M511210200

Recent Progress Towards Pharmaceutical Applications of Disulfide-Rich Cyclic Peptides

Publisher: Bentham Science Publishers Ltd.

Date: 09-2013

DOI: 10.2174/13892037113149990069

Abstract: Cyclotides and conotoxins are two classes of disulfide-rich peptides that occur in plants and animals respectively and are the major focus of study in our laboratory. In the last three years there has been significant progress in studies of these two classes of compounds and in this article we provide an overview of the findings from our laboratory in this period. Highlights include the discovery of cyclotides in the Fabaceae and Solanaceae plant families, members of which are widely used in human nutrition, and the discovery of new classes of cyclotide precursors. These discoveries confirm the widespread distribution of cyclotides in the plant kingdom and the ersity of precursor proteins involved in their biosynthesis. Other studies have delineated the mode of action of naturally occurring cyclotides and have demonstrated the versatility of synthetic cyclotides as stable protein engineering frameworks, with applications in drug design. Conotoxins continue to be a rich source of inspiration for drug design programs, and we summarize here a range of recent studies from our laboratory focusing on the development of novel synthetic strategies and the delineation of structure-activity relationships. A major highlight was the development of an orally active cyclized conotoxin derivative that is highly efficacious in a rat model of neuropathic pain. Overall the studies described herein provide much encouragement for continuing efforts to develop peptides as drugs.

Low-Molecular-Weight Peptidic and Cyclic Antagonists of the Receptor for the Complement Factor C5a

Publisher: American Chemical Society (ACS)

Date: 07-05-1999

DOI: 10.1021/JM9806594

Inside Cover: Application and Structural Analysis of Triazole‐Bridged Disulfide Mimetics in Cyclic Peptides (Angew. Chem. Int. Ed. 28/2020)

Publisher: Wiley

Date: 26-05-2020

DOI: 10.1002/ANIE.202006713

Cyclic peptides arising by evolutionary parallelism via asparaginyl-endopeptidase-mediated biosynthesis

Publisher: Oxford University Press (OUP)

Date: 07-2012

DOI: 10.1105/TPC.112.099085

Nicotiana alata Defensin Chimeras Reveal Differences in the Mechanism of Fungal and Tumor Cell Killing and an Enhanced Antifungal Variant.

Publisher: American Society for Microbiology

Date: 10-2016

DOI: 10.1128/AAC.01479-16

Abstract: The plant defensin NaD1 is a potent antifungal molecule that also targets tumor cells with a high efficiency. We examined the features of NaD1 that contribute to these two activities by producing a series of chimeras with NaD2, a defensin that has relatively poor activity against fungi and no activity against tumor cells. All plant defensins have a common tertiary structure known as a cysteine-stabilized α-β motif which consists of an α helix and a triple-stranded β-sheet stabilized by four disulfide bonds. The chimeras were produced by replacing loops 1 to 7, the sequences between each of the conserved cysteine residues on NaD1, with the corresponding loops from NaD2. The loop 5 swap replaced the sequence motif (SKILRR) that mediates tight binding with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] and is essential for the potent cytotoxic effect of NaD1 on tumor cells. Consistent with previous reports, there was a strong correlation between PI(4,5)P 2 binding and the tumor cell killing activity of all of the chimeras. However, this correlation did not extend to antifungal activity. Some of the loop swap chimeras were efficient antifungal molecules, even though they bound poorly to PI(4,5)P 2 , suggesting that additional mechanisms operate against fungal cells. Unexpectedly, the loop 1B swap chimera was 10 times more active than NaD1 against filamentous fungi. This led to the conclusion that defensin loops have evolved as modular components that combine to make antifungal molecules with variable mechanisms of action and that artificial combinations of loops can increase antifungal activity compared to that of the natural variants.

Anchor Residues Guide Form and Function in Grafted Peptides

Publisher: Wiley

Date: 02-05-2019

DOI: 10.1002/ANGE.201901572

Membrane-binding properties of gating modifier and pore-blocking toxins: Membrane interaction is not a prerequisite for modification of channel gating

Publisher: Elsevier BV

Date: 04-2016

DOI: 10.1016/J.BBAMEM.2016.02.002

Abstract: Many venom peptides are potent and selective inhibitors of voltage-gated ion channels, including channels that are validated therapeutic targets for treatment of a wide range of human diseases. However, the development of novel venom-peptide-based therapeutics requires an understanding of their mechanism of action. In the case of voltage-gated ion channels, venom peptides act either as pore blockers that bind to the extracellular side of the channel pore or gating modifiers that bind to one or more of the membrane-embedded voltage sensor domains. In the case of gating modifiers, it has been debated whether the peptide must partition into the membrane to reach its binding site. In this study, we used surface plasmon resonance, fluorescence spectroscopy and molecular dynamics to directly compare the lipid-binding properties of two gating modifiers (μ-TRTX-Hd1a and ProTx-I) and two pore blockers (ShK and KIIIA). Only ProTx-I was found to bind to model membranes. Our results provide further evidence that the ability to insert into the lipid bilayer is not a requirement to be a gating modifier. In addition, we characterised the surface of ProTx-I that mediates its interaction with neutral and anionic phospholipid membranes and show that it preferentially interacts with anionic lipids.

Peptide quantification by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry: Investigations of the cyclotide kalata B1 in biological fluids

Publisher: Elsevier BV

Date: 10-2005

DOI: 10.1016/J.CHROMA.2005.07.094

Abstract: A rapid method has been developed for the quantification of the prototypic cyclotide kalata B1 in water and plasma utilizing matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. The unusual structure of the cyclotides means that they do not ionise as readily as linear peptides and as a result of their low ionisation efficiency, traditional LC/MS analyses were not able to reach the levels of detection required for the quantification of cyclotides in plasma for pharmacokinetic studies. MALDI-TOF-MS analysis showed linearity (R2 > 0.99) in the concentration range 0.05-10 microg/mL with a limit of detection of 0.05 microg/mL (9 fmol) in plasma. This paper highlights the applicability of MALDI-TOF mass spectrometry for the rapid and sensitive quantification of peptides in biological s les without the need for extensive extraction procedures.

Solution structures in aqueous SDS micelles of two amyloid β peptides of aβ(1-28) mutated at the α-secretase cleavage site (K16E, K16F)

Publisher: Elsevier BV

Date: 06-2000

DOI: 10.1006/JSBI.2000.4267

Cyclotide interactions with the nematode external surface

Publisher: American Society for Microbiology

Date: 05-2010

DOI: 10.1128/AAC.01306-09

Abstract: Cyclotides are a large family of cyclic cystine knot-containing plant peptides that have anthelminthic activities against Haemonchus contortus and Trichostrongylus colubriformis , two important gastrointestinal nematodes of sheep. In this study, we investigated the interaction of the prototypic cyclotide kalata B1 with the external surface of H. contortus larvae and adult worms. We show that cyclotides do not need to be ingested by the worms to exert their toxic effects but that an interaction with the external surface alone is toxic. Evidence for this was the toxicity toward adult worms in the presence of a chemically induced pharyngeal ligature and toxicity of cyclotides toward nonfeeding larval life stages. Uptake of tritiated inulin in ligated adult worms was increased in the presence of cyclotide, suggesting that cyclotides increase the permeability of the external membranes of adult nematodes. Polyethylene glycols of various sizes showed protective effects on the nonfeeding larval life stage, as well as in hemolytic activity assays, suggesting that discrete pores are formed in the membrane surfaces by cyclotides and that these can be blocked by polyethylene glycols of appropriate size. This increased permeability is consistent with recently reported effects of cyclotides on membranes in which kalata B1 was demonstrated to form pores and cause leakage of vesicle/cellular contents. Our data, together with known size constraints on the movement of permeants across nematode cuticle layers, suggest that one action of the cyclotides involves an interaction with the lipid-rich epicuticle layer at the surface of the worm.

Evolutionary Origins of a Bioactive Peptide Buried within Preproalbumin

Publisher: Oxford University Press (OUP)

Date: 03-2014

DOI: 10.1105/TPC.114.123620

Abstract: The de novo evolution of proteins is now considered a frequented route for biological innovation, but the genetic and biochemical processes that lead to each newly created protein are often poorly documented. The common sunflower (Helianthus annuus) contains the unusual gene PawS1 (Preproalbumin with SFTI-1) that encodes a precursor for seed storage albumin however, in a region usually discarded during albumin maturation, its sequence is matured into SFTI-1, a protease-inhibiting cyclic peptide with a motif homologous to unrelated inhibitors from legumes, cereals, and frogs. To understand how PawS1 acquired this additional peptide with novel biochemical functionality, we cloned PawS1 genes and showed that this dual destiny is over 18 million years old. This new family of mostly backbone-cyclic peptides is structurally erse, but the protease-inhibitory motif was restricted to peptides from sunflower and close relatives from its subtribe. We describe a widely distributed, potential evolutionary intermediate PawS-Like1 (PawL1), which is matured into storage albumin, but makes no stable peptide despite possessing residues essential for processing and cyclization from within PawS1. Using sequences we cloned, we retrodict the likely stepwise creation of PawS1's additional destiny within a simple albumin precursor. We propose that relaxed selection enabled SFTI-1 to evolve its inhibitor function by converging upon a successful sequence and structure.

Understanding the Structure/Activity Relationships of the Iron Regulatory Peptide Hepcidin

Publisher: Elsevier BV

Date: 03-2011

DOI: 10.1016/J.CHEMBIOL.2010.12.009

Cyclotides as grafting frameworks for protein engineering and drug design applications

Publisher: Wiley

Date: 09-2013

DOI: 10.1002/BIP.22284

Abstract: Cyclotides are a family of naturally occurring backbone‐cyclized macrocyclic mini‐proteins from plants that have a knotted trio of intramolecular disulfide bonds. Their structural features imbue cyclotides with extraordinary stability against degradation at elevated temperatures or in the presence of proteolytic enzymes. The plasticity of their intracysteine loop sequences is exemplified by the more than 250 natural cyclotides sequenced to date, and this tolerance to sequence variation, along with their erse bioactivities, underpins the suitability of the cyclic cystine knot motif as a valuable drug design scaffold and research tool for protein engineering studies. Here, we review the recent literature on applications of cyclotides for the stabilization of peptide epitopes and related protein engineering studies. Possible future directions in this field are also described. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 480–491, 2013.

Cycloviolacin H4, a Hydrophobic Cyclotide fromViola hederaceae

Publisher: American Chemical Society (ACS)

Date: 14-12-2005

DOI: 10.1021/NP050317I

Abstract: Cycloviolacin H4, a new macrocyclic miniprotein comprising 30 amino acid residues, was isolated from the underground parts of the Australian native violet Viola hederaceae. Its sequence, cyclo-(CAESCVWIPCTVTALLGCSCSNNVCYNGIP), was determined by nanospray tandem mass spectrometry and quantitative amino acid analysis. A knotted disulfide arrangement, which was designated as a cyclic cystine knot motif and characteristic to all known cyclotides, is proposed for stabilizing the molecular structure and folding. The cyclotide is classified in the bracelet subfamily of cyclotides due to the absence of a cis-Pro peptide bond in the circular peptide backbone. A model of its three-dimensional structure was derived based on the template of the homologous cyclotide vhr1 (Trabi et al. Plant Cell 2004, 16, 2204-2216). Cycloviolacin H4 exhibits the most potent hemolytic activity in cyclotides reported so far, and this activity correlates with the size of a surface-exposed hydrophobic patch. This work has thus provided insight into the factors that modulate the cytotoxic properties of cyclotides.

The Engineering of an Orally Active Conotoxin for the Treatment of Neuropathic Pain

Publisher: Wiley

Date: 08-06-2010

DOI: 10.1002/ANIE.201000620

Amino Acid Scanning at P5' within the Bowman-Birk Inhibitory Loop Reveals Specificity Trends for Diverse Serine Proteases.

Publisher: American Chemical Society (ACS)

Date: 19-03-2019

DOI: 10.1021/ACS.JMEDCHEM.9B00211

Abstract: Sunflower trypsin inhibitor-1 (SFTI-1) is a 14-amino acid cyclic peptide that shares an inhibitory loop with a sequence and structure similar to a larger family of serine protease inhibitors, the Bowman-Birk inhibitors. Here, we focus on the P5' residue in the Bowman-Birk inhibitory loop and produce a library of SFTI variants to characterize the P5' specificity of 11 different proteases. We identify seven amino acids that are generally preferred by these enzymes and also correlate with P5' sequence ersity in naturally occurring Bowman-Birk inhibitors. Additionally, we show that several enzymes have ergent specificities that can be harnessed in engineering studies. By optimizing the P5' residue, we improve the potency or selectivity of existing inhibitors for kallikrein-related peptidase 5 and show that a variant with substitutions at 7 of the scaffold's 14 residues retains a similar structure to SFTI-1. These findings provide new insights into P5' specificity requirements for the Bowman-Birk inhibitory loop.

A Comparison of the Self-association Behavior of the Plant Cyclotides Kalata B1 and Kalata B2 via Analytical Ultracentrifugation

Publisher: Elsevier BV

Date: 2004

DOI: 10.1074/JBC.M306826200

Drug susceptibility profiling of Australian Burkholderia species as models for developing melioidosis therapeutics

Publisher: Cold Spring Harbor Laboratory

Date: 27-01-2020

DOI: 10.1101/2020.01.21.914846

Abstract: Melioidosis is a neglected tropical disease caused by the Gram-negative soil bacterium Burkholderia pseudomallei. Current treatment regimens are prolonged and costly, and acquired antimicrobial resistance has been reported for all currently used antibiotics. Efforts to develop new treatments for melioidosis are h ered by the risks associated with handling pathogenic B. pseudomallei , which restricts research to facilities with Biosafety Level (BSL) 3 containment. Closely related Burkholderia species that are less pathogenic can be investigated under less stringent BSL 2 containment. We hypothesized that near-neighbour Burkholderia species could be used as model organisms for developing therapies that would also be effective against B. pseudomallei . We used microbroth dilution assays to compare the susceptibility of three Australian B. pseudomallei isolates and five near-neighbour Burkholderia species – B. humptydooensis, B. thailandensis, B. oklahomensis, B territorii and B. stagnalis – to antibiotics currently used to treat melioidosis, and general-use antibacterial agents. We also established the susceptibility profiles of B. humptydooensis and B. territorii to 400 compounds from the Medicines for Malaria Venture Pathogen Box. From these comparisons, we observed a high degree of similarity in the susceptibility profiles of B. pseudomallei and near-neighbour species B. humptydooensis, B. thailandensis, B. oklahomensis and B. territorii. Less pathogenic Australian Burkholderia species B. humptydooensis, B. thailandensis, B. oklahomensis and B. territorii are excellent model organisms for developing potential new therapies for melioidosis.

Is the Mirror Image a True Reflection? Intrinsic Membrane Chirality Modulates Peptide Binding

Publisher: American Chemical Society (ACS)

Date: 25-11-2019

DOI: 10.1021/JACS.9B11194

Abstract: Peptides with pharmaceutical activities are attractive drug leads, and knowledge of their mode-of-action is essential for translation into the clinic. Comparison of native and enantiomeric peptides has long been used as a powerful approach to discriminate membrane- or receptor-mediated modes-of-action on the basis of the assumption that interactions with cell membranes are independent of peptide chirality. Here, we revisit this paradigm with the cyclotide kalata B1, a drug scaffold with intrinsic membrane-binding activity whose enantiomer is less potent than native peptide. To investigate this chirality dependence, we compared peptide-lipid binding using mirror image model membranes. We synthesized phospholipids with non-natural chirality and demonstrate that native kalata B1 binds with higher affinity to phospholipids with chirality found in eukaryotic membranes. This study shows for the first time that the chiral environment of lipid bilayers can modulate the function of membrane-active peptides and challenges the view that peptide-lipid interactions are achiral.

Inhibition of Neuronal Nicotinic Acetylcholine Receptor Subtypes by α-Conotoxin GID and Analogues

Publisher: Elsevier BV

Date: 02-2009

DOI: 10.1074/JBC.M804950200

Discovery and mechanistic studies of cytotoxic cyclotides from the medicinal herb Hybanthus enneaspermus

Publisher: Elsevier BV

Date: 08-2020

DOI: 10.1074/JBC.RA120.012627

Isolation and characterization of peptides from Momordica cochinchinensis seeds

Publisher: American Chemical Society (ACS)

Date: 30-07-2009

DOI: 10.1021/NP900174N

Abstract: The plant Momordica cochinchinensis has traditionally been used in Chinese medicine to treat a variety of illnesses. A range of bioactive molecules have been isolated from this plant, including peptides, which are the focus of this study. Here we report the isolation and characterization of two novel peptides, MCoCC-1 and MCoCC-2, containing 33 and 32 amino acids, respectively, which are toxic against three cancer cell lines. The two peptides are highly homologous to one another, but show no sequence similarity to known peptides. Elucidation of the three-dimensional structure of MCoCC-1 suggests the presence of a cystine knot motif, also found in a family of trypsin inhibitor peptides from this plant. However, unlike its structural counterparts, MCoCC-1 does not inhibit trypsin. MCoCC-1 has a well-defined structure, characterized mainly by a triple-stranded antiparallel beta-sheet, but unlike the majority of cystine knot proteins MCoCC-1 contains a disordered loop presumably as a result of flexibility in a localized region of the molecule. Of the cell lines tested, MCoCC-1 is the most toxic against a human melanoma cell line (MM96L) and is nonhemolytic to human erythrocytes. The role of these peptides within the plant remains to be determined.

Cellular Uptake and Cytosolic Delivery of a Cyclic Cystine Knot Scaffold

Publisher: American Chemical Society (ACS)

Date: 19-02-2020

DOI: 10.26434/CHEMRXIV.11860155.V1

Abstract: Cyclotides are macrocyclic peptides that have exceptionally stable structures and been reported to penetrate cells, making them promising scaffolds for the delivery of peptide inhibitory sequences to target intracellular proteins. However, their cellular uptake and cytosolic localization have been poorly understood until now, which has limited their therapeutic potential. In this study, the recently developed chloroalkane penetration assay was combined with established assays to characterize the cellular uptake and cytosolic delivery of the prototypic cyclotide, kalata B1. We show that kalata B1 enters the cytosol at low efficiency, but introducing various epitopes, including a single hydrophobic amino acid, into its loop 6 significantly improved its cytosolic delivery. Our results provide a foundation for the further development of a structurally unique class of scaffolds for the delivery of therapeutic cargoes into cells.

Importance of the Cell Membrane on the Mechanism of Action of Cyclotides

Publisher: American Chemical Society (ACS)

Date: 03-02-2012

DOI: 10.1021/CB200395F

Abstract: Their distinctive structures, erse range of bioactivities, and potential for pharmaceutical or agricultural applications make cyclotides an intriguing family of cyclic peptides. Together with the physiological role in plant host defense, cyclotides possess antimicrobial, anticancer, and anti-HIV activities. In all of the reported activities, cell membranes seem to be the primary target for cyclotide binding. This article examines recent literature on cyclotide-membrane studies and highlights the hypothesis that the activity of cyclotides is dependent on their affinity for lipid bilayers and enhanced by the presence of specific lipids, i.e., phospholipids containing phosphatidylethanolamine headgroups. There is growing evidence that the lipid composition of target cell membranes dictates the amount of cyclotides bound to the cell and the extent of their activity. After membrane targeting and insertion in the bilayer core, cyclotides induce disruption of membranes by a pore formation mechanism. This proposed mechanism of action is supported by biophysical studies with model membranes and by studies on natural biological membranes of known lipid compositions.

Insecticidal diversity of butterfly pea (Clitoria ternatea) accessions

Publisher: Elsevier BV

Date: 05-2020

DOI: 10.1016/J.INDCROP.2020.112214

Creating a specialist protein resource network: a meeting report for the protein bioinformatics and community resources retreat: Figure 1.

Publisher: Oxford University Press (OUP)

Date: 2015

DOI: 10.1093/DATABASE/BAV063

Front Cover: Cyclisation of Disulfide-Rich Conotoxins in Drug Design Applications (Eur. J. Org. Chem. 21/2016)

Publisher: Wiley

Date: 07-2016

DOI: 10.1002/EJOC.201670211

Structural and Functional Analysis of Human Liver‐Expressed Antimicrobial Peptide 2

Publisher: Wiley

Date: 15-09-2010

DOI: 10.1002/CBIC.201000400

Abstract: Human liver-expressed antimicrobial peptide 2 (LEAP-2) is a cationic antimicrobial peptide (CAMP) believed to have a protective role against bacterial infection. Little is known about the structure-activity relationships of LEAP-2 or its mechanism of action. In this study we describe the structure of LEAP-2, analyze its interaction with model membranes, and relate them to the antimicrobial activity of the peptide. The structure of LEAP-2, determined by NMR spectroscopy, reveals a compact central core with disorder at the N and C termini. The core comprises a β-hairpin and a 3(10)- helix that are braced by disulfide bonds between Cys17-28 and Cys23-33 and further stabilized by a network of hydrogen bonds. Membrane-affinity studies show that LEAP-2 membrane binding is governed by electrostatic attractions, which are sensitive to ionic strength. Truncation studies show that the C-terminal region of LEAP-2 is irrelevant for membrane binding, whereas the N-terminal (hydrophobic domain) and core regions (cationic domain) are essential. Bacterial-growth-inhibition assays reveal that the antimicrobial activity of LEAP-2 correlates with membrane affinity. Interestingly, the native and reduced forms of LEAP-2 have similar membrane affinity and antimicrobial activities this suggests that disulfide bonds are not essential for the bactericidal activity. This study reveals that LEAP-2 has a novel fold for a CAMP and suggests that although LEAP-2 exhibits antimicrobial activity under low-salt conditions, there is likely to be another physiological role for the peptide.

Erratum: Ribosomally synthesized and post-translationally modified peptide natural products: Overview and recommendations for a universal nomenclature (Natural Product Reports (2013) 30 (108-160) DOI:10.1039/C2NP20085F)

Publisher: Royal Society of Chemistry (RSC)

Date: 2013

DOI: 10.1039/C3NP90040A

Design, synthesis, and characterization of a single-chain peptide antagonist for the relaxin-3 receptor RXFP3

Publisher: American Chemical Society (ACS)

Date: 08-03-2011

DOI: 10.1021/JA110567J

Abstract: Relaxin-3 is a two-chain disulfide-rich peptide that is the ancestral member of the relaxin peptide family and, together with its G protein-coupled receptor RXFP3, is highly expressed in the brain. Strong evolutionary conservation of relaxin-3 suggests a critical biological function and recent studies have demonstrated modulation of sensory, neuroendocrine, metabolic, and cognitive systems. However, detailed studies of central relaxin-3-RXFP3 signaling have until now been severely h ered by the lack of a readily available high-affinity antagonist for RXFP3. Previous studies have utilized a complex two-chain chimeric relaxin peptide, R3(BΔ23-27)R/I5, in which a truncated relaxin-3 B-chain carrying an additional C-terminal Arg residue was combined with the insulin-like peptide 5 (INSL5) A-chain. In this study we demonstrate that, by replacing the native Cys in this truncated relaxin-3 B-chain with Ser, a single-chain linear peptide of 23 amino acids that retains high-affinity antagonism for RXFP3 can be achieved. In vivo studies demonstrate that this peptide, R3 B1-22R, antagonized relaxin-3/RXFP3 induced increases in feeding in rats after intracerebroventricular injection. Thus, R3 B1-22R represents an excellent tool for biological studies probing relaxin pharmacology and a lead molecule for the development of synthetically tractable, single-chain RXFP3 modulators for clinical use.

Application and Structural Analysis of Triazole‐Bridged Disulfide Mimetics in Cyclic Peptides

Publisher: Wiley

Date: 18-05-2020

DOI: 10.1002/ANIE.202003435

Engineered Conotoxin Differentially Blocks and Discriminates Rat and Human α7 Nicotinic Acetylcholine Receptors

Publisher: American Chemical Society (ACS)

Date: 27-04-2021

DOI: 10.1021/ACS.JMEDCHEM.0C02079

µ-Conotoxins Targeting the Human Voltage-Gated Sodium Channel Subtype Na_V1.7

Publisher: MDPI AG

Date: 30-08-2022

DOI: 10.3390/TOXINS14090600

Abstract: µ-Conotoxins are small, potent, peptide voltage-gated sodium (NaV) channel inhibitors characterised by a conserved cysteine framework. Despite promising in vivo studies indicating analgesic potential of these compounds, selectivity towards the therapeutically relevant subtype NaV1.7 has so far been limited. We recently identified a novel µ-conotoxin, SxIIIC, which potently inhibits human NaV1.7 (hNaV1.7). SxIIIC has high sequence homology with other µ-conotoxins, including SmIIIA and KIIIA, yet shows different NaV channel selectivity for mammalian subtypes. Here, we evaluated and compared the inhibitory potency of µ-conotoxins SxIIIC, SmIIIA and KIIIA at hNaV channels by whole-cell patch-cl electrophysiology and discovered that these three closely related µ-conotoxins display unique selectivity profiles with significant variations in inhibitory potency at hNaV1.7. Analysis of other µ-conotoxins at hNaV1.7 shows that only a limited number are capable of inhibition at this subtype and that differences between the number of residues in loop 3 appear to influence the ability of µ-conotoxins to inhibit hNaV1.7. Through mutagenesis studies, we confirmed that charged residues in this region also affect the selectivity for hNaV1.4. Comparison of µ-conotoxin NMR solution structures identified differences that may contribute to the variance in hNaV1.7 inhibition and validated the role of the loop 1 extension in SxIIIC for improving potency at hNaV1.7, when compared to KIIIA. This work could assist in designing µ-conotoxin derivatives specific for hNaV1.7.

A Continent of Plant Defense Peptide Diversity: Cyclotides in AustralianHybanthus(Violaceae)

Publisher: Oxford University Press (OUP)

Date: 30-09-2005

DOI: 10.1105/TPC.105.034678

Abstract: Cyclotides are plant-derived miniproteins that have the unusual features of a head-to-tail cyclized peptide backbone and a knotted arrangement of disulfide bonds. It had been postulated that they might be an especially large family of host defense agents, but this had not yet been tested by field data on cyclotide variation in wild plant populations. In this study, we s led Australian Hybanthus (Violaceae) to gain an insight into the level of variation within populations, within species, and between species. A wealth of cyclotide ersity was discovered: at least 246 new cyclotides are present in the 11 species s led, and 26 novel sequences were characterized. A new approach to the discovery of cyclotide sequences was developed based on the identification of a conserved sequence within a signal sequence in cyclotide precursors. The number of cyclotides in the Violaceae is now estimated to be & . Cyclotide physicochemical profiles were shown to be a useful taxonomic feature that reflected species and their morphological relationships. The novel sequences provided substantial insight into the tolerance of the cystine knot framework in cyclotides to amino acid substitutions and will facilitate protein engineering applications of this framework.

Conotoxins and their potential pharmaceutical applications

Publisher: Wiley

Date: 03-1999

DOI: 10.1002/(SICI)1098-2299(199903/04)46:3/4<219::AID-DDR7>3.0.CO;2-S

Yeast-based bioproduction of disulfide-rich peptides and their cyclization via asparaginyl endopeptidases

Publisher: Springer Science and Business Media LLC

Date: 17-02-2021

DOI: 10.1038/S41596-020-00483-0

Complete mitochondrial genome sequence of Indian medium carp, Labeo gonius (Hamilton, 1822) and its comparison with other related carp species.

Publisher: Informa UK Limited

Date: 28-12-2017

DOI: 10.3109/19401736.2015.1106517

Abstract: In the present study, the complete mitochondrial genome sequence of Labeo gonius is reported using PGM sequencer (Ion Torrent). The complete mitogenome of L. gonius is obtained by the de novo sequences assembly of genomic reads using the Torrent Mapping Alignment Program (TMAP) which is 16 614 bp in length. The mitogenome of L. gonius comprised of 13 protein-coding genes, 22 tRNAs, 2 rRNA genes, and D-loop as control region along with gene order and organization, being similar to most of other fish mitogenomes of NCBI databases. The mitogenome in the present study has 99% similarity to the complete mitogenome sequence of Labeo fimbriatus, as reported earlier. The phylogenetic analysis of Cypriniformes depicted that their mitogenomes are closely related to each other. The complete mitogenome sequence of L. gonius would be helpful in understanding the population genetics, phylogenetics, and evolution of Indian Carps.

Correction to Chemical Re-engineering of Chlorotoxin Improves Bioconjugation Properties for Tumor Imaging and Targeted Therapy

Publisher: American Chemical Society (ACS)

Date: 21-11-2013

DOI: 10.1021/JM4016119

Structural plasticity of the cyclic-cystine-knot framework: implications for biological activity and drug design

Publisher: Portland Press Ltd.

Date: 27-01-2006

DOI: 10.1042/BJ20051691

Abstract: The cyclotide family of plant proteins is of interest because of their unique topology, which combines a head-to-tail cyclic backbone with an embedded cystine knot, and because their remarkable chemical and biological properties make them ideal candidates as grafting templates for biologically active peptide epitopes. The present study describes the first steps towards exploiting the cyclotide framework by synthesizing and structurally characterizing two grafted analogues of the cyclotide kalata B1. The modified peptides have polar or charged residues substituted for residues that form part of a surface-exposed hydrophobic patch that plays a significant role in the folding and biological activity of kalata B1. Both analogues retain the native cyclotide fold, but lack the undesired haemolytic activity of their parent molecule, kalata B1. This finding confirms the tolerance of the cyclotide framework to residue substitutions and opens up possibilities for the substitution of biologically active peptide epitopes into the framework.

Application and Structural Analysis of Triazole‐Bridged Disulfide Mimetics in Cyclic Peptides

Publisher: Wiley

Date: 18-05-2020

DOI: 10.1002/ANGE.202003435

A comparative study of extraction methods reveals preferred solvents for cystine knot peptide isolation from Momordica cochinchinensis seeds

Publisher: Elsevier BV

Date: 06-2014

DOI: 10.1016/J.FITOTE.2014.02.016

Abstract: MCoTI-I and MCoTI-II (short for Momordica cochinchinensis Trypsin Inhibitor-I and -II, respectively) are attractive candidates for developing novel intracellular-targeting drugs because both are exceptionally stable and can internalize into cells. These seed-derived cystine knot peptides are ex les of how natural product discovery efforts can lead to biomedical applications. However, discovery efforts are sometimes h ered by the limited availability of seed materials, highlighting the need for efficient extraction methods. In this study, we assessed five extraction methods using M. cochinchinensis seeds, a source of well-characterized cystine knot peptides. The most efficient extraction of nine known cystine knot peptides was achieved by a method based on acetonitrile/water/formic acid (25:24:1), followed by methods based on sodium acetate (20 mM, pH 5.0), ammonium bicarbonate (5 mM, pH 8.0), and boiling water. On average, the yields obtained by these four methods were more than 250-fold higher than that obtained using dichloromethane/methanol (1:1) extraction, a previously applied standard method. Extraction using acetonitrile/water/formic acid (25:24:1) yielded the highest number of reconstructed masses within the majority of plant-derived cystine knot peptide mass range but only accounted for around 50% of the total number of masses, indicating that any single method may result in under-s ling. Applying acetonitrile/water/formic acid (25:24:1), boiling water, and ammonium bicarbonate (5 mM, pH 8.0) extractions either successively or discretely significantly increased the s ling number. Overall, acetonitrile/water/formic acid (25:24:1) can facilitate efficient extraction of cystine-knot peptides from M. cochinchinensis seeds but for discovery purposes the use of a combination of extraction methods is recommended where practical.

Structure−Activity Studies of Conantokins as Human N-Methyl-d-aspartate Receptor Modulators,

Publisher: American Chemical Society (ACS)

Date: 02-1999

DOI: 10.1021/JM981052Q

Calcium-Mediated Allostery of the EGF Fold

Publisher: American Chemical Society (ACS)

Date: 05-2018

DOI: 10.1021/ACSCHEMBIO.8B00291

Interaction of Synthetic Human SLURP-1 with the Nicotinic Acetylcholine Receptors

Publisher: Springer Science and Business Media LLC

Date: 30-11-2017

DOI: 10.1038/S41598-017-16809-0

Abstract: Human SLURP-1 is a secreted protein of the Ly6/uPAR/three-finger neurotoxin family that co-localizes with nicotinic acetylcholine receptors (nAChRs) and modulates their functions. Conflicting biological activities of SLURP-1 at various nAChR subtypes have been based on heterologously produced SLURP-1 containing N- and/or C-terminal extensions. Here, we report the chemical synthesis of the 81 amino acid residue human SLURP-1 protein, characterization of its 3D structure by NMR, and its biological activity at nAChR subtypes. Radioligand assays indicated that synthetic SLURP-1 did not compete with [ 125 I]-α-bungarotoxin (α-Bgt) binding to human neuronal α7 and Torpedo californica muscle-type nAChRs, nor to mollusk acetylcholine binding proteins (AChBP). Inhibition of human α7-mediated currents only occurred in the presence of the allosteric modulator PNU120596. In contrast, we observed robust SLURP-1 mediated inhibition of human α3β4, α4β4, α3β2 nAChRs, as well as human and rat α9α10 nAChRs. SLURP-1 inhibition of α9α10 nAChRs was accentuated at higher ACh concentrations, indicating an allosteric binding mechanism. Our results are discussed in the context of recent studies on heterologously produced SLURP-1 and indicate that N-terminal extensions of SLURP-1 may affect its activity and selectivity on its targets. In this respect, synthetic SLURP-1 appears to be a better probe for structure-function studies.

Synthesis and Structure Determination by NMR of a Putative Vacuolar Targeting Peptide and Model of a Proteinase Inhibitor from Nicotiana alata^,

Publisher: American Chemical Society (ACS)

Date: 1996

DOI: 10.1021/BI952228I

Evaluation of the in Vivo Aphrodisiac Activity of a Cyclotide Extract from Hybanthus enneaspermus

Publisher: American Chemical Society (ACS)

Date: 09-12-2020

DOI: 10.1021/ACS.JNATPROD.0C01045

Characterising the Subsite Specificity of Urokinase‐Type Plasminogen Activator and Tissue‐Type Plasminogen Activator using a Sequence‐Defined Peptide Aldehyde Library

Publisher: Wiley

Date: 24-10-2018

DOI: 10.1002/CBIC.201800395

Abstract: Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are two serine proteases that contribute to initiating fibrinolysis by activating plasminogen. uPA is also an important tumour-associated protease due to its role in extracellular matrix remodelling. Overexpression of uPA has been identified in several different cancers and uPA inhibition has been reported as a promising therapeutic strategy. Although several peptide-based uPA inhibitors have been developed, the extent to which uPA tolerates different tetrapeptide sequences that span the P1-P4 positions remains to be thoroughly explored. In this study, we screened a sequence-defined peptide aldehyde library against uPA and tPA. Preferred sequences from the library screen yielded potent inhibitors for uPA, led by Ac-GTAR-H (K

Accurate de novo design of hyperstable constrained peptides

Publisher: Springer Science and Business Media LLC

Date: 14-09-2016

DOI: 10.1038/NATURE19791

Design and Synthesis of Truncated EGF-A Peptides that Restore LDL-R Recycling in the Presence of PCSK9 In Vitro

Publisher: Elsevier BV

Date: 02-2014

DOI: 10.1016/J.CHEMBIOL.2013.11.014

Abstract: Disrupting the binding interaction between proprotein convertase (PCSK9) and the epidermal growth factor-like domain A (EGF-A domain) in the low-density lipoprotein receptor (LDL-R) is a promising strategy to promote LDL-R recycling and thereby lower circulating cholesterol levels. In this study, truncated 26 amino acid EGF-A analogs were designed and synthesized, and their structures were analyzed in solution and in complex with PCSK9. The most potent peptide had an increased binding affinity for PCSK9 (KD = 0.6 μM) compared with wild-type EGF-A (KD = 1.2 μM), and the ability to increase LDL-R recycling in the presence of PCSK9 in a cell-based assay.

Antimicrobial peptides in plants.

Publisher: CABI

Date: 2010

DOI: 10.1079/9781845936570.0040

Abstract: is chapter provides an overview of plant antimicrobial peptides. It mainly focuses on one particular class of plant defence peptides, namely the cyclotides, which have been discovered over the last decade in plants from the Rubiaceae , Violaceae and Cucurbitaceae families. Cyclotides have a head-to-tail cyclized peptide backbone and a cystine knot motif formed from their six conserved cysteine residues, which makes them exceptionally stable. This chapter describes their isolation and characterization, structure and biosynthesis, and applications. The structural stability of cyclotides makes them excellent scaffolds for the engineering of novel therapeutic proteins. Advances in methods for the production of cyclotides and their potential clinical applications are also described.

Plant-Derived Decapeptide OSIP108 Interferes with Candida albicans Biofilm Formation without Affecting Cell Viability

Publisher: American Society for Microbiology

Date: 05-2014

DOI: 10.1128/AAC.01274-13

Abstract: We previously identified a decapeptide from the model plant Arabidopsis thaliana , OSIP108, which is induced upon fungal pathogen infection. In this study, we demonstrated that OSIP108 interferes with biofilm formation of the fungal pathogen Candida albicans without affecting the viability or growth of C. albicans cells. OSIP108 displayed no cytotoxicity against various human cell lines. Furthermore, OSIP108 enhanced the activity of the antifungal agents hotericin B and caspofungin in vitro and in vivo in a Caenorhabditis elegans-C. albicans biofilm infection model. These data point to the potential use of OSIP108 in combination therapy with conventional antifungal agents. In a first attempt to unravel its mode of action, we screened a library of 137 homozygous C. albicans mutants, affected in genes encoding cell wall proteins or transcription factors important for biofilm formation, for altered OSIP108 sensitivity. We identified 9 OSIP108-tolerant C. albicans mutants that were defective in either components important for cell wall integrity or the yeast-to-hypha transition. In line with these findings, we demonstrated that OSIP108 activates the C. albicans cell wall integrity pathway and that its antibiofilm activity can be blocked by compounds inhibiting the yeast-to-hypha transition. Furthermore, we found that OSIP108 is predominantly localized at the C. albicans cell surface. These data point to interference of OSIP108 with cell wall-related processes of C. albicans , resulting in impaired biofilm formation.

A Chemoenzymatic Approach To Produce a Cyclic Analogue of the Analgesic Drug MVIIA (Ziconotide)

Publisher: Wiley

Date: 31-05-2023

DOI: 10.1002/ANGE.202302812

Abstract: Ziconotide (ω‐conotoxin MVIIA) is an approved analgesic for the treatment of chronic pain. However, the need for intrathecal administration and adverse effects have limited its widespread application. Backbone cyclization is one way to improve the pharmaceutical properties of conopeptides, but so far chemical synthesis alone has been unable to produce correctly folded and backbone cyclic analogues of MVIIA. In this study, an asparaginyl endopeptidase (AEP)‐mediated cyclization was used to generate backbone cyclic analogues of MVIIA for the first time. Cyclization using six‐ to nine‐residue linkers did not perturb the overall structure of MVIIA, and the cyclic analogues of MVIIA showed inhibition of voltage‐gated calcium channels (Ca V 2.2) and substantially improved stability in human serum and stimulated intestinal fluid. Our study reveals that AEP transpeptidases are capable of cyclizing structurally complex peptides that chemical synthesis cannot achieve and paves the way for further improving the therapeutic value of conotoxins.

Enzyme Mechanism and Function of a Novel Plant PDI Involved in the Oxidative Folding of Cystine Knot Defense Peptides

Publisher: Springer New York

Date: 2009

DOI: 10.1007/978-0-387-73657-0_13

Disulfide Folding Pathways of Cystine Knot Proteins

Publisher: Elsevier BV

Date: 02-2003

DOI: 10.1074/JBC.M210492200

Towards a generic prototyping approach for therapeutically-relevant peptides and proteins in a cell-free translation system

Publisher: Springer Science and Business Media LLC

Date: 11-01-2022

DOI: 10.1038/S41467-021-27854-9

Abstract: Advances in peptide and protein therapeutics increased the need for rapid and cost-effective polypeptide prototyping. While in vitro translation systems are well suited for fast and multiplexed polypeptide prototyping, they suffer from misfolding, aggregation and disulfide-bond scrambling of the translated products. Here we propose that efficient folding of in vitro produced disulfide-rich peptides and proteins can be achieved if performed in an aggregation-free and thermodynamically controlled folding environment. To this end, we modify an E. coli -based in vitro translation system to allow co-translational capture of translated products by affinity matrix. This process reduces protein aggregation and enables productive oxidative folding and recycling of misfolded states under thermodynamic control. In this study we show that the developed approach is likely to be generally applicable for prototyping of a wide variety of disulfide-constrained peptides, macrocyclic peptides with non-native bonds and antibody fragments in amounts sufficient for interaction analysis and biological activity assessment.

A systematic approach to document cyclotide distribution in plant species from genomic, transcriptomic, and peptidomic analysis

Publisher: Wiley

Date: 09-2013

DOI: 10.1002/BIP.22258

Abstract: Cyclotides are a large family of plant peptides characterized by their cyclic cystine knot composed of a circular backbone and three disulfide bonds that impart exceptional stability. They, and several acyclic variants, have been isolated from plants within the Rubiaceae, Violaceae, Cucurbitaceae, Fabaceae, Solanaceae, and Poaceae families. A variety of chemical and genetic approaches have been applied for the discovery and characterization of cyclotides. As investigations of cyclotide expression, distribution, and phylogeny rapidly increase, the authors have proposed the inclusion of information pertaining to plant species that have been analyzed but do not appear to express cyclotides into the CyBase database. CyBase is dedicated to providing web tools and information about cyclic peptides and proteins to the scientific community. Including detailed information about s ling and analysis parameters of plant species that have been investigated but not published elsewhere should assist in the process of selecting species for establishing new cyclotide discovery projects, as well as for detailed reanalysis using alternative technical approaches. In summary, the collection and deposition of all plant species that have been examined (whether cyclotides have been found or not) would help to impart a deeper understanding of cyclotide discovery, evolution, and physiological function. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 433–437, 2013.

Sunflower Trypsin Inhibitor‐1 (SFTI‐1): Sowing Seeds in the Fields of Chemistry and Biology

Publisher: Wiley

Date: 30-11-2020

DOI: 10.1002/ANIE.202006919

PTRF/cavin-1 neutralizes non-caveolar caveolin-1 microdomains in prostate cancer

Publisher: Springer Science and Business Media LLC

Date: 12-08-2013

DOI: 10.1038/ONC.2013.315

Abstract: Caveolin-1 has a complex role in prostate cancer and has been suggested to be a potential biomarker and therapeutic target. As mature caveolin-1 resides in caveolae, invaginated lipid raft domains at the plasma membrane, caveolae have been suggested as a tumor-promoting signaling platform in prostate cancer. However, caveola formation requires both caveolin-1 and cavin-1 (also known as PTRF polymerase I and transcript release factor). Here, we examined the expression of cavin-1 in prostate epithelia and stroma using tissue microarray including normal, non-malignant and malignant prostate tissues. We found that caveolin-1 was induced without the presence of cavin-1 in advanced prostate carcinoma, an expression pattern mirrored in the PC-3 cell line. In contrast, normal prostate epithelia expressed neither caveolin-1 nor cavin-1, while prostate stroma highly expressed both caveolin-1 and cavin-1. Utilizing PC-3 cells as a suitable model for caveolin-1-positive advanced prostate cancer, we found that cavin-1 expression in PC-3 cells inhibits anchorage-independent growth, and reduces in vivo tumor growth and metastasis in an orthotopic prostate cancer xenograft mouse model. The expression of α-smooth muscle actin in stroma along with interleukin-6 (IL-6) in cancer cells was also decreased in tumors of mice bearing PC-3-cavin-1 tumor cells. To determine whether cavin-1 acts by neutralizing caveolin-1, we expressed cavin-1 in caveolin-1-negative prostate cancer LNCaP and 22Rv1 cells. Caveolin-1 but not cavin-1 expression increased anchorage-independent growth in LNCaP and 22Rv1 cells. Cavin-1 co-expression reversed caveolin-1 effects in caveolin-1-positive LNCaP cells. Taken together, these results suggest that caveolin-1 in advanced prostate cancer is present outside of caveolae, because of the lack of cavin-1 expression. Cavin-1 expression attenuates the effects of non-caveolar caveolin-1 microdomains partly via reduced IL-6 microenvironmental function. With circulating caveolin-1 as a potential biomarker for advanced prostate cancer, identification of the molecular pathways affected by cavin-1 could provide novel therapeutic targets.

¹H‐NMR structural studies of a cystine‐linked peptide containing residues 71–93 of transthyretin and effects of a Ser84 substitution implicated in familial amyloidotic polyneuropathy

Publisher: Wiley

Date: 06-1999

DOI: 10.1046/J.1432-1327.1999.00423.X

Abstract: The Ile-->Ser84 substitution in the thyroid hormone transport protein transthyretin is one of over 50 variations found to be associated with familial amyloid polyneuropathy, a hereditary type of lethal amyloidosis. Using a peptide analogue of the loop containing residue 84 in transthyretin, we have examined the putative local structural effects of this substitution using 1H-NMR spectroscopy. The peptide, containing residues 71-93 of transthyretin with its termini linked via a disulfide bond, was found to possess the same helix-turn motif as in the corresponding region of the crystallographically derived structure of transthyretin in 20% trifluoroethanol (TFE) solution. It therefore, represents a useful model with which to examine the effects of amyloidogenic substitutions. In a peptide analogue containing the Ile84-->Ser substitution it was found that the substitution does not greatly disrupt the overall three-dimensional structure, but leads to minor local differences at the turn in which residue 84 is involved. Coupling constant and NOE measurements indicate that the helix-turn motif is still present, but differences in chemical shifts and amide-exchange rates reflect a small distortion. This is in keeping with observations that several other mutant forms of transthyretin display similar subunit interactions and those that have been structurally analysed possess a near native structure. We propose that the Ser84 mutation induces only subtle perturbations to the transthyretin structure which predisposes the protein to amyloid formation.

Using the MCoTI-II Cyclotide Scaffold To Design a Stable Cyclic Peptide Antagonist of SET, a Protein Overexpressed in Human Cancer

Publisher: American Chemical Society (ACS)

Date: 06-01-2016

DOI: 10.1021/ACS.BIOCHEM.5B00529

Abstract: The SET protein is a promising drug target in cancer therapy, because of its ability to inhibit the function of the tumor suppressor gene protein phosphatase 2A (PP2A). COG peptides, derived from apolipoprotein E (apoE), are potent antagonists of SET they induce cytotoxicity in cancer cells upon binding to intracellular SET and modulate the nuclear factor kappa B (NF-κB) signaling pathway. However, the therapeutic potential of COG peptides is limited, because of their poor proteolytic stability and low bioavailability. In this study, the COG peptide, COG1410, was stabilized by grafting it onto the ultrastable cyclic peptide scaffold, Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II). The grafted MCoTI-II peptides were cytotoxic to a cancer cell line and showed high stability in human serum. The most potent grafted MCoTI-II peptide inhibited lipopolysaccharide (LPS)-mediated activation of NF-κB in murine macrophages. Overall, this study demonstrates the application of the MCoTI-II scaffold for the development of stable peptide drugs for cancer therapy.

The structure of a two-disulfide intermediate assists in elucidating the oxidative folding pathway of a cyclic cystine knot protein

Publisher: Elsevier BV

Date: 06-2008

DOI: 10.1016/J.STR.2008.02.023

Abstract: We have determined the three-dimensional structure of a two-disulfide intermediate (Cys(8)-Cys(20), Cys(14)-Cys(26)) on the oxidative folding pathway of the cyclotide MCoTI-II. Cyclotides have a range of bioactivities and, because of their exceptional stability, have been proposed as potential molecular scaffolds for drug design applications. The three-dimensional structure of the stable two-disulfide intermediate shows for the most part identical secondary and tertiary structure to the native state. The only exception is a flexible loop, which is collapsed onto the protein core in the native state, whereas in the intermediate it is more loosely associated with the remainder of the protein. The results suggest that the native fold of the peptide does not represent the free energy minimum in the absence of the Cys(1)-Cys(18) disulfide bridge and that although there is not a large energy barrier, the peptide must transiently adopt an energetically unfavorable state before the final disulfide can form.

Solution Structure of BSTI:  A New Trypsin Inhibitor from Skin Secretions of Bombina bombina^,

Publisher: American Chemical Society (ACS)

Date: 17-03-2001

DOI: 10.1021/BI002623V

Abstract: The three-dimensional solution structure of BSTI, a trypsin inhibitor from the European frog Bombina bombina, has been solved using (1)H NMR spectroscopy. The 60 amino acid protein contains five disulfide bonds, which were unambiguously determined to be Cys (4--38), Cys (13--34), Cys (17--30), Cys (21--60), and Cys (40--54) by experimental restraints and subsequent structure calculations. The main elements of secondary structure are four beta-strands, arranged as two small antiparallel beta-sheets. The overall fold of BSTI is disk shaped and is characterized by the lack of a hydrophobic core. The presumed active site is located on a loop comprising residues 21--34, which is a relatively disordered region similar to that seen in many other protease inhibitors. However, the overall fold is different to other known protease inhibitors with the exception of a small family of inhibitors isolated from nematodes of the family Ascaris and recently also from the haemolymph of Apis mellifera. BSTI may thus be classified as a new member of this recently discovered family of protease inhibitors.

Characterization of a Bioactive Acyclotide from Palicourea rigida

Publisher: American Chemical Society (ACS)

Date: 03-11-2016

DOI: 10.1021/ACS.JNATPROD.6B00270

Abstract: The extraction and purification of parigidin-br3, a cyclotide analogue belonging to the "bracelet" subfamily, from Palicourea rigida leaves is discussed. Unlike conventional cyclotides, parigidin-br3 has free N- and C-termini, as identified by MALDI-TOF/TOF analysis and confirmed by gene structure elucidation, and is one of a small number of acyclotides discovered during recent years. Parigidin-br3 showed cytotoxic activity against MCF-7 (breast cancer) and CACO2 (colorectal adenocarcinoma) cells, with IC

Structure determination of the three disulfide bond isomers of α-conotoxin GI: A model for the role of disulfide bonds in structural stability

Publisher: Elsevier BV

Date: 05-1998

DOI: 10.1006/JMBI.1998.1701

Abstract: The three possible disulfide bonded isomers of alpha-conotoxin GI have been selectively synthesised and their structures determined by 1H NMR spectroscopy. alpha-Conotoxin GI derives from the venom of Conus geographus and is a useful neuropharmacological tool as it selectively binds to the nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel involved in nerve signal transmission. The peptide has the sequence ECCNPACGRHYSC-NH2, and the three disulfide bonded isomers are referred to as GI(2-7 -13), GI(2-13 -7) and GI(2-3 -13). The NMR structure for the native isomer GI(2-7 -13) is of excellent quality, with a backbone pairwise RMSD of 0.16 A for a family of 35 structures, and comprises primarily a distorted 310 helix between residues 5 to 11. The two non-native isomers exhibit multiple conformers in solution, with the major populated forms being different in structure both from each other and from the native form. Structure-activity relationships for the native GI(2-7 -13) as well as the role of the disulfide bonds on folding and stability of the three isomers are examined. It is concluded that the disulfide bonds in alpha-conotoxin GI play a crucial part in determining both the structure and stability of the peptide. A trend for increased conformational heterogeneity was observed in the order of GI(2-7 -13)<GI(2-13 -7)<GI(2-3 -13). It was found that the peptide bond joining Cys2 to Cys3 in GI(2-3 -13) is predominantly trans, rather than cis as theoretically predicted. These structural data are used to interpret the varying nAChR binding of the non-native forms.A model for the binding of native GI(2-7 -13) to the mammalian nAChR is proposed, with an alpha-subunit binding face made up of Cys2, Asn4, Pro5, Ala6 and Cys7 and a selectivity face, comprised of Arg9 and His10. These two faces orient the molecule between the alpha and delta subunits of the receptor. The structure of the CCNPAC sequence of the native GI(2-7 -13) is compared to the structure of the identical sequence from the toxic domain of heat-stable enterotoxins, which forms part of the receptor binding region of the enterotoxins, but which has a different disulfide connectivity.

Heparan sulfate regulates IL-21 bioavailability and signal strength that control germinal center B cell selection and differentiation

Publisher: American Association for the Advancement of Science (AAAS)

Date: 24-02-2023

DOI: 10.1126/SCIIMMUNOL.ADD1728

Abstract: In antibody responses, mutated germinal center B (B GC ) cells are positively selected for reentry or differentiation. As the products from GCs, memory B cells and antibody-secreting cells (ASCs) support high-affinity and long-lasting immunity. Positive selection of B GC cells is controlled by signals received through the B cell receptor (BCR) and follicular helper T (T FH ) cell–derived signals, in particular costimulation through CD40. Here, we demonstrate that the T FH cell effector cytokine interleukin-21 (IL-21) joins BCR and CD40 in supporting B GC selection and reveal that strong IL-21 signaling prioritizes ASC differentiation in vivo. B GC cells, compared with non-B GC cells, show significantly reduced IL-21 binding and attenuated signaling, which is mediated by low cellular heparan sulfate (HS) sulfation. Mechanistically, N-deacetylase and N-sulfotransferase 1 (Ndst1)–mediated N-sulfation of HS in B cells promotes IL-21 binding and signal strength. Ndst1 is down-regulated in B GC cells and up-regulated in ASC precursors, suggesting selective desensitization to IL-21 in B GC cells. Thus, specialized biochemical regulation of IL-21 bioavailability and signal strength sets a balance between the stringency and efficiency of GC selection.

Discovery and Characterization of Cyclotides from Rinorea Species

Publisher: American Chemical Society (ACS)

Date: 02-11-2018

DOI: 10.1021/ACS.JNATPROD.8B00572

Abstract: Cyclotides are macrocyclic cystine-knotted peptides most commonly found in the Violaceae plant family. Although Rinorea is the second-largest genera within the Violaceae family, few studies have examined whether or not they contain cyclotides. To further our understanding of cyclotide ersity and evolution, we examined the cyclotide content of two Rinorea species found in Southeast Asia: R. virgata and R. bengalensis. Seven cyclotides were isolated from R. virgata (named Rivi1-7), and a known cyclotide (cT10) was found in R. bengalensis. Loops 2, 5, and 6 of Rivi1-4 contained sequences not previously seen in corresponding loops of known cyclotides, thereby expanding our understanding of the ersity of cyclotides. In addition, the sequence of loop 2 of Rivi3 and Rivi4 were identical to some related noncyclic "acyclotides" from the Poaceae plant family. As only acyclotides, but not cyclotides, have been reported in monocotyledons thus far, our findings support an evolutionary link between monocotyledon-derived ancestral cyclotide precursors and dicotyledon-derived cyclotides. Furthermore, Rivi2 and Rivi3 had comparable cytotoxic activities to the most cytotoxic cyclotide known to date: cycloviolacin O2 from Viola odorata yet, unlike cycloviolacin O2, they did not show hemolytic activity. Therefore, these cyclotides represent novel scaffolds for use in future anticancer drug design.

Design of substrate-based BCR-ABL kinase inhibitors using the cyclotide scaffold

Publisher: Springer Science and Business Media LLC

Date: 12-08-2015

DOI: 10.1038/SREP12974

Abstract: The constitutively active tyrosine kinase BCR-ABL is the underlying cause of chronic myeloid leukemia (CML). Current CML treatments rely on the long-term use of tyrosine kinase inhibitors (TKIs), which target the ATP binding site of BCR-ABL. Over the course of treatment, 20–30% of CML patients develop TKI resistance, which is commonly attributed to point mutations in the drug-binding region. We design a new class of peptide inhibitors that target the substrate-binding site of BCR-ABL by grafting sequences derived from abltide, the optimal substrate of Abl kinase, onto a cell-penetrating cyclotide MCoTI-II. Three grafted cyclotides show significant Abl kinase inhibition in vitro in the low micromolar range using a novel kinase inhibition assay. Our work also demonstrates that a reengineered MCoTI-II with abltide sequences grafted in both loop 1 and 6 inhibits the activity of [T315I]Abl in vitro , a mutant Abl kinase harboring the “gatekeeper” mutation which is notorious for being multidrug resistant. Results from serum stability and cell internalization studies confirm that the MCoTI-II scaffold provides enzymatic stability and cell-penetrating properties to the lead molecules. Taken together, our study highlights that reengineered cyclotides incorporating abltide-derived sequences are promising substrate-competitive inhibitors for Abl kinase and the T315I mutant.

New indole alkaloids from the bark of Alstonia scholaris

Publisher: American Chemical Society (ACS)

Date: 29-07-2004

DOI: 10.1021/NP0498612

Abstract: A new indole alkaloid, akuammiginone (1), and a new glycosidic indole alkaloid, echitamidine-N-oxide 19-O-beta-d-glucopyranoside (2), together with the five known alkaloids, echitaminic acid (3), echitamidine N-oxide (4), N(b)-demethylalstogustine N-oxide (5), akuammicine N-oxide (6), and N(b)-demethylalstogustine (7), were isolated from the trunk bark of Alstonia scholaris collected in Timor, Indonesia. The structures of all compounds were elucidated by spectroscopic methods. This is the first report of compounds 3-5and 7 in A. scholaris. Some NMR assignments of the known compounds were revised.

Structure–Activity Studies Reveal the Molecular Basis for GABA_B-Receptor Mediated Inhibition of High Voltage-Activated Calcium Channels by α-Conotoxin Vc1.1

Publisher: American Chemical Society (ACS)

Date: 10-05-2018

DOI: 10.1021/ACSCHEMBIO.8B00190

Abstract: α-Conotoxins are disulfide-bonded peptides from cone snail venoms and are characterized by their affinity for nicotinic acetylcholine receptors (nAChR). Several α-conotoxins with distinct selectivity for nAChR subtypes have been identified as potent analgesics in animal models of chronic pain. However, a number of α-conotoxins have been shown to inhibit N-type calcium channel currents in rodent dissociated dorsal root ganglion (DRG) neurons via activation of G protein-coupled GABA

Conformational Analysis of LYS(11−36), a Peptide Derived from the β-Sheet Region of T4 Lysozyme, in TFE and SDS

Publisher: American Chemical Society (ACS)

Date: 09-1997

DOI: 10.1021/BI970730S

Truncated Glucagon-like Peptide-1 and Exendin-4 α-Conotoxin pl14a Peptide Chimeras Maintain Potency and α-Helicity and Reveal Interactions Vital for cAMP Signaling in Vitro

Publisher: Elsevier BV

Date: 07-2016

DOI: 10.1074/JBC.M116.724542

Manipulation of a spider peptide toxin alters its affinity for lipid bilayers and potency and selectivity for voltage-gated sodium channel subtype 1.7

Publisher: Elsevier BV

Date: 04-2020

DOI: 10.1074/JBC.RA119.012281

Discovery and applications of naturally occurring cyclic peptides

Publisher: Elsevier BV

Date: 03-2011

DOI: 10.1016/J.DDTEC.2011.07.005

Gene coevolution and regulation lock cyclic plant defence peptides to their targets

Publisher: Wiley

Date: 15-12-2016

DOI: 10.1111/NPH.13789

Abstract: Plants have evolved many strategies to protect themselves from attack, including peptide toxins that are ribosomally synthesized and thus adaptable directly by genetic polymorphisms. Certain toxins in Clitoria ternatea (butterfly pea) are cyclic cystine‐knot peptides of c . 30 residues, called cyclotides, which have co‐opted the plant's albumin‐1 gene family for their production. How butterfly pea albumin‐1 genes were commandeered and how these cyclotides are utilized in defence remain unclear. The role of cyclotides in host plant ecology and biotechnological applications requires exploration. We characterized the sequence ersity and expression dynamics of precursor and processing proteins implicated in butterfly pea cyclotide biosynthesis by expression profiling through RNA‐sequencing ( RNA ‐seq). Peptide‐enriched extracts from various organs were tested for activity against insect‐like membranes and the model nematode Caenorhabditis elegans . We found that the evolution and deployment of cyclotides involved their ersification to exhibit different chemical properties and expression between organs facing different defensive challenges. Cyclotide‐enriched fractions from soil‐contacting organs were effective at killing nematodes, whereas similar enriched fractions from aerial organs contained cyclotides that exhibited stronger interactions with insect‐like membrane lipids. Cyclotides are employed as versatile and combinatorial mediators of defence in C. ternatea and have specialized to affect different classes of attacking organisms.

Distribution and Evolution of Circular Miniproteins in Flowering Plants

Publisher: Oxford University Press (OUP)

Date: 09-2008

DOI: 10.1105/TPC.108.062331

Abstract: Cyclotides are disulfide-rich miniproteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. Cyclotides have been found only in two plant families: in every analyzed species of the violet family (Violaceae) and in few species of the coffee family (Rubiaceae). In this study, we analyzed & Rubiaceae species and confirmed the presence of cyclotides in 22 species. Additionally, we analyzed & species in related plant families to Rubiaceae and Violaceae and report the occurrence of cyclotides in the Apocynaceae. We further report new cyclotide sequences that provide insights into the mechanistic basis of cyclotide evolution. On the basis of the phylogeny of cyclotide-bearing plants and the analysis of cyclotide precursor gene sequences, we hypothesize that cyclotide evolution occurred independently in various plant families after the ergence of Asterids and Rosids (∼125 million years ago). This is strongly supported by recent findings on the in planta biosynthesis of cyclotides, which involves the serendipitous recruitment of ubiquitous proteolytic enzymes for cyclization. We further predict that the number of cyclotides within the Rubiaceae may exceed tens of thousands, potentially making cyclotides one of the largest protein families in the plant kingdom.

Effects of Cyclization on Stability, Structure, and Activity of α-Conotoxin RgIA at the α9α10 Nicotinic Acetylcholine Receptor and GABA_B Receptor

Publisher: American Chemical Society (ACS)

Date: 15-09-2011

DOI: 10.1021/JM201060R

Abstract: α-Conotoxin RgIA is of interest as a lead in the development of drugs for neuropathic pain. It modulates the α9α10 nicotinic acetylcholine receptor (nAChR) and the GABA(B) receptor, both of which are implicated in antinociception. However, because of its peptidic nature, RgIA is potentially susceptible to generic problems encountered by peptide-based drugs of poor oral bioavailability, short biological half-life, and low stability. Here, we improved the biopharmaceutical properties of RgIA by backbone cyclization using 3-7 residue peptidic linkers. Cyclization with a six-residue linker does not perturb the overall structure of RgIA, improves selectivity for the GABA(B) receptor over the α9α10 nAChR, and improves stability in human serum. The results provide insights to further improve the therapeutic properties of RgIA and other conotoxins being considered as drug leads and confirm that cyclization is a readily applicable strategy to improve the stability of peptides with proximate N- and C-termini.

Peptide Macrocyclization by a Bifunctional Endoprotease

Publisher: Elsevier BV

Date: 05-2015

DOI: 10.1016/J.CHEMBIOL.2015.04.010

Abstract: Proteases usually cleave peptides, but under some conditions, they can ligate them. Seeds of the common sunflower contain the 14-residue, backbone-macrocyclic peptide sunflower trypsin inhibitor 1 (SFTI-1) whose maturation from its precursor has a genetic requirement for asparaginyl endopeptidase (AEP). To provide more direct evidence, we developed an in situ assay and used (18)O-water to demonstrate that SFTI-1 is excised and simultaneously macrocyclized from its linear precursor. The reaction is inefficient in situ, but a newfound breakdown pathway can mask this inefficiency by reducing the internal disulfide bridge of any acyclic-SFTI to thiols before degrading it. To confirm AEP can directly perform the excision/ligation, we produced several recombinant plant AEPs in E. coli, and one from jack bean could catalyze both a typical cleavage reaction and cleavage-dependent, intramolecular transpeptidation to create SFTI-1. We propose that the evolution of ligating endoproteases enables plants like sunflower and jack bean to stabilize bioactive peptides.

Delineation of the unbinding pathway of α-conotoxin ImI from the α7 nicotinic acetylcholine receptor

Publisher: American Chemical Society (ACS)

Date: 18-05-2012

DOI: 10.1021/JP301352D

Abstract: α-Conotoxins potently and specifically inhibit isoforms of nicotinic acetylcholine receptors (nAChRs) and are used as molecular probes and as drugs or drug leads. Interactions occurring during binding and unbinding events are linked to binding kinetics, and knowledge of these interactions could help in the development of α-conotoxins as drugs. Here, the unbinding process for the prototypical α-conotoxin ImI/α7-nAChR system was investigated theoretically, and three exit routes were identified using random accelerated molecular dynamics simulations. The route involving the smallest conformation perturbation was further ided into three subpathways, which were studied using steered molecular dynamics simulations. Of the three subpathways, two had better experimental support and lower potential of mean force, indicating that they might be s led more frequently. Additionally, these subpathways were supported by previous experimental studies. Several pairwise interactions, including a cation-π interaction and charge and hydrogen bond interactions, were identified as potentially playing important roles in the unbinding event.

Synthesis and Structural Analysis of the N-Terminal Domain of the Thyroid Hormone-Binding Protein Transthyretin

Publisher: Walter de Gruyter GmbH

Date: 10-01-2002

DOI: 10.1515/CCLM.2002.212

Design, synthesis, and characterization of cyclic analogues of the iron regulatory peptide hormone hepcidin

Publisher: Wiley

Date: 09-2013

DOI: 10.1002/BIP.22350

Abstract: The peptide hormone hepcidin is a key regulator of iron homeostasis in vertebrates. Hepcidin acts by binding to ferroportin, the sole known iron exporter, causing it to be internalized and thus trapping iron within the cell. Dysregulation of hepcidin concentrations is associated with a range of iron‐related diseases and hepcidin‐based therapeutics could be developed as candidate treatments for these diseases. However peptide‐based drugs, despite their many advantages, are often limited by their susceptibility to degradation within the body. Here we describe the design, synthesis and characterization of a series of backbone cyclized hepcidin analogues as an approach to produce stable hepcidin‐based leads. The cyclic peptides were shown by NMR to be structurally analogous to native hepcidin. Comparison of the stability of hepcidin with one of the cyclic analogues in human serum revealed that 77% of the cyclic peptide but only 18% of linear hepcidin remained after 24 h. The cyclic peptides were tested for their ability to induce internalization of GFP‐ferroportin in vitro but were all found to be inactive. This study demonstrates that backbone cyclization of disulfide‐rich peptides is a suitable approach for increasing stability. However, careful consideration of a number of factors, including location of important residues and their bioactive conformation, is required to generate biologically active lead molecules. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 519–526, 2013.

Der Sonnenblumen‐Trypsin‐Inhibitor 1 (SFTI‐1) in der Chemie und Biologie

Publisher: Wiley

Date: 30-11-2020

DOI: 10.1002/ANGE.202006919

Circular proteins from Melicytus (Violaceae) refine the conserved protein and gene architecture of cyclotides

Publisher: Royal Society of Chemistry (RSC)

Date: 2009

DOI: 10.1039/B823020J

Abstract: Cyclotides are cyclic disulfide rich mini-proteins found in various Rubiaceae (coffee family), Violaceae (violet family) and Cucurbitaceae (squash family) plant species. Within the Violaceae, cyclotides have been found in numerous species of the genus Viola as well as species from two other genera, namely Hybanthus and Leonia. This is the first in-depth report of cyclotides in the genus Melicytus (Violaceae). We present the chromatographic profiles of extracts of eight Melicytus species and one Melicytus hybrid that were found to contain these circular peptides. We isolated and characterised five novel cyclotides (mra1 to mra5) from the aerial parts of a common New Zealand tree, Melicytus ramiflorus. All five peptides show the characteristics of the bracelet subfamily of cyclotides. Furthermore, we isolated 17 non-redundant cDNA clones from the leaves of Melicytus ramiflorus encoding cyclotide prepropeptides. This detailed report on the presence of cyclotides in several species of the genus Melicytus further strengthens our hypothesis that cyclotides are ubiquitous in Violaceae family plants and provides additional insight into the biochemical processing mechanisms that produce the cyclic protein backbone of this unique family of ultra-stable plant proteins.

Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis

Publisher: Proceedings of the National Academy of Sciences

Date: 04-09-2001

DOI: 10.1073/PNAS.191366898

Abstract: Several members of the Rubiaceae and Violaceae families produce a series of cyclotides or macrocyclic peptides of 29–31 amino acids with an embedded cystine knot. We aim to understand the mechanism of synthesis of cyclic peptides in plants and have isolated a cDNA clone that encodes the cyclotide kalata B1 as well as three other clones for related cyclotides from the African plant Oldenlandia affinis . The cDNA clones encode prepropeptides with a 20-aa signal sequence, an N-terminal prosequence of 46–68 amino acids and one, two, or three cyclotide domains separated by regions of about 25 aa. The corresponding cyclotides have been isolated from plant material, indicating that the cyclotide domains are excised and cyclized from all four predicted precursor proteins. The exact processing site is likely to lie on the N-terminal side of the strongly conserved GlyLeuPro or SerLeuPro sequence that flanks both sides of the cyclotide domain. Cyclotides have previously been assigned an antimicrobial function here we describe a potent inhibitory effect on the growth and development of larvae from the Lepidopteran species Helicoverpa punctigera.

Conotoxin modulation of voltage-gated sodium channels.

Publisher: Elsevier BV

Date: 2007

DOI: 10.1016/J.BIOCEL.2007.08.017

Abstract: The rising phase of the action potential in excitable cells is mediated by voltage-gated sodium channels (VGSCs), of which there are nine mammalian subtypes with distinct tissue distribution and biophysical properties. The involvement of certain VGSC subtypes in disease states such as pain and epilepsy highlights the need for agents that modulate VGSCs in a subtype-specific manner. Conotoxins from marine snails of the Conus genus constitute a promising source of such modulators, since these peptide toxins have evolved to become selective for various membrane receptors, ion channels and transporters in excitable cells. This review covers the structure and function of three classes of conopeptides that modulate VGSCs: the pore-blocking mu-conotoxins, the delta-conotoxins which delay or inhibit VGSC inactivation, and the microO-conotoxins which inhibit VGSC Na+ conductance independent of the tetrodotoxin binding site. Some of these toxins have potential therapeutic and research applications, in particular the microO-conotoxins, which may develop into potential drug leads for the treatment of pain states.

Identification of survival-promoting OSIP108 peptide variants and their internalization in human cells

Publisher: Elsevier BV

Date: 2017

DOI: 10.1016/J.MAD.2016.07.013

Abstract: The plant-derived decapeptide OSIP108 increases tolerance of yeast and human cells to apoptosis-inducing agents, such as copper and cisplatin. We performed a whole amino acid scan of OSIP108 and conducted structure-activity relationship studies on the induction of cisplatin tolerance (CT) in yeast. The use of cisplatin as apoptosis-inducing trigger in this study should be considered as a tool to better understand the survival-promoting nature of OSIP108 and not for purposes related to anti-cancer treatment. We found that charged residues (Arg, His, Lys, Glu or Asp) or a Pro on positions 4-7 improved OSIP108 activity by 10% or more. The variant OSIP108[G7P] induced the most pronounced tolerance to toxic concentrations of copper and cisplatin in yeast and/or HepG2 cells. Both OSIP108 and OSIP108[G7P] were shown to internalize equally into HeLa cells, but at a higher rate than the inactive OSIP108[E10A], suggesting that the peptides can internalize into cells and that OSIP108 activity is dependent on subsequent intracellular interactions. In conclusion, our studies demonstrated that tolerance/survival-promoting properties of OSIP108 can be significantly improved by single amino acid substitutions, and that these properties are dependent on (an) intracellular target(s), yet to be determined.

α-Conotoxin [S9A]TxID Potently Discriminates between α3β4 and α6/α3β4 Nicotinic Acetylcholine Receptors

Publisher: American Chemical Society (ACS)

Date: 21-06-2017

DOI: 10.1021/ACS.JMEDCHEM.7B00546

Scanning Mutagenesis of α-Conotoxin Vc1.1 Reveals Residues Crucial for Activity at the α9α10 Nicotinic Acetylcholine Receptor

Publisher: Elsevier BV

Date: 07-2009

DOI: 10.1074/JBC.M109.015339

Nematicidal Activity of Cyclotides: Toxicity Against Caenorhabditis elegans

Publisher: American Chemical Society (ACS)

Date: 26-04-2023

DOI: 10.1021/ACS.JNATPROD.2C01124

VARIABLE-TEMPERATURE AND PH STUDIES OF A DNA MINOR-GROOVE BINDER, HOECHST-32985, BY H-1-NMR SPECTROSCOPY

Publisher: Wiley

Date: 09-1994

DOI: 10.1002/MRC.1260320903

Altered Metabolism of Growth Hormone Receptor Mutant Mice: A Combined NMR Metabonomics and Microarray Study

Publisher: Public Library of Science (PLoS)

Date: 23-07-2008

DOI: 10.1371/JOURNAL.PONE.0002764

Difficult Macrocyclizations:  New Strategies for Synthesizing Highly Strained Cyclic Tetrapeptides

Publisher: American Chemical Society (ACS)

Date: 07-2003

DOI: 10.1021/OL034907O

Abstract: [reaction: see text] Cyclic tetrapeptides are an intriguing class of natural products. To synthesize highly strained cyclic tetrapeptides we developed a macrocyclization strategy that involves the inclusion of 2-hydroxy-6-nitrobenzyl (HnB) group at the N-terminus and in the "middle" of the sequence. The N-terminal auxiliary performs a ring closure/ring contraction role, and the backbone auxiliary promotes cis amide bonds to facilitate the otherwise difficult ring contraction. Following this route, the all-L cyclic tetrapeptide cyclo-[Tyr-Arg-Phe-Ala] was successfully prepared.

Solution structure, membrane interactions, and protein binding partners of the tetraspanin Sm-TSP-2, a vaccine antigen from the human blood fluke schistosoma mansoni

Publisher: Elsevier BV

Date: 03-2014

DOI: 10.1074/JBC.M113.531558

Molecular basis for the resistance of an insect chymotrypsin to a potato type II proteinase inhibitor

Publisher: Proceedings of the National Academy of Sciences

Date: 09-08-2010

DOI: 10.1073/PNAS.1009327107

Abstract: Plants produce a variety of proteinase inhibitors (PIs) that have a major function in defense against insect herbivores. In turn, insects have developed strategies to minimize the effect of dietary PIs on digestion. We have discovered that Helicoverpa larvae that survive consumption of a multidomain serine PI from Nicotiana alata (NaPI) contain high levels of a chymotrypsin that is not inhibited by NaPI. Here we describe the isolation of this NaPI-resistant chymotrypsin and an NaPI-susceptible chymotrypsin from Helicoverpa larvae, together with their corresponding cDNAs. We investigated the mechanism of resistance by mutating selected positions of the NaPI-susceptible chymotrypsin using the corresponding amino acids of the NaPI-resistant chymotrypsin. Four critical residues that conferred resistance to NaPI were identified. Molecular modeling revealed that a Phe→Leu substitution at position 37 in the chymotrypsin results in the loss of important binding contacts with NaPI. Identification of the molecular mechanisms that contribute to PI resistance in insect digestive proteases will enable us to develop better inhibitors for the control of lepidopteran species that are major agricultural pests worldwide.

Protein disulfide isomerase: the structure of oxidative folding.

Publisher: Elsevier BV

Date: 08-2006

DOI: 10.1016/J.TIBS.2006.06.001

Abstract: Cellular functions hinge on the ability of proteins to adopt their correct folds, and misfolded proteins can lead to disease. Here, we focus on the proteins that catalyze disulfide bond formation, a step in the oxidative folding pathway that takes place in specialized cellular compartments. In the endoplasmic reticulum of eukaryotes, disulfide formation is catalyzed by protein disulfide isomerase (PDI) by contrast, prokaryotes produce a family of disulfide bond (Dsb) proteins, which together achieve an equivalent outcome in the bacterial periplasm. The recent crystal structure of yeast PDI has increased our understanding of the function and mechanism of PDI. Comparison of the structure of yeast PDI with those of bacterial DsbC and DsbG reveals some similarities but also striking differences that suggest directions for future research aimed at unraveling the catalytic mechanism of disulfide bond formation in the cell.

Insecticidal plant cyclotides and related cystine knot toxins

Publisher: Elsevier BV

Date: 03-2007

DOI: 10.1016/J.TOXICON.2006.11.018

Abstract: Cyclotides are small disulphide-rich peptides found in plants from the violet (Violaceae), coffee (Rubiaceae) and cucurbit (Cucurbitaceae) families. They have the distinguishing structural features of a macrocyclic peptide backbone and a cystine knot made up of six conserved cysteine residues, which makes cyclotides exceptionally stable. In idual plants express a suite of cyclotides in a wide range of tissue types, including leaves, flowers, stems and roots and it is thought that their natural function in plants is as defence agents. This proposal is supported by their high expression levels in plants and their toxic and growth retardant activity in feeding trials against Helicoverpa spp. insect pests. This review describes the structures and activities of cyclotides with specific reference to their insecticidal activity and compares them with structurally similar cystine knot proteins from peas (Pisum sativum) and an amaranthus crop plant (Amaranthus hypocondriancus). More broadly, cystine knot proteins are common in a wide range of organisms from fungi to mammals, and it appears that this interesting structural motif has evolved independently in different organisms as a stable protein framework that has a variety of biological functions.

Three-dimensional solution structure of μ-conotoxin GIIIB, a specific blocker of skeletal muscle sodium channels

Publisher: American Chemical Society (ACS)

Date: 1996

DOI: 10.1021/BI960073O

A Chemoenzymatic Approach To Produce a Cyclic Analogue of the Analgesic Drug MVIIA (Ziconotide)

Publisher: Wiley

Date: 31-05-2023

DOI: 10.1002/ANIE.202302812

Abstract: Ziconotide (ω‐conotoxin MVIIA) is an approved analgesic for the treatment of chronic pain. However, the need for intrathecal administration and adverse effects have limited its widespread application. Backbone cyclization is one way to improve the pharmaceutical properties of conopeptides, but so far chemical synthesis alone has been unable to produce correctly folded and backbone cyclic analogues of MVIIA. In this study, an asparaginyl endopeptidase (AEP)‐mediated cyclization was used to generate backbone cyclic analogues of MVIIA for the first time. Cyclization using six‐ to nine‐residue linkers did not perturb the overall structure of MVIIA, and the cyclic analogues of MVIIA showed inhibition of voltage‐gated calcium channels (Ca V 2.2) and substantially improved stability in human serum and stimulated intestinal fluid. Our study reveals that AEP transpeptidases are capable of cyclizing structurally complex peptides that chemical synthesis cannot achieve and paves the way for further improving the therapeutic value of conotoxins.

Interactions of Globular and Ribbon [γ4E]GID with α4β2 Neuronal Nicotinic Acetylcholine Receptor

Publisher: MDPI AG

Date: 26-08-2021

DOI: 10.3390/MD19090482

Abstract: The α4β2 nAChR is implicated in a range of diseases and disorders including nicotine addiction, epilepsy and Parkinson’s and Alzheimer’s diseases. Designing α4β2 nAChR selective inhibitors could help define the role of the α4β2 nAChR in such disease states. In this study, we aimed to modify globular and ribbon α-conotoxin GID to selectively target the α4β2 nAChR through competitive inhibition of the α4(+)β2(−) or α4(+)α4(−) interfaces. The binding modes of the globular α-conotoxin [γ4E]GID with rat α3β2, α4β2 and α7 nAChRs were deduced using computational methods and were validated using published experimental data. The binding mode of globular [γ4E]GID at α4β2 nAChR can explain the experimental mutagenesis data, suggesting that it could be used to design GID variants. The predicted mutational energy results showed that globular [γ4E]GID is optimal for binding to α4β2 nAChR and its activity could not likely be further improved through amino-acid substitutions. The binding mode of ribbon GID with the (α4)3(β2)2 nAChR was deduced using the information from the cryo-electron structure of (α4)3(β2)2 nAChR and the binding mode of ribbon AuIB. The program FoldX predicted the mutational energies of ribbon [γ4E]GID at the α4(+)α4(−) interface, and several ribbon[γ4E]GID mutants were suggested to have desirable properties to inhibit (α4)3(β2)2 nAChR.

Effects of Cyclization on Peptide Backbone Dynamics

Publisher: American Chemical Society (ACS)

Date: 17-12-2015

DOI: 10.1021/ACS.JPCB.5B11085

Abstract: Despite the widespread use of cyclization as a structure optimization tool in peptide chemistry, little is known about the effect of cyclization on peptide internal dynamics. In this work, we used a combination of multifield NMR relaxation and molecular dynamics techniques to study both monocyclic and polycyclic peptides that have promising biopharmaceutical properties, namely, VH, SFTI-1, and cVc1.1, and their less constrained analogues to study the effects of backbone cyclization (which forms a macrocycle) and disulfide-bond cyclization (which forms internal cycles). We confirmed that backbone cyclization contributes to the rigidity of the monocyclic VH. Interestingly, however, backbone cyclization of the bicyclic SFTI-1 had a limited effect on rigidity, with changes in internal dynamics localized around the ligation site. This suggests that the disulfide bond, which creates an internal cycle, has an insulating effect, protecting the internal cycle from external motional effects. An insulating effect was also observed for the polycyclic cVc1.1: The rigidity of the core was not enhanced by macrocyclization. Additionally, we found that disulfide bonds provide a greater contribution to overall rigidity than macrocyclization. Overall, our results suggest that, although backbone cyclization can improve rigidity, there is a complex interplay between dynamics and cyclization, particularly for polycyclic systems.

The three‐dimensional structure of the analgesic α‐conotoxin, RgIA

Publisher: Wiley

Date: 31-01-2008

DOI: 10.1016/J.FEBSLET.2008.01.027

Abstract: The alpha-conotoxin RgIA is a selective antagonist of the alpha9alpha10 nicotinic acetylcholine receptor and has been shown to be a potent analgesic and reduces nerve injury associated inflammation. RgIA was chemically synthesized and found to fold into two disulfide isomers, globular and ribbon. The native globular isomer inhibited ACh-evoked currents reversibly in oocytes expressing rat alpha9alpha10 nAChRs but the ribbon isomer was inactive. We determined the three-dimensional structure of RgIA using NMR methods to assist in elucidating the molecular role of RgIA in analgesia and inflammation.

Blockade of neuronal α7-nAchR by α-Conotoxin ImI explained by computational scanning and energy calculations

Publisher: Public Library of Science (PLoS)

Date: 03-03-2011

DOI: 10.1371/JOURNAL.PCBI.1002011

Discovery and structures of the cyclotides: novel macrocyclic peptides from plants

Publisher: Springer Science and Business Media LLC

Date: 05-2001

DOI: 10.1007/BF02446507

Conotoxin-Based Leads in Drug Design

Publisher: Wiley

Date: 05-10-2011

DOI: 10.1002/9783527636730.CH5

Cyclotides Suppress Human T-Lymphocyte Proliferation by an Interleukin 2-Dependent Mechanism

Publisher: Public Library of Science (PLoS)

Date: 26-06-2013

DOI: 10.1371/JOURNAL.PONE.0068016

Chemical synthesis and biosynthesis of the cyclotide family of circular proteins

Publisher: Wiley

Date: 09-2006

DOI: 10.1080/15216540600889532

Abstract: Cyclotides are a recently discovered class of proteins that have a characteristic head-to-tail cyclized backbone stabilized by a knotted arrangement of three disulfide bonds. They are exceptionally resistant to chemical, enzymatic and thermal treatments because of their unique structural scaffold. Cyclotides have a range of bio-activities, including uterotonic, anti-HIV, anti-bacterial and cytotoxic activity but their insecticidal properties suggest that their natural physiological role is in plant defense. They are genetically encoded as linear precursors and subsequently processed to produce mature cyclic peptides but the mechanism by which this occurs remains unknown. Currently most cyclotides are obtained via direct extraction from plants in the Rubiaceae and Violaceae families. To facilitate the screening of cyclotides for structure-activity studies and to exploit them in drug design or agricultural applications a convenient route for the synthesis of cyclotides is vital. In this review the current chemical, recombinant and biosynthetic routes to the production of cyclotides are discussed.

Interaction of Tarantula Venom Peptide ProTx-II with Lipid Membranes Is a Prerequisite for Its Inhibition of Human Voltage-gated Sodium Channel Na(V)1.7

Publisher: Elsevier BV

Date: 08-2016

DOI: 10.1074/JBC.M116.729095

Atypical α-conotoxin LtIA from Conus litteratus targets a novel microsite of the α3β2 nicotinic receptor

Publisher: Elsevier BV

Date: 04-2010

DOI: 10.1074/JBC.M109.079012

Structure-activity relationships of ω-conotoxins MVIIA, MVIIC and 14 loop splice hybrids at N and P/Q-type calcium channels 1 1Edited by P. E. Wright

Publisher: Elsevier BV

Date: 06-1999

DOI: 10.1006/JMBI.1999.2817

Accurate de novo design of membrane-traversing macrocycles

Publisher: Elsevier BV

Date: 09-2022

DOI: 10.1016/J.CELL.2022.07.019

Plant cyclotides disrupt epithelial cells in the midgut of lepidopteran larvae

Publisher: Proceedings of the National Academy of Sciences

Date: 29-01-2008

DOI: 10.1073/PNAS.0710338104

Abstract: Several members of the Rubiaceae and Violaceae plant families produce a series of cyclotides or macrocyclic peptides of 28–37 aa with an embedded cystine knot. The cyclic peptide backbone together with the knotted and strongly braced structure confers exceptional chemical and biological stability that has attracted attention for potential pharmaceutical applications. Cyclotides display a erse range of biological activities, such as uterotonic action, anti-HIV activity, and neurotensin antagonism. In plants, their primary role is probably protection from insect attack. Ingestion of the cyclotide kalata B1 severely retards the growth of larvae from the Lepidopteran species Helicoverpa armigera . We examined the gut of these larvae after consumption of kalata B1 by light, scanning, and transmission electron microscopy. We established that kalata B1 induces disruption of the microvilli, blebbing, swelling, and ultimately rupture of the cells of the gut epithelium. The histology of this response is similar to the response of H. armigera larvae to the Bacillus thuringiensis delta-endotoxin, which is widely used to control these insect pests of crops such as cotton.

Synthesis, Racemic X-ray Crystallographic, and Permeability Studies of Bioactive Orbitides from Jatropha Species.

Publisher: American Chemical Society (ACS)

Date: 22-10-2018

DOI: 10.1021/ACS.JNATPROD.8B00447

Abstract: Orbitides are small cyclic peptides with a erse range of therapeutic bioactivities. They are produced by many plant species, including those of the Jatropha genus. Here, the objective was to provide new structural information on orbitides to complement the growing knowledge base on orbitide sequences and activities by focusing on three Jatropha orbitides: ribifolin (1), pohlianin C (7), and jatrophidin (12). To determine three-dimensional structures, racemic crystallography, an emerging structural technique that enables rapid crystallization of biomolecules by combining equal amounts of the two enantiomers, was used. The high-resolution structure of ribifolin (0.99 Å) was elucidated from its racemate and showed it was identical to the structure crystallized from its l-enantiomer only (1.35 Å). Racemic crystallography was also used to elucidate high-resolution structures of pohlianin C (1.20 Å) and jatrophidin (1.03 Å), for which there was difficulty forming crystals without using racemic mixtures. The structures were used to interpret membrane permeability data in PAMPA and a Caco-2 cell assay, showing they had poor permeability. Overall, the results show racemic crystallography can be used to obtain high-resolution structures of orbitides and is useful when enantiopure s les are difficult to crystallize or solution structures from NMR are of low resolution.

The Absolute Structural Requirement for a Proline in the P3′-position of Bowman-Birk Protease Inhibitors Is Surmounted in the Minimized SFTI-1 Scaffold

Publisher: Elsevier BV

Date: 08-2006

DOI: 10.1074/JBC.M601426200

Captivity induces a sweeping and sustained genomic response in a starfish

Publisher: Wiley

Date: 11-04-2023

DOI: 10.1111/MEC.16947

Abstract: Marine animals in the wild are often difficult to access, so they are studied in captivity. However, the implicit assumption that physiological processes of animals in artificial environments are not different from those in the wild has rarely been tested. Here, we investigate the extent to which an animal is impacted by captivity by comparing global gene expression in wild and captive crown‐of‐thorns starfish (COTS). In a preliminary analysis, we compared transcriptomes of three external tissues obtained from multiple wild COTS with a single captive COTS maintained in aquaria for at least 1 week. On average, an astonishingly large 24% of the coding sequences in the genome were differentially expressed. This led us to conduct a replicated experiment to test more comprehensively the impact of captivity on gene expression. Specifically, a comparison of 13 wild with 8 captive COTS coelomocyte transcriptomes revealed significant differences in the expression of 20% of coding sequences. Coelomocyte transcriptomes in captive COTS remain different from those in wild COTS for more than 30 days and show no indication of reverting back to a wild state (i.e. no evidence of acclimation). Genes upregulated in captivity include those involved in oxidative stress and energy metabolism, whereas genes downregulated are involved in cell signalling. These changes in gene expression indicate that being translocated and maintained in captivity has a marked impact on the physiology and health of these echinoderms. This study suggests that caution should be exercised when extrapolating results from captive aquatic invertebrates to their wild counterparts.

A Novel Plant Protein-disulfide Isomerase Involved in the Oxidative Folding of Cystine Knot Defense Proteins

Publisher: Elsevier BV

Date: 07-2007

DOI: 10.1074/JBC.M700018200

Cyclic peptide scaffold with ability to stabilize and deliver a helical cell-impermeable cargo across membranes of cultured cancer cells

Publisher: Royal Society of Chemistry (RSC)

Date: 2020

DOI: 10.1039/D0CB00099J

Abstract: A new helix-loop-helix peptide scaffold with dual ability to transport cargo across cancer cell membranes and disrupt mitochondrial membrane function.

Total Synthesis of the Analgesic Conotoxin MrVIB through Selenocysteine‐Assisted Folding

Publisher: Wiley

Date: 31-05-2011

DOI: 10.1002/ANIE.201101642

Backbone cyclization of analgesic conotoxin GeXIVA facilitates direct folding of the ribbon isomer.

Publisher: Elsevier BV

Date: 10-2017

DOI: 10.1074/JBC.M117.808386

Highly Potent and Selective Plasmin Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold Attenuate Fibrinolysis in Plasma

Publisher: American Chemical Society (ACS)

Date: 06-12-2019

DOI: 10.1021/ACS.JMEDCHEM.8B01139

Efficient Enzymatic Ligation of Inhibitor Cystine Knot Spider Venom Peptides: Using Sortase A To Form Double-Knottins That Probe Voltage-Gated Sodium Channel Na_V1.7

Publisher: American Chemical Society (ACS)

Date: 27-08-2018

DOI: 10.1021/ACS.BIOCONJCHEM.8B00505

Abstract: Gating modifier toxins from spider venom are disulfide-rich peptides that typically comprise a stabilizing inhibitor cystine knot (ICK). These knottin peptides are being pursued as therapeutic leads for a range of conditions linked to transmembrane proteins. Recently, double-knottin peptides discovered in spider venom and produced by recombinant expression have provided insights into the pharmacology of transmembrane channels. Here, we use chemoenzymatic ligation to produce double-knottins to probe the effect of bivalent modulation on the voltage-gated sodium channel subtype 1.7 (Na

Semienzymatic cyclization of disulfide-rich peptides using sortase A

Publisher: Elsevier BV

Date: 03-2014

DOI: 10.1074/JBC.M113.539262

Selective Removal of Individual Disulfide Bonds within a Potato Type II Serine Proteinase Inhibitor from Nicotiana alata Reveals Differential Stabilization of the Reactive-Site Loop

Publisher: Elsevier BV

Date: 2010

DOI: 10.1016/J.JMB.2009.11.031

Abstract: The 53-amino-acid trypsin inhibitor 1 from Nicotiana alata (T1) belongs to the potato type II family also known as the PinII family of proteinase inhibitors, one of the major families of canonical proteinase inhibitors. T1 contains four disulfide bonds, two of which (C4-C41 and C8-C37) stabilize the reactive-site loop. To investigate the influence of these two disulfide bonds on the structure and function of potato II inhibitors, we constructed two variants of T1, C4A/C41A-T1 and C8A/C37A-T1, in which these two disulfide bonds were in idually removed and replaced by alanine residues. Trypsin inhibition assays show that wild-type T1 has a K(i) of <5 nM, C4A/C41A-T1 has a weaker K(i) of approximately 350 nM, and the potency of the C8A/C37A variant is further decreased to a K(i) of approximately 1.8 microM. To assess the influence of the disulfide bonds on the structure of T1, we determined the structure and dynamics of both disulfide variants by NMR spectroscopy. The structure of C4A/C41A-T1 and the litude of intrinsic flexibility in the reactive-site loop resemble that of the wild-type protein closely, despite the lack of the C4-C41 disulfide bond, whereas the timescale of motions is markedly decreased. The rescue of the structure despite loss of a disulfide bond is due to a previously unrecognized network of interactions, which stabilizes the structure of the reactive-site loop in the region of the missing disulfide bond, while allowing intrinsic motions on a fast (picosecond-nanosecond) timescale. In contrast, no comparable interactions are present around the C8-C37 disulfide bond. Consequently, the reactive-site loop becomes disordered and highly flexible in the structure of C8A/C37A-T1, making it unable to bind to trypsin. Thus, the reactive-site loop of T1 is stabilized differently by the C8-C37 and C4-C41 disulfide bonds. The C8-C37 disulfide bond is essential for the inhibitory activity of T1, whereas the C4-C41 disulfide bond is not as critical for maintaining the three-dimensional structure and function of the molecule but is responsible for maintaining flexibility of the reactive-site loop on a microsecond-nanosecond timescale.

Structure of Petunia hybrida Defensin 1, a Novel Plant Defensin with Five Disulfide Bonds

Publisher: American Chemical Society (ACS)

Date: 20-06-2003

DOI: 10.1021/BI034379O

Abstract: The structure of a novel plant defensin isolated from the flowers of Petunia hybrida has been determined by (1)H NMR spectroscopy. P. hybrida defensin 1 (PhD1) is a basic, cysteine-rich, antifungal protein of 47 residues and is the first ex le of a new subclass of plant defensins with five disulfide bonds whose structure has been determined. PhD1 has the fold of the cysteine-stabilized alphabeta motif, consisting of an alpha-helix and a triple-stranded antiparallel beta-sheet, except that it contains a fifth disulfide bond from the first loop to the alpha-helix. The additional disulfide bond is accommodated in PhD1 without any alteration of its tertiary structure with respect to other plant defensins. Comparison of its structure with those of classic, four-disulfide defensins has allowed us to identify a previously unrecognized hydrogen bond network that is integral to structure stabilization in the family.

Cyclotides: Natural, circular plant peptides that possess significant activity against gastrointestinal nematode parasites of sheep

Publisher: American Chemical Society (ACS)

Date: 22-04-2008

DOI: 10.1021/BI800223Y

Abstract: The cyclotides are a novel family of backbone-cyclized cystine-knot containing peptides from plants that have been shown to possess insecticidal activity against Helicoverpa larvae, an important pest of corn and cotton. In the current study, we investigated the in vitro effects of the cyclotides on the viability of egg, larval, and adult life stages of two species of economically important gastrointestinal nematode parasites of livestock, Hemonchus contortus and Trichostrongylus colubriformis. The cyclotides showed significant activity in inhibiting development of nematode larvae and motility of adult worms. Activities were comparable to some currently used anthelmintic compounds in these in vitro assay systems. A series of alanine mutants of the prototypic cyclotide kalata B1 were assayed against larvae to determine regions of the peptide responsible for activity. It was observed that anthelmintic activity was dramatically reduced as a consequence of the mutation of a large number of residues that are found clustered on one surface. Activities toward larvae were equivalent in the naturally occurring L-isomer of kalata B1 and a synthetic all-D-isomer, indicating that there is no chiral requirement for anthelmintic activity. The clustering of important residues and the lack of chiral selectivity further support the proposed mode of action of the cyclotides, which involves a membrane-based interaction rather than an interaction at a specific receptor. The cyclotide-induced leakage of a fluorescent dye from vesicles used as a model membrane mimetic further confirms the membrane lytic ability of cyclotides. The relative potency of kalata B1 and kalata B2 in causing membrane leakage is consistent with the order of their anthelmintic activity. These results demonstrate that the cyclotides show potential for use in the control of gastrointestinal nematode parasites.

Cyclotides associate with leaf vasculature and are the products of a novel precursor in Petunia (Solanaceae)

Publisher: Elsevier BV

Date: 08-2012

DOI: 10.1074/JBC.M112.370841

In Planta Discovery and Chemical Synthesis of Bracelet Cystine Knot Peptides from Rinorea bengalensis

Publisher: American Chemical Society (ACS)

Date: 11-02-2021

DOI: 10.1021/ACS.JNATPROD.0C01065

Structure and metal binding studies of the second copper binding domain of the Menkes ATPase

Publisher: Elsevier BV

Date: 09-2003

DOI: 10.1016/J.JSB.2003.08.008

Abstract: Biological utilisation of copper requires that the metal, in its ionic forms, be meticulously transported, inserted into enzymes and regulatory proteins, and excess be excreted. To understand the trafficking process, it is crucial that the structures of the proteins involved in the varied processes be resolved. To investigate copper binding to a family of structurally related copper-binding proteins, we have characterised the second Menkes N-terminal domain (MNKr2). The structure, determined using 1H and 15N heteronuclear NMR, of the reduced form of MNKr2 has revealed two alpha-helices lying over a single beta-sheet and shows that the binding site, a Cys(X)2Cys pair, is located on an exposed loop. 1H-15N HSQC experiments demonstrate that binding of Cu(I) causes changes that are localised to conserved residues adjacent to the metal binding site. Residues in this area are important to the delivery of copper by the structurally related Cu(I) chaperones. Complementary site-directed mutagenesis of the adjacent residues has been used to probe the structural roles of conserved residues.

Conotoxins: Chemistry and Biology

Publisher: American Chemical Society (ACS)

Date: 21-10-2019

DOI: 10.1021/ACS.CHEMREV.9B00207

Abstract: The venom of the marine predatory cone snails (genus

Cyclotides from Brazilian Palicourea sessilis and Their Effects on Human Lymphocytes

Publisher: American Chemical Society (ACS)

Date: 05-01-2021

DOI: 10.1021/ACS.JNATPROD.0C01069

Comparative analysis of cyclotide-producing plant cell suspensions presents opportunities for cyclotide plant molecular farming

Publisher: Elsevier BV

Date: 03-2022

DOI: 10.1016/J.PHYTOCHEM.2021.113053

Abstract: Cyclotides are a class of ribosomally-synthesized plant peptides that function in plants as a defense against insects and fungal pathogens. Their unique structure comprises a cyclized peptide backbone threaded by three disulfide bonds, that imparts structural stability, a desirable quality for peptide-based therapeutics or insecticides. Producing these peptides synthetically is challenging due to the amount of chemical waste produced and inefficiency of folding certain cyclotides. Thus, it is desirable to develop a means to access cyclotide biosynthesis in their native hosts, cultured in defined conditions, at both laboratory and commercial scale. Here we developed suspension cell cultures from two species previously unexplored for cyclotide production in suspension cells, Clitoria ternatea L., Hybanthus enneaspermus F. Muell., as well as with Oldenlandia affinis (Roem. & Schult.) DC., a species reported previously to accumulate cyclotides in cell suspensions. We assessed the growth rate, cyclotide production and gene expression for the various species. We found that while many cyclotides had reduced expression in Oldenlandia affinis suspension cells when compared to plant organs, those in Clitoria ternatea and Hybanthus enneaspermus maintained or increased expression levels. The cyclotides that continued to be expressed in suspension cultures shared similar sequence and biophysical properties as a group, regardless of phylogenetic origin of the host. Of particular interest was the discovery of inducibility by NaCl of cyclotide expression in O. affinis, cycloviolacin O2 expression in O. affinis, and the scale up of cycloviolacin O2 production in H. enneaspermus. Together the results presented here highlight the utility of plant cell suspensions as modalities to produce macrocyclic peptides.

Development of a Selective Peptide κ-Opioid Receptor Antagonist by Late-Stage Functionalization with Cysteine Staples

Publisher: American Chemical Society (ACS)

Date: 26-08-2023

DOI: 10.1021/ACS.JMEDCHEM.3C00426

A H-1-NMR DETERMINATION OF THE SOLUTION STRUCTURE OF THE A-CHAIN OF INSULIN - COMPARISON WITH THE CRYSTAL-STRUCTURE AND AN EXAMINATION OF THE ROLE OF SOLVENT

Publisher: Elsevier BV

Date: 12-1994

DOI: 10.1016/0167-4838(94)90182-1

Abstract: The 1H-NMR chemical shift assignments for the oxidized A-chain of bovine insulin have been determined in aqueous and 30% trifluoroethanol/water solutions. Analysis of the observed medium-range nuclear Overhauser effects indicates that in aqueous solution significant populations of the peptide exist, with a 3(10)-helical conformation over residues 12-17. This region corresponds to helix A (13-20) in the crystal structure of the 2 Zn insulin hexamer. In 30% TFE solution, the NOE data are supportive of a random coil conformation throughout the peptide.

CD and NMR determination of the solution structure of a peptide corresponding to T4 lysozyme residues 38–51

Publisher: Elsevier BV

Date: 07-1995

DOI: 10.1016/0167-4838(95)00045-V

Abstract: Solid phase methods have been used to synthesise a peptide corresponding to residues 38-51 of T4 lysozyme. The peptide, LYS(38-51), encompasses helix B in the crystal structure of T4 lysozyme. CD and 1H-NMR analysis showed that the peptide was unstructured in aqueous solution but adopted a helical conformation in the more hydrophobic environment provided by 50% TFE and SDS micelles. The solution structure derived from the NMR data was similar to that of the helix in the X-ray structure, although there was some fraying at the N-terminus.

Harnessing cyclotides to design and develop novel peptide GPCR ligands

Publisher: Royal Society of Chemistry (RSC)

Date: 2020

DOI: 10.1039/D0CB00062K

Abstract: Cyclotides are plant-derived cyclic peptides that have emerged as promising scaffold molecules for designing peptide-based therapeutics. Cyclotide engineering may lead to the development of novel ligands of G protein-coupled receptors with improved pharmacological properties.

The three-dimensional solution structure of mini-M conotoxin BtIIIA reveals a disconnection between disulfide connectivity and peptide fold

Publisher: Elsevier BV

Date: 06-2013

DOI: 10.1016/J.BMC.2013.02.012

Abstract: Conotoxins are bioactive peptides from the venoms of marine snails and have been ided into several superfamilies based on homologies in their precursor sequences. The M-superfamily conotoxins can be further ided into five branches based on the number of residues in the third loop of the peptide sequence. Recently two M-1 branch conotoxins (tx3a and mr3e) with a C1-C5, C2-C4, C3-C6 disulfide connectivity and one M-2 branch conotoxin (mr3a) with a C1-C6, C2-C4, C3-C5 disulfide connectivity were described. Here we report the disulfide connectivity, chemical synthesis and the three-dimensional NMR structure of the novel 14-residue conotoxin BtIIIA, extracted from the venom of Conus betulinus. It has the same disulfide connectivity as mr3a, which puts it in the M-2 branch conotoxins but has a distinctly different structure from other M-2 branch conotoxins. 105 NOE distance restraints and seven dihedral angle restraints were used for the structure calculations. The three-dimensional structure was determined with CYANA based on torsion angle dynamics and refinement in a water solvent box was carried out with CNS. Fifty structures were calculated and the 20 lowest energy structures superimposed with a RMSD of 0.49±0.16 Å. Even though it has the M-2 branch disulfide connectivity, BtIIIA was found to have a 'flying bird' backbone motif depiction that is found in the M-1 branch conotoxin mr3e. This study shows that conotoxins with the same cysteine framework can have different disulfide connectivities and different peptide folds.

Solution structure, aggregation behavior, and flexibility of human relaxin-2

Publisher: American Chemical Society (ACS)

Date: 15-01-2015

DOI: 10.1021/CB500918V

Abstract: Relaxin is a member of the relaxin/insulin peptide hormone superfamily and is characterized by a two-chain structure constrained by three disulfide bonds. Relaxin is a pleiotropic hormone and involved in a number of physiological and pathogenic processes, including collagen and cardiovascular regulation and tissue remodelling during pregnancy and cancer. Crystallographic and ultracentrifugation experiments have revealed that the human form of relaxin, H2 relaxin, self-associates into dimers, but the significance of this is poorly understood. Here, we present the NMR structure of a monomeric, amidated form of H2 relaxin and compare its features and behavior in solution to those of native H2 relaxin. The overall structure of H2 relaxin is retained in the monomeric form. H2 relaxin amide is fully active at the relaxin receptor RXFP1 and thus dimerization is not required for biological activity. Analysis of NMR chemical shifts and relaxation parameters identified internal motion in H2 relaxin at the pico-nanosecond and milli-microsecond time scales, which is commonly seen in other relaxin and insulin peptides and might be related to function.

Native peptide folding dominates over stereoelectronic effects of prolyl hydroxylation in loop 5 of the macrocyclic peptide kalata B1

Publisher: Elsevier BV

Date: 10-2014

DOI: 10.1016/J.TET.2014.06.058

Development of Novel Melanocortin Receptor Agonists Based on the Cyclic Peptide Framework of Sunflower Trypsin Inhibitor-1

Publisher: American Chemical Society (ACS)

Date: 31-03-2018

DOI: 10.1021/ACS.JMEDCHEM.8B00170

Dissecting the Oxidative Folding of Circular Cystine Knot Miniproteins

Publisher: Mary Ann Liebert Inc

Date: 05-2009

DOI: 10.1089/ARS.2008.2295

Abstract: Cyclotides are plant proteins with exceptional stability owing to the presence of a cyclic backbone and three disulfide bonds arranged in a cystine knot motif. Accordingly, they have been proposed as templates to stabilize bioactive epitopes in drug-design applications. The two main subfamilies, referred to as the Möbius and bracelet cyclotides, require dramatically different in vitro folding conditions to achieve the native fold. To determine the underlying elements that influence cyclotide folding, we examined the in vitro folding of a suite of hybrid cyclotides based on combination of the Möbius cyclotide kalata B1 and the bracelet cyclotide cycloviolacin O1. The folding pathways of the two cyclotide subfamilies were found to be different and influenced by specific residues within intercysteine loops 2 and 6. Two changes in these loops, a substitution in loop 2 and an addition in loop 6, enabled the folding of a cycloviolacin O1 analogue under conditions in which folding does not occur in vitro for the native peptide. A key intermediate contains a native-like hairpin structure that appears to be a nucleation locus early in the folding process. Overall, these mechanistic findings on the folding of cyclotides are potentially valuable for the design of new drug leads.

Identification and structural characterization of novel cyclotide with activity against an insect pest of sugar cane

Publisher: Elsevier BV

Date: 2012

DOI: 10.1074/JBC.M111.294009

Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase.

Publisher: Springer Science and Business Media LLC

Date: 18-12-2015

DOI: 10.1038/NCOMMS10199

Abstract: Cyclotides are erse plant backbone cyclized peptides that have attracted interest as pharmaceutical scaffolds, but fundamentals of their biosynthetic origin remain elusive. Backbone cyclization is a key enzyme-mediated step of cyclotide biosynthesis and confers a measure of stability on the resultant cyclotide. Furthermore, cyclization would be desirable for engineered peptides. Here we report the identification of four asparaginyl endopeptidases (AEPs), proteases implicated in cyclization, from the cyclotide-producing plant Oldenlandia affinis. We recombinantly express Oa AEP1 b and find it functions preferably as a cyclase by coupling C-terminal cleavage of propeptide substrates with backbone cyclization. Interestingly, Oa AEP1 b cannot cleave at the N-terminal site of O. affinis cyclotide precursors, implicating additional proteases in cyclotide biosynthesis. Finally, we demonstrate the broad utility of this enzyme by cyclization of peptides unrelated to cyclotides. We propose that recombinant Oa AEP1 b is a powerful tool for use in peptide engineering applications where increased stability of peptide products is desired.

α-Selenoconotoxins, a New Class of Potent α7 Neuronal Nicotinic Receptor Antagonists

Publisher: Elsevier BV

Date: 05-2006

DOI: 10.1074/JBC.M512419200

Structural Insights into the Role of the Cyclic Backbone in a Squash Trypsin Inhibitor

Publisher: Elsevier BV

Date: 12-2013

DOI: 10.1074/JBC.M113.528240

Cyclic Penta- and Hexaleucine Peptides without N-Methylation Are Orally Absorbed

Publisher: American Chemical Society (ACS)

Date: 07-08-2014

DOI: 10.1021/ML5002823

The Synthesis, Structural Characterization, and Receptor Specificity of the α-Conotoxin Vc1.1

Publisher: Elsevier BV

Date: 08-2006

DOI: 10.1074/JBC.M604550200

The Future of Peptide-based Drugs

Publisher: Wiley

Date: 17-12-2013

DOI: 10.1111/CBDD.12055

Abstract: The suite of currently used drugs can be ided into two categories - traditional 'small molecule' drugs with typical molecular weights of 5000 Da that are not orally bioavailable and need to be delivered via injection. Due to their small size, conventional small molecule drugs may suffer from reduced target selectivity that often ultimately manifests in human side-effects, whereas protein therapeutics tend to be exquisitely specific for their targets due to many more interactions with them, but this comes at a cost of low bioavailability, poor membrane permeability, and metabolic instability. The time has now come to reinvestigate new drug leads that fit between these two molecular weight extremes, with the goal of combining advantages of small molecules (cost, conformational restriction, membrane permeability, metabolic stability, oral bioavailability) with those of proteins (natural components, target specificity, high potency). This article uses selected ex les of peptides to highlight the importance of peptide drugs, some potential new opportunities for their exploitation, and some difficult challenges ahead in this field.

Two new classes of conopeptides inhibit the α1-adrenoceptor and noradrenaline transporter

Publisher: Springer Science and Business Media LLC

Date: 06-08-2001

DOI: 10.1038/NN0901-902

Hormone-like conopeptides – new tools for pharmaceutical design

Publisher: Royal Society of Chemistry (RSC)

Date: 2020

DOI: 10.1039/D0MD00173B

Abstract: Marine cone snails produce an array of hormone-like peptides in their venom, providing bioactive leads for developing peptide drugs and small molecule mimetics.

The alpine violet, Viola biflora, is a rich source of cyclotides with potent cytotoxicity

Publisher: Elsevier BV

Date: 02-2008

DOI: 10.1016/J.PHYTOCHEM.2007.10.023

Abstract: The cyclotides are currently the largest known family of head-to-tail cyclic proteins. The complex structure of these small plant proteins, which consist of approximately 30 amino acid residues, contains both a circular peptide backbone and a cystine knot, the combination of which produces the cyclic cystine knot motif. To date, cyclotides have been found in plants from the Rubiaceae, Violaceace and Cucurbitaceae families, and are believed to be part of the host defence system. In addition to their insecticidal effect, cyclotides have also been shown to be cytotoxic, anti-HIV, antimicrobial and haemolytic agents. In this study, we show that the alpine violet Viola biflora (Violaceae) is a rich source of cyclotides. The sequences of 11 cyclotides, vibi A-K, were determined by isolation and MS/MS sequencing of proteins and screening of a cDNA library of V. biflora in parallel. For the cDNA screening, a degenerate primer against a conserved (AAFALPA) motif in the cyclotide precursor ER signal sequence yielded a series of predicted cyclotide sequences that were correlated to those of the isolated proteins. There was an apparent discrepancy between the results of the two strategies as only one of the isolated proteins could be identified as a cDNA clone. Finally, to correlate amino acid sequence to cytotoxic potency, vibi D, E, G and H were analysed using a fluorometric microculture cytotoxicity assay using a lymphoma cell line. The IC(50)-values of the bracelet cyclotides vibi E, G and H ranged between 0.96 and 5.0 microM while the Möbius cyclotide vibi D was not cytotoxic at 30 microM.

Enhanced Activity against Multidrug-Resistant Bacteria through Coapplication of an Analogue of Tachyplesin I and an Inhibitor of the QseC/B Signaling Pathway

Publisher: American Chemical Society (ACS)

Date: 31-01-2020

DOI: 10.1021/ACS.JMEDCHEM.9B01563

Abstract: Tachyplesin I (TPI) is a cationic β-hairpin antimicrobial peptide with broad-spectrum, potent antimicrobial activity. In this study, the all d-amino acid analogue of TPI (TPAD) was synthesized, and its structure and activity were determined. TPAD has comparable antibacterial activity to TPI on 14 bacterial strains, including four drug-resistant bacteria. Importantly, TPAD has significantly improved stability against enzymatic degradation and decreased hemolytic activity compared to TPI, indicating that it has better therapeutic potential. The induction of bacterial resistance using low concentrations of TPAD resulted in the activation of the QseC/B two-component system. Deletion of this system resulted in at least five-fold improvement of TPAD activity, and the combined use of TPAD with LED209, a QseC/B inhibitor, significantly enhanced the bactericidal effect against three classes of multidrug-resistant bacteria.

Structure of a putative ancestral protein encoded by a single sequence repeat from a multidomain proteinase inhibitor gene fromNicotiana alata

Publisher: Elsevier BV

Date: 07-1999

DOI: 10.1016/S0969-2126(99)80103-8

Abstract: The ornamental tobacco Nicotiana alata produces a series of proteinase inhibitors (PIs) that are derived from a 43 kDa precursor protein, NaProPI. NaProPI contains six highly homologous repeats that fold to generate six separate structural domains, each corresponding to one of the native PIs. An unusual feature of NaProPI is that the structural domains lie across adjacent repeats and that the sixth PI domain is generated from fragments of the first and sixth repeats. Although the homology of the repeats suggests that they may have arisen from gene duplication, the observed folding does not appear to support this. This study of the solution structure of a single NaProPI repeat (aPI1) forms a basis for unravelling the mechanism by which this protein may have evolved. The three-dimensional structure of aPI1 closely resembles the triple-stranded antiparallel beta sheet observed in each of the native PIs. The five-residue sequence Glu-Glu-Lys-Lys-Asn, which forms the linker between the six structural domains in NaProPI, exists as a disordered loop in aPI1. The presence of this loop in aPI1 results in a loss of the characteristically flat and disc-like topography of the native inhibitors. A single repeat from NaProPI is capable of folding into a compact globular domain that displays native-like PI activity. Consequently, it is possible that a similar single-domain inhibitor represents the ancestral protein from which NaProPI evolved.

Solution structure of methionine-oxidized amyloid β-peptide (1-40). Does oxidation affect conformational switching?

Publisher: American Chemical Society (ACS)

Date: 22-08-1998

DOI: 10.1021/BI9810757

Dual-targeting anti-angiogenic cyclic peptides as potential drug leads for cancer therapy

Publisher: Springer Science and Business Media LLC

Date: 13-10-2016

DOI: 10.1038/SREP35347

Abstract: Peptide analogues derived from bioactive hormones such as somatostatin or certain growth factors have great potential as angiogenesis inhibitors for cancer applications. In an attempt to combat emerging drug resistance many FDA-approved anti-angiogenesis therapies are co-administered with cytotoxic drugs as a combination therapy to target multiple signaling pathways of cancers. However, cancer therapies often encounter limiting factors such as high toxicities and side effects. Here, we combined two anti-angiogenic epitopes that act on different pathways of angiogenesis into a single non-toxic cyclic peptide framework, namely MCoTI-II ( Momordica cochinchinensis trypsin inhibitor-II), and subsequently assessed the anti-angiogenic activity of the novel compound. We hypothesized that the combination of these two epitopes would elicit a synergistic effect by targeting different angiogenesis pathways and result in improved potency, compared to that of a single epitope. This novel approach has resulted in the development of a potent, non-toxic, stable and cyclic analogue with nanomolar potency inhibition in in vitro endothelial cell migration and in vivo chorioallantoic membrane angiogenesis assays. This is the first report to use the MCoTI-II framework to develop a 2-in-1 anti-angiogenic peptide, which has the potential to be used as a form of combination therapy for targeting a wide range of cancers.

Design of a Cyclotide Antagonist of Neuropilin-1 and -2 That Potently Inhibits Endothelial Cell Migration

Publisher: American Chemical Society (ACS)

Date: 12-04-2013

DOI: 10.1021/CB4000585

Abstract: Neuropilin-1 and -2 are critical regulators of angiogenesis, lymphangiogenesis, and cell survival as receptors for multiple growth factors. Disulfide-rich peptides that antagonize the growth factor receptors neuropilin-1 and neuropilin-2 were developed using bacterial display libraries. Peptide ligands specific for the VEGFA binding site on neuropilin-1 were identified by screening a library of disulfide-rich peptides derived from the thermostable, protease-resistant cyclotide kalata B1. First generation ligands were subjected to one cycle of affinity maturation to yield acyclic peptides with affinities of 40-60 nM and slow dissociation rate constants (∼1 × 10(-3) s(-1)). Peptides exhibited equivalent affinities for human and mouse neuropilin-1 and cross-reacted with human neuropilin-2 with lower affinity. A C-to-N cyclized variant (cyclotide) of one neuropilin ligand retained high affinity, exhibited increased protease resistance, and conferred improved potency for inhibiting endothelial cell migration in vitro (EC50 ≈ 100 nM). These results demonstrate that potent, target-specific cyclotides can be created by evolutionary design and that backbone cyclization can confer improved pharmacological properties.

Papain-like cysteine proteases prepare plant cyclic peptide precursors for cyclization

Publisher: Proceedings of the National Academy of Sciences

Date: 03-04-2019

DOI: 10.1073/PNAS.1901807116

Abstract: Ribosomally synthesized and post-translationally modified peptides are natural products that hold great promise for a range of medical and biotechnological applications. However, the cost-effective heterologous production of these peptides is h ered by a poor understanding of their biosynthesis. Cyclotides, plant-derived, disulfide-knotted, head-to-tail cyclic peptides, exhibit exceptional stability and great amenability for amino acid substitutions and insertions. Although much effort has been invested toward understanding cyclotide biosynthesis, details of the key proteolytic step before cyclization remained elusive for two decades. We used an activity-guided approach to discover the enzymes involved. Our characterization of these enzymes will enable efficient approaches for heterologous cyclotide production.

Anthelmintic activity of cyclotides: In vitro studies with canine and human hookworms

Publisher: Elsevier BV

Date: 02-2009

DOI: 10.1016/J.ACTATROPICA.2008.11.003

Abstract: Hookworm infection is a leading cause of maternal and child morbidity in countries of the tropics and subtropics, as well as being an important parasite in companion-animal medicine. The cyclotides are a novel family of cyclic cystine knot containing peptides from plants that have been shown to possess anthelmintic activity against Haemonchus contortus and Trichostrongylus colubriformis, two important gastrointestinal nematodes of sheep. In the current study we demonstrated the in vitro effects of three representative cyclotides, kalata B1, kalata B6 and cycloviolacin O14, on the viability of larval and adult life stages of the dog hookworm Ancylostoma caninum, and larvae of the human hookworm Necator americanus. The cyclotides showed significant anthelmintic activity towards both hookworm species. The different cyclotides showed similar patterns of relative activity as that seen previously with the livestock nematode species. This study demonstrates that cyclotides have promising activity in vitro against important parasites of companion animals and humans.

Discovery and Characterization of a Linear Cyclotide from Viola odorata: Implications for the Processing of Circular Proteins

Publisher: Elsevier BV

Date: 04-2006

DOI: 10.1016/J.JMB.2006.01.051

Abstract: Cyclotides are mini-proteins of 28-37 amino acid residues that have the unusual feature of a head-to-tail cyclic backbone surrounding a cystine knot. This molecular architecture gives the cyclotides heightened resistance to thermal, chemical and enzymatic degradation and has prompted investigations into their use as scaffolds in peptide therapeutics. There are now more than 80 reported cyclotide sequences from plants in the families Rubiaceae, Violaceae and Cucurbitaceae, with a wide variety of biological activities observed. However, potentially limiting the development of cyclotide-based therapeutics is a lack of understanding of the mechanism by which these peptides are cyclized in vivo. Until now, no linear versions of cyclotides have been reported, limiting our understanding of the cyclization mechanism. This study reports the discovery of a naturally occurring linear cyclotide, violacin A, from the plant Viola odorata and discusses the implications for in vivo cyclization of peptides. The elucidation of the cDNA clone of violacin A revealed a point mutation that introduces a stop codon, which inhibits the translation of a key Asn residue that is thought to be required for cyclization. The three-dimensional solution structure of violacin A was determined and found to adopt the cystine knot fold of native cyclotides. Enzymatic stability assays on violacin A indicate that despite an increase in the flexibility of the structure relative to cyclic counterparts, the cystine knot preserves the overall stability of the molecule.

A Synthetic Mirror Image of Kalata B1 Reveals that Cyclotide Activity Is Independent of a Protein Receptor

Publisher: Wiley

Date: 16-09-2011

DOI: 10.1002/CBIC.201100450

Modified horseshoe crab peptides target and kill bacteria inside host cells

Publisher: Springer Science and Business Media LLC

Date: 31-12-2022

DOI: 10.1007/S00018-021-04041-Z

Abstract: Bacteria that occupy an intracellular niche can evade extracellular host immune responses and antimicrobial molecules. In addition to classic intracellular pathogens, other bacteria including uropathogenic Escherichia coli (UPEC) can adopt both extracellular and intracellular lifestyles. UPEC intracellular survival and replication complicates treatment, as many therapeutic molecules do not effectively reach all components of the infection cycle. In this study, we explored cell-penetrating antimicrobial peptides from distinct structural classes as alternative molecules for targeting bacteria. We identified two β-hairpin peptides from the horseshoe crab, tachyplesin I and polyphemusin I, with broad antimicrobial activity toward a panel of pathogenic and non-pathogenic bacteria in planktonic form. Peptide analogs [I11A]tachyplesin I and [I11S]tachyplesin I maintained activity toward bacteria, but were less toxic to mammalian cells than native tachyplesin I. This important increase in therapeutic window allowed treatment with higher concentrations of [I11A]tachyplesin I and [I11S]tachyplesin I, to significantly reduce intramacrophage survival of UPEC in an in vitro infection model. Mechanistic studies using bacterial cells, model membranes and cell membrane extracts, suggest that tachyplesin I and polyphemusin I peptides kill UPEC by selectively binding and disrupting bacterial cell membranes. Moreover, treatment of UPEC with sublethal peptide concentrations increased zinc toxicity and enhanced innate macrophage antimicrobial pathways. In summary, our combined data show that cell-penetrating peptides are attractive alternatives to traditional small molecule antibiotics for treating UPEC infection, and that optimization of native peptide sequences can deliver effective antimicrobials for targeting bacteria in extracellular and intracellular environments.

Cyclic MrIA:  A Stable and Potent Cyclic Conotoxin with a Novel Topological Fold that Targets the Norepinephrine Transporter

Publisher: American Chemical Society (ACS)

Date: 07-10-2006

DOI: 10.1021/JM060299H

Abstract: Conotoxins, disulfide-rich peptides from the venom of cone snails, have created much excitement over recent years due to their potency and specificity for ion channels and their therapeutic potential. One recently identified conotoxin, MrIA, a 13-residue member of the chi-conotoxin family, inhibits the human norepinephrine transporter (NET) and has potential applications in the treatment of pain. In the current study, we show that the beta-hairpin structure of native MrIA is retained in a synthetic cyclic version, as is biological activity at the NET. Furthermore, the cyclic version has increased resistance to trypsin digestion relative to the native peptide, an intriguing result because the cleavage site for the trypsin is not close to the cyclization site. The use of peptides as drugs is generally h ered by susceptibility to proteolysis, and so, the increase in enzymatic stability against trypsin observed in the current study may be useful in improving the therapeutic potential of MrIA. Furthermore, the structure reported here for cyclic MrIA represents a new topology among a growing number of circular disulfide-rich peptides.

An Integrated Molecular Grafting Approach for the Design of Keap1-Targeted Peptide Inhibitors

Publisher: American Chemical Society (ACS)

Date: 21-06-2021

DOI: 10.1021/ACSCHEMBIO.1C00388

The chemistry and biology of human relaxin-3

Publisher: Wiley

Date: 05-2005

DOI: 10.1196/ANNALS.1282.008

Abstract: A novel member of the human relaxin subclass of the insulin superfamily was recently discovered during a genomics database search and named relaxin-3. Like human relaxin-1 and relaxin-2, relaxin-3 is predicted to consist of a two-chain structure and three disulfide bonds in a disposition identical to that of insulin. To undertake detailed biophysical and biological characterization of the peptide, its chemical synthesis was undertaken. In contrast to human relaxin-1 and relaxin-2, however, relaxin-3 could not be successfully prepared by simple combination of the in idual chains, thus necessitating recourse to the use of a regioselective disulfide bond formation strategy. Solid phase synthesis of the separate, selectively S-protected A and B chains followed by their purification and the subsequent stepwise formation of each of the three disulfides led to the successful acquisition of human relaxin-3. Comprehensive chemical characterization confirmed both the correct chain orientation and the integrity of the synthetic product. Relaxin-3 was found to bind to and activate native relaxin receptors in vitro and stimulate water drinking through central relaxin receptors in vivo. Recent studies have demonstrated that relaxin-3 will bind to and activate human LGR7, but not LGR8, in vitro. Secondary structural analysis showed it to adopt a less ordered confirmation than either relaxin-1 or relaxin-2, reflecting the presence in the former of a greater percentage of nonhelical forming amino acids. NMR spectroscopy and simulated annealing calculations were used to determine the three-dimensional structure of relaxin-3 and to identify key structural differences between the human relaxins.

Engineered protease inhibitors based on sunflower trypsin inhibitor-1 (SFTI-1) provide insights into the role of sequence and conformation in Laskowski mechanism inhibition

Publisher: Portland Press Ltd.

Date: 06-07-2015

DOI: 10.1042/BJ20150412

Abstract: Laskowski inhibitors regulate serine proteases by an intriguing mode of action that involves deceiving the protease into synthesizing a peptide bond. Studies exploring naturally occurring Laskowski inhibitors have uncovered several structural features that convey the inhibitor's resistance to hydrolysis and exceptional binding affinity. However, in the context of Laskowski inhibitor engineering, the way that various modifications intended to fine-tune an inhibitor's potency and selectivity impact on its association and dissociation rates remains unclear. This information is important as Laskowski inhibitors are becoming increasingly used as design templates to develop new protease inhibitors for pharmaceutical applications. In this study, we used the cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), as a model system to explore how the inhibitor's sequence and structure relate to its binding kinetics and function. Using enzyme assays, MD simulations and NMR spectroscopy to study SFTI variants with erse sequence and backbone modifications, we show that the geometry of the binding loop mainly influences the inhibitor's potency by modulating the association rate, such that variants lacking a favourable conformation show dramatic losses in activity. Additionally, we show that the inhibitor's sequence (including both the binding loop and its scaffolding) influences its potency and selectivity by modulating both the association and the dissociation rates. These findings provide new insights into protease inhibitor function and design that we apply by engineering novel inhibitors for classical serine proteases, trypsin and chymotrypsin and two kallikrein-related peptidases (KLK5 and KLK14) that are implicated in various cancers and skin diseases.

Characterization of a Novel α-Conotoxin from Conus textile That Selectively Targets α6/α3β2β3 Nicotinic Acetylcholine Receptors

Publisher: Elsevier BV

Date: 2013

DOI: 10.1074/JBC.M112.427898

ConoServer: Updated content, knowledge, and discovery tools in the conopeptide database

Publisher: Oxford University Press (OUP)

Date: 03-11-2012

DOI: 10.1093/NAR/GKR886

Are α9α10 nicotinic acetylcholine receptors a pain target for α-conotoxins?

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 05-09-2007

DOI: 10.1124/MOL.107.040568

Abstract: The synthetic alpha-conotoxin Vc1.1 is a small disulfide bonded peptide currently in development as a treatment for neuropathic pain. Unlike Vc1.1, the native post-translationally modified peptide vc1a does not act as an analgesic in vivo in rat models of neuropathic pain. It has recently been proposed that the primary target of Vc1.1 is the alpha9alpha10 nicotinic acetylcholine receptor (nAChR). We show that Vc1.1 and its post-translationally modified analogs vc1a, [P6O]Vc1.1, and [E14gamma]Vc1.1 are equally potent at inhibiting ACh-evoked currents mediated by alpha9alpha10 nAChRs. This suggests that alpha9alpha10 nAChRs are unlikely to be the molecular mechanism or therapeutic target of Vc1.1 for the treatment of neuropathic pain.

Synthesis of cyclic disulfide-rich peptides

Publisher: Humana Press

Date: 2013

DOI: 10.1007/978-1-62703-544-6_6

Abstract: In this chapter we describe two SPPS approaches for producing cyclic disulfide-rich peptides in our laboratory, including cyclotides from plants, cyclic conotoxins from cone snail venoms, chlorotoxin from scorpion venom, and the sunflower trypsin inhibitor peptide, SFTI-1.

The solution structure of C1-T1, a two-domain proteinase inhibitor derived from a circular precursor protein from Nicotiana alata11Edited by P. E. Wright

Publisher: Elsevier BV

Date: 02-2001

DOI: 10.1006/JMBI.2000.4318

Efficient enzymatic cyclization of an inhibitory cystine knot-containing peptide.

Publisher: Wiley

Date: 09-08-2016

DOI: 10.1002/BIT.25993

The role of disulfide bonds in structure and activity of chlorotoxin

Publisher: Future Science Ltd

Date: 10-2014

DOI: 10.4155/FMC.14.93

Abstract: Background: Chlorotoxin is a small scorpion peptide that inhibits glioma cell migration. We investigated the importance of a major component of chlorotoxin's chemical structure – four disulfide bonds – to its tertiary structure and biological function. Results: Five disulfide bond analogs of chlorotoxin were synthesized, with l-α-aminobutyric acid residues replacing each or all of the disulfide bonds. Chemical oxidation and circular dichroism experiments revealed that Cys III-VII and Cys V-VIII were essential for native structure formation. Cys I-IV and Cys II-VI were important for stability of enzymatic proteolysis but not for the inhibition of human umbilical vein endothelial cell migration. Conclusion: The disulfide bonds of chlorotoxin are important for its structure and stability and have a minor role in its activity against cell migration.

Oxidative Folding of Cyclic Cystine Knot Proteins

Publisher: Mary Ann Liebert Inc

Date: 2008

DOI: 10.1089/ARS.2007.1849

Abstract: Cyclic cystine knot proteins are small but topologically complex molecules that occur naturally in plants and have a wide range of bioactivities that make them interesting from a pharmaceutical perspective. Their remarkable stability is dependent on the correct formation of a knotted arrangement of disulfide bonds. This review reports on studies that have deciphered the pathways to the "tying of the knot." These studies have involved a range of biophysical techniques and suggest that the major intermediate species presented on these pathways are two disulfide native species, which are not necessarily the precursors of the native protein. Structural elucidations of one analogue and one such intermediate have been reported, and they both show highly native-like conformation and native disulfide bond connectivity. Cyclic cystine knot formation has also been shown to be assisted by protein disulfide isomerase. The points summarized in this review will be important to consider in the design of novel pharmaceutically interesting biomolecules based on the cyclic cystine knot motif, which has shown potential as a molecular scaffold because of its exceptional stability.

A Novel Quantitative Kinase Assay Using Bacterial Surface Display and Flow Cytometry

Publisher: Public Library of Science (PLoS)

Date: 15-11-2013

DOI: 10.1371/JOURNAL.PONE.0080474

An N-capping asparagine–lysine–proline (NKP) motif contributes to a hybrid flexible/stable multifunctional peptide scaffold

Publisher: Royal Society of Chemistry (RSC)

Date: 2022

DOI: 10.1039/D1SC06998E

Abstract: An unusual N-capping asparagine-lysine-proline (5NKP7) motif yields a coil/N-cap/α-helix multifunctional scaffold in a computer-made peptide selective for anionic surfaces and with anticancer, antibacterial, antibiofilm, anti-infective ( in vivo ), and immunomodulatory potential.

Antimicrobial Peptide Mimetics Based on a Diphenylacetylene Scaffold: Synthesis, Conformational Analysis, and Activity

Publisher: Wiley

Date: 15-09-2020

DOI: 10.1002/CMDC.202000474

A novel α4/7‐conotoxin LvIA from Conus lividus that selectively blocks α3β2 vs. α6/α3β2β3 nicotinic acetylcholine receptors

Publisher: Wiley

Date: 07-01-2014

DOI: 10.1096/FJ.13-244103

The cyclic cystine knot miniprotein MCoTI-II is internalized into cells by macropinocytosis

Publisher: Elsevier BV

Date: 2007

DOI: 10.1016/J.BIOCEL.2007.06.016

Abstract: The cyclotides are macrocyclic knotted proteins characterized by a compact topology and exceptional stability. Accordingly it has been hypothesized that they may be useful as protein engineering frameworks for the stabilization and delivery of bioactive peptide sequences. This study examined the internalization of cyclotides into mammalian cells, a vital step for the delivery of bioactive peptide sequences to intracellular targets. Although the entry of various linear peptides into cells has been reported previously, this is the first report of internalization of a macrocyclic peptide. Cell uptake was examined for representatives of two cyclotide subfamilies the first was MCoTI-II, a member of the trypsin inhibitor subfamily, which was internalized by a macrophage and breast cancer cell line and the second, the prototypic cyclotide kalata B1 from the Möbius subfamily, which remained extracellular. Biotin labeled MCoTI-II entered macrophages by macropinocytosis, resulting in vesicular encapsulation without trafficking to lysosomes for degradation. The ready uptake, coupled with low cytotoxicity, indicates that MCoTI-II has the potential to transport grafted bioactivities to intracellular targets, making it a potentially valuable framework in drug design applications.

A novel two-chain proteinase inhibitor generated by circularization of a multidomain precursor protein

Publisher: Springer Science and Business Media LLC

Date: 06-1999

DOI: 10.1038/9293

Abstract: Female reproductive tissues of the ornamental tobacco amass high levels of serine proteinase inhibitors (PIs) for protection against pests and pathogens. These PIs are produced from a precursor protein composed of six repeats each with a protease reactive site. Here we show that proteolytic processing of the precursor generates five single-chain PIs and a remarkable two-chain inhibitor formed by disulfide-bond linkage of N- and C-terminal peptide fragments. Surprisingly, PI precursors adopt this circular structure regardless of the number of inhibitor domains, suggesting this bracelet-like conformation is characteristic of the widespread potato inhibitor II (Pot II) protein family.

Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design

Publisher: Proceedings of the National Academy of Sciences

Date: 18-11-2013

DOI: 10.1073/PNAS.1311183110

Abstract: G protein-coupled receptors (GPCRs) are promising drug targets: % of the currently marketed drugs elicit their actions by binding to these transmembrane receptors. However, only ∼10% of all GPCRs are targeted by approved drugs. Resorting to plant-derived compounds catalogued by ethnopharmacological analyses may increase this repertoire. We provide a proof of concept by analyzing the uterotonic action of an herbal remedy used in traditional African medicine. We identified cyclic peptides, investigated the molecular mechanisms underlying their uterotonic activity, and report an oxytocic plant peptide that modulates the human oxytocin/vasopressin receptors. This naturally occurring peptide served as a template for the design of an oxytocin-like nonapeptide with enhanced receptor selectivity, highlighting the potential of cyclotides for the discovery of peptide-based GPCR ligands.

Anchor Residues Guide Form and Function in Grafted Peptides.

Publisher: Wiley

Date: 02-05-2019

DOI: 10.1002/ANIE.201901572

Abstract: Loops at protein-protein interfaces are a rich source of peptide leads that have high specificity and low toxicity. Although such peptides typically need to be constrained to overcome thermodynamic and metabolic limitations, design guidelines to obtain a successfully constrained peptides, and thus facilitate the transition from loop to drug, are relatively poorly formulated. In this work, we surveyed the structures of interface loops and found the position of the terminal residues to be a key determinant of conformation. We used this knowledge to improve the process of molecular grafting, a valuable approach for constraining and stabilising peptides by fusing them to a suitable scaffold. We show that an informed choice of where a loop is "anchored" to a scaffold improves its form and function. This knowledge can help guide the choice of loop and its matching scaffold, and thus increase the success rate for designing stable and potent peptide drug leads.

Insights into Processing and Cyclization Events Associated with Biosynthesis of the Cyclic Peptide Kalata B1

Publisher: Elsevier BV

Date: 08-2012

DOI: 10.1074/JBC.M112.347823

Exploring experimental and computational markers of cyclic peptides: Charting islands of permeability

Publisher: Elsevier BV

Date: 06-2015

DOI: 10.1016/J.EJMECH.2015.04.049

Abstract: An increasing number of macrocyclic peptides that cross biological membranes are being reported, suggesting that it might be possible to develop peptides into orally bioavailable therapeutics however, current understanding of what makes macrocyclic peptides cell permeable is still limited. Here, we synthesized 62 cyclic hexapeptides and characterized their permeability using in vitro assays commonly used to predict in vivo absorption rates, i.e. the Caco-2 and PAMPA assays. We correlated permeability with experimentally measured parameters of peptide conformation obtained using rapid methods based on chromatography and nuclear magnetic resonance spectroscopy. Based on these correlations, we propose a model describing the interplay between peptide permeability, lipophilicity and hydrogen bonding potential. Specifically, peptides with very high permeability have high lipophilicity and few solvent hydrogen bond interactions, whereas peptides with very low permeability have low lipophilicity or many solvent interactions. Our model is supported by molecular dynamics simulations of the cyclic peptides calculated in explicit solvent, providing a structural basis for the observed correlations. This prospective exploration into biomarkers of peptide permeability has the potential to unlock wider opportunities for development of peptides into drugs.

Solving the α-conotoxin folding problem: Efficient selenium-directed on-resin generation of more potent and stable nicotinic acetylcholine receptor antaqonists

Publisher: American Chemical Society (ACS)

Date: 17-02-2010

DOI: 10.1021/JA910602H

Abstract: Alpha-conotoxins are tightly folded miniproteins that antagonize nicotinic acetylcholine receptors (nAChR) with high specificity for erse subtypes. Here we report the use of selenocysteine in a supported phase method to direct native folding and produce alpha-conotoxins efficiently with improved biophysical properties. By replacing complementary cysteine pairs with selenocysteine pairs on an hiphilic resin, we were able to chemically direct all five structural subclasses of alpha-conotoxins exclusively into their native folds. X-ray analysis at 1.4 A resolution of alpha-selenoconotoxin PnIA confirmed the isosteric character of the diselenide bond and the integrity of the alpha-conotoxin fold. The alpha-selenoconotoxins exhibited similar or improved potency at rat diaphragm muscle and alpha3beta4, alpha7, and alpha1beta1 deltagamma nAChRs expressed in Xenopus oocytes plus improved disulfide bond scrambling stability in plasma. Together, these results underpin the development of more stable and potent nicotinic antagonists suitable for new drug therapies, and highlight the application of selenocysteine technology more broadly to disulfide-bonded peptides and proteins.

Structure-activity analysis of truncated albumin-binding domains suggests new lead constructs for potential therapeutic delivery

Publisher: Elsevier BV

Date: 08-2020

DOI: 10.1074/JBC.RA120.014168

Linearization of a Naturally Occurring Circular Protein Maintains Structure but Eliminates Hemolytic Activity^,

Publisher: American Chemical Society (ACS)

Date: 16-05-2003

DOI: 10.1021/BI027323N

Abstract: Cyclotides are a recently discovered family of disulfide rich proteins from plants that contain a circular protein backbone. They are exceptionally stable, as exemplified by their use in native medicine of the prototypic cyclotide kalata B1. The peptide retains uterotonic activity after the plant from which it is derived is boiled to make a medicinal tea. The circular backbone is thought to be in part responsible for the stability of the cyclotides, and to investigate its role in determining structure and biological activity, an acyclic derivative, des-(24-28)-kalata B1, was chemically synthesized and purified. This derivative has five residues removed from the 29-amino acid circular backbone of kalata B1 in a loop region corresponding to a processing site in the biosynthetic precursor protein. Two-dimensional NMR spectra of the peptide were recorded, assigned, and used to identify a series of distance, angle, and hydrogen bonding restraints. These were in turn used to determine a representative family of solution structures. Of particular interest was a determination of the structural similarities and differences between des-(24-28)-kalata B1 and native kalata B1. Although the overall three-dimensional fold remains very similar to that of the native circular protein, removal of residues 24-28 of kalata B1 causes disruption of some structural features that are important to the overall stability. Furthermore, loss of hemolytic activity is associated with backbone truncation and linearization.

The emerging landscape of peptide-based inhibitors of PCSK9

Publisher: Elsevier BV

Date: 08-2021

DOI: 10.1016/J.ATHEROSCLEROSIS.2021.06.903

Isolation and Characterization of Novel Cyclotides from Viola hederaceae

Publisher: Elsevier BV

Date: 06-2005

DOI: 10.1074/JBC.M501737200

RegIIA

Publisher: Elsevier BV

Date: 02-2012

DOI: 10.1016/J.BCP.2011.11.006

Abstract: Neuronal nicotinic acetylcholine receptors (nAChRs) play pivotal roles in the central and peripheral nervous systems. They are implicated in disease states such as Parkinson's disease and schizophrenia, as well as addictive processes for nicotine and other drugs of abuse. Modulation of specific nAChRs is essential to understand their role in the CNS. α-Conotoxins, disulfide-constrained peptides isolated from the venom of cone snails, potently inhibit nAChRs. Their selectivity varies markedly depending upon the specific nAChR subtype/α-conotoxin pair under consideration. Thus, α-conotoxins are excellent probes to evaluate the functional roles of nAChRs subtypes. We isolated an α4/7-conotoxin (RegIIA) from the venom of Conus regius. Its sequence was determined by Edman degradation and confirmed by sequencing the cDNA of the protein precursor. RegIIA was synthesized using solid phase methods and native and synthetic RegIIA were functionally tested using two-electrode voltage cl recording on nAChRs expressed in Xenopus laevis oocytes. RegIIA is among the most potent antagonist of the α3β4 nAChRs found to date and is also active at α3β2 and α7 nAChRs. The 3D structure of RegIIA reveals the typical folding of most α4/7-conotoxins. Thus, while structurally related to other α4/7 conotoxins, RegIIA has an exquisite balance of shape, charge, and polarity exposed in its structure to potently block the α3β4 nAChRs.

Key Residues in the Nicotinic Acetylcholine Receptor β2 Subunit Contribute to α-Conotoxin LvIA Binding

Publisher: Elsevier BV

Date: 04-2015

DOI: 10.1074/JBC.M114.632646

Computational and functional mapping of human and rat α6β4 nicotinic acetylcholine receptors reveals species-specific ligand-binding motifs

Publisher: American Chemical Society (ACS)

Date: 02-2021

DOI: 10.1021/ACS.JMEDCHEM.0C01973

Cellular Uptake and Cytosolic Delivery of a Cyclic Cystine Knot Scaffold

Publisher: American Chemical Society (ACS)

Date: 21-04-2020

DOI: 10.1021/ACSCHEMBIO.0C00297

Molecular engineering of conotoxins: The importance of loop size to α-conotoxin structure and function

Publisher: American Chemical Society (ACS)

Date: 28-08-2008

DOI: 10.1021/JM800278K

Abstract: Alpha-conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). The majority of currently characterized alpha-conotoxins have a 4/7 loop size, and the major features of neuronal alpha-conotoxins include a globular disulfide connectivity and a helical structure centered around the third of their four cysteine residues. In this study, a novel "molecular pruning" approach was undertaken to define the relationship between loop size, structure, and function of alpha-conotoxins. This involved the systematic truncation of the second loop in the alpha-conotoxin [A10L]PnIA [4/7], a potent antagonist of the alpha7 nAChR. The penalty for truncation was found to be decreased conformational stability and increased susceptibility to disulfide bond scrambling. Truncation down to 4/4[A10L]PnIA maintained helicity and did not significantly reduce electrophysiological activity at alpha7 nAChRs, whereas 4/3[A10L]PnIA lost both alpha7 nAChR activity and helicity. In contrast, all truncated analogues lost approximately 100-fold affinity at the AChBP, a model protein for the extracellular domain of the nAChR. Docking simulations identified several hydrogen bonds lost upon truncation that provide an explanation for the reduced affinities observed at the alpha7 nAChR and AChBP.

Diversity in the disulfide folding pathways of cystine knot peptides

Publisher: Springer Science and Business Media LLC

Date: 09-2003

DOI: 10.1007/BF02442584

Chemical synthesis, 3D structure, and ASIC binding site of the toxin mambalgin-2

Publisher: Wiley

Date: 09-12-2014

DOI: 10.1002/ANIE.201308898

Abstract: Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid-sensing ion channels (ASICs). The 57-residue polypeptide mambalgin-2 (Ma-2) was synthesized by using a combination of solid-phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three-finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma-2 on wild-type and mutant ASIC1a receptors allowed us to identify α-helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma-2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure-activity relationship (SAR) studies and further development of this promising analgesic peptide.

Improving the Selectivity of Engineered Protease Inhibitors: Optimizing the P2 Prime Residue Using a Versatile Cyclic Peptide Library

Publisher: American Chemical Society (ACS)

Date: 05-10-2015

DOI: 10.1021/ACS.JMEDCHEM.5B01148

Abstract: Standard mechanism inhibitors are attractive design templates for engineering reversible serine protease inhibitors. When optimizing interactions between the inhibitor and target protease, many studies focus on the nonprimed segment of the inhibitor's binding loop (encompassing the contact β-strand). However, there are currently few methods for screening residues on the primed segment. Here, we designed a synthetic inhibitor library (based on sunflower trypsin inhibitor-1) for characterizing the P2' specificity of various serine proteases. Screening the library against 13 different proteases revealed unique P2' preferences for trypsin, chymotrypsin, matriptase, plasmin, thrombin, four kallikrein-related peptidases, and several clotting factors. Using this information to modify existing engineered inhibitors yielded new variants that showed considerably improved selectivity, reaching up to 7000-fold selectivity over certain off-target proteases. Our study demonstrates the importance of the P2' residue in standard mechanism inhibition and unveils a new approach for screening P2' substitutions that will benefit future inhibitor engineering studies.

Identification and characterization of a new family of cell-penetrating peptides: Cyclic cell-penetrating peptides

Publisher: Elsevier BV

Date: 10-2011

DOI: 10.1074/JBC.M111.264424

Mining the genome of Arabidopsis thaliana as a basis for the identification of novel bioactive peptides involved in oxidative stress tolerance

Publisher: Oxford University Press (OUP)

Date: 16-09-2013

DOI: 10.1093/JXB/ERT295

Abstract: Although evidence has accumulated on the role of plant peptides in the response to external conditions, the number of peptide-encoding genes in the genome is still underestimated. Using tiling arrays, we identified 176 unannotated transcriptionally active regions (TARs) in Arabidopsis thaliana that were induced upon oxidative stress generated by the herbicide paraquat (PQ). These 176 TARs could be translated into 575 putative oxidative stress-induced peptides (OSIPs). A high-throughput functional assay was used in the eukaryotic model organism Saccharomyces cerevisiae allowing us to test for bioactive peptides that increase oxidative stress tolerance. In this way, we identified three OSIPs that, upon overexpression in yeast, resulted in a significant rise in tolerance to hydrogen peroxide (H2O2). For one of these peptides, the decapeptide OSIP108, exogenous application to H2O2-treated yeast also resulted in significantly increased survival. OSIP108 is contained within a pseudogene and is induced in A. thaliana leaves by both the reactive oxygen species-inducer PQ and the necrotrophic fungal pathogen Botrytis cinerea. Moreover, infiltration and overexpression of OSIP108 in A. thaliana leaves resulted in increased tolerance to treatment with PQ. In conclusion, the identification and characterization of OSIP108 confirms the validity of our high-throughput approach, based on tiling array analysis in A. thaliana and functional screening in yeast, to identify bioactive peptides.

Rational domestication of a plant-based recombinant expression system expands its biosynthetic range

Publisher: Cold Spring Harbor Laboratory

Date: 10-12-2021

DOI: 10.1101/2021.12.09.472022

Abstract: Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead and an orally active analgesic are demonstrated.

Development of a μO-conotoxin analogue with improved lipid membrane interactions and potency for the analgesic sodium channel NaV1.8

Publisher: Elsevier BV

Date: 05-2016

DOI: 10.1074/JBC.M116.721662

Synthesis of an Analog of the Thyroid Hormone-binding Protein Transthyretin via Regioselective Chemical Ligation

Publisher: Elsevier BV

Date: 07-2001

DOI: 10.1074/JBC.M101228200

Exploring the Sequence Diversity of Cyclotides from Vietnamese Viola Species

Publisher: American Chemical Society (ACS)

Date: 21-05-2020

DOI: 10.1021/ACS.JNATPROD.9B01218

Optimization of the cyclotide framework to improve cell penetration properties

Publisher: Frontiers Media SA

Date: 09-02-2015

DOI: 10.3389/FPHAR.2015.00017

Structural insights into the function of relaxins

Publisher: Wiley

Date: 04-2009

DOI: 10.1111/J.1749-6632.2009.03833.X

Abstract: The relaxin peptide hormones are members of the insulin superfamily and share a structural fold that is characterized by two peptide chains which are cross-braced by three disulfide bonds. On this framework, various amino acid side chains are presented, allowing specific interactions with different receptors. The relaxin receptors belong to two unrelated classes of G-protein-coupled receptors, but interestingly they are not selective for a single relaxin peptide. Relaxin-3, which is considered to be an extreme ex le of the relaxin family, can activate receptors from both classes and in fact interacts to some degree with all four receptors identified to date. To deduce how changes in the primary sequence can fine-tune the overall structure and thus the ability to interact with the various receptors, we have studied a range of relaxin-like peptides using solution nuclear magnetic resonance analysis. Three-dimensional structures of relaxin-3, insulin-like peptide 3 (INSL3), and INSL5 were determined and revealed a number of interesting features. All peptides showed a significant amount of line-broadening in certain regions, in particular around the intra-A-chain disulfide bond, suggesting that despite the disulfide bonds the fold is rather dynamic. Although the peptides share a common structural core there are significant differences, particularly around the termini. The structural data in combination with mutational studies provide valuable insights into the structure-activity relationships of relaxins.

Inhibitors of β-amyloid formation based on the β-secretase cleavage site

Publisher: Elsevier BV

Date: 02-2000

DOI: 10.1006/BBRC.2000.2098

Novel ω-conotoxins from Conus catus discriminate among neuronal calcium channel subtypes

Publisher: Elsevier BV

Date: 11-2000

DOI: 10.1074/JBC.M002252200

Identification of Initiation Sites for T4 Lysozyme Folding Using CD and NMR Spectroscopy of Peptide Fragments

Publisher: American Chemical Society (ACS)

Date: 21-04-2000

DOI: 10.1021/BI000070I

Abstract: Using CD and 2D (1)H NMR spectroscopy, we have identified potential initiation sites for the folding of T4 lysozyme by examining the conformational preferences of peptide fragments corresponding to regions of secondary structure. CD spectropolarimetry showed most peptides were unstructured in water, but adopted partial helical conformations in TFE and SDS solution. This was also consistent with the (1)H NMR data which showed that the peptides were predominantly disordered in water, although in some cases, nascent or small populations of partially folded conformations could be detected. NOE patterns, coupling constants, and deviations from random coil Halpha chemical shift values complemented the CD data and confirmed that many of the peptides were helical in TFE and SDS micelles. In particular, the peptide corresponding to helix E in the native enzyme formed a well-defined helix in both TFE and SDS, indicating that helix E potentially forms an initiation site for T4 lysozyme folding. The data for the other peptides indicated that helices D, F, G, and H are dependent on tertiary interactions for their folding and/or stability. Overall, the results from this study, and those of our earlier studies, are in agreement with modeling and HD-deuterium exchange experiments, and support an hierarchical model of folding for T4 lysozyme.

Determination of the Solution Structures of Conantokin-G and Conantokin-T by CD and NMR Spectroscopy

Publisher: Elsevier BV

Date: 1997

DOI: 10.1074/JBC.272.4.2291

An environmentally sustainable biomimetic production of cyclic disulfide-rich peptides

Publisher: Royal Society of Chemistry (RSC)

Date: 2020

DOI: 10.1039/D0GC01366H

Abstract: An environmentally sustainable production platform for a variety of correctly folded cyclic disulfide-rich peptides with enhanced yields.

Cyclotides are a component of the innate defense of Oldenlandia affinis

Publisher: Wiley

Date: 2010

DOI: 10.1002/BIP.21419

Abstract: Cyclotides are small cysteine-rich plant peptides similar in size and processing to the defensins. Long-term growth of the Rubiaceae family plant Oldenlandia affinis under different conditions reveals a erse cyclotide gene and peptide expression profile, including tissue specificity, suggesting that different cyclotides are regulated differently both spatially and in response to the environment. To determine whether cyclotide precursor gene regulation was dynamic we exposed O. affinis to a range of abiotic, biotic, and hormonal stimuli and monitored Oak1-4 expression over a 48-h period. Unlike some defensins, the genes for cyclotide precursor proteins Oak1-4 did not display dynamic change, indicating that they contribute to the basal defense of O. affinis. Despite this lack of dynamism, the cyclotide profile of plants grown on plates differed markedly from field-grown plants and so prompted attempts to discover novel cyclotides and precursor genes. The two most abundant cyclotides from plate-grown O. affinis were sequenced and one was found to be an unusual linear cyclotide derivative, kalata B20-lin. Degenerate PCR of plate-grown O. affinis obtained five novel cyclotide genes including Oak9 which encodes for kalata B20-lin and appears to have arisen by the presence of a premature stop codon.

Discovery of Cyclotides in the Fabaceae Plant Family Provides New Insights into the Cyclization, Evolution, and Distribution of Circular Proteins

Publisher: American Chemical Society (ACS)

Date: 20-01-2011

DOI: 10.1021/CB100388J

Abstract: Cyclotides are plant proteins whose defining structural features are a head-to-tail cyclized backbone and three interlocking disulfide bonds, which in combination are known as a cyclic cystine knot. This unique structural motif confers cyclotides with exceptional resistance to proteolysis. Their endogenous function is thought to be as plant defense agents, associated with their insecticidal and larval growth-inhibitory properties. However, in addition, an array of pharmaceutically relevant biological activities has been ascribed to cyclotides, including anti-HIV, anthelmintic, uterotonic, and antimicrobial effects. So far, >150 cyclotides have been elucidated from members of the Rubiaceae, Violaceae, and Cucurbitaceae plant families, but their wider distribution among other plant families remains unclear. Clitoria ternatea (Butterfly pea) is a member of plant family Fabaceae and through its usage in traditional medicine to aid childbirth bears similarity to Oldenlandia affinis, from which many cyclotides have been isolated. Using a combination of nanospray and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) analyses, we examined seed extracts of C. ternatea and discovered cyclotides in the Fabaceae, the third-largest family of flowering plants. We characterized 12 novel cyclotides, thus expanding knowledge of cyclotide distribution and evolution within the plant kingdom. The discovery of cyclotides containing novel sequence motifs near the in planta cyclization site has provided new insights into cyclotide biosynthesis. In particular, MS analyses of the novel cyclotides from C. ternatea suggest that Asn to Asp variants at the cyclization site are more common than previously recognized. Moreover, this study provides impetus for the examination of other economically and agriculturally significant species within Fabaceae, now the largest plant family from which cyclotides have been described.

Discovery and Characterization of Cyclic and Acyclic Trypsin Inhibitors from Momordica dioica.

Publisher: American Chemical Society (ACS)

Date: 23-01-2019

DOI: 10.1021/ACS.JNATPROD.8B00716

Abstract: Momordica trypsin inhibitors (TIs) such as those isolated from the seeds of the gấc fruit, Momordica cochinchinensis (MCoTI-I and MCoTI-II), are widely used as scaffolds for drug design studies. To more effectively exploit these molecules in the development of therapeutics, there is a need for wider discovery of the natural sequence ersity among TIs from other species in the Momordica subfamily. Here we report the discovery of the encoding gene and six TIs from the seeds of the spiny gourd, Momordica dioica, four of which possess novel sequences (Modi 1, 3, 5, and 6) and two (Modi 2 and 4) of which are known peptides (TI-14, TI-17) previously identified in Momordica subangulata. Modi 6 is an acyclic peptide featuring a pyrrolidone carboxylic acid modification, whereas the remaining five TIs are cyclic. All Modi peptides display similar overall structures and trypsin inhibitory activities. No toxicity was observed for these peptides when tested against cancer and insect cells. All Modi peptides were exceptionally stable over 24 h in human serum, indicating a dual strategy to stabilize the peptides in nature, either head-to-tail cyclization or N-pyrolation, which suggests these peptides might be excellent candidates as scaffolds for epitope stabilization in drug design studies.

Structure of the R3/I5 chimeric relaxin peptide, a selective GPCR135 and GPCR142 agonist

Publisher: Elsevier BV

Date: 08-2008

DOI: 10.1074/JBC.M800489200

ConoServer, a database for conopeptide sequences and structures

Publisher: Oxford University Press (OUP)

Date: 06-12-2008

DOI: 10.1093/BIOINFORMATICS/BTM596

Abstract: Summary: ConoServer is a new database dedicated to conopeptides, a large family of peptides found in the venom of marine snails of the genus Conus. These peptides have an exceptional ersity of sequences and chemical modifications and their ability to block ion channels makes them important as drug leads and tools for physiological studies. ConoServer uses standardized names and a genetic and structural classification scheme to present data retrieved from SwissProt, GenBank, the Protein DataBank and the literature. The ConoServer web site incorporates specialized features like the graphic display of post-translational modifications that are extensively present in conopeptides. Currently, ConoServer manages 1214 nucleic sequences (from 54 Conus species), 2258 proteic sequences (from 66 Conus species) and 99 3D structures. Availability: research1t.imb.uq.edu.au/conoserver/ Contact: d.craik@imb.uq.edu.au

Sunflower trypsin inhibitor-1, proteolytic studies on a trypsin inhibitor peptide and its analogs.

Publisher: Wiley

Date: 2010

DOI: 10.1002/BIP.21415

Abstract: Sunflower trypsin inhibitor-1 (SFTI-1) is a 14 amino acid cyclic peptide from sunflower seeds, which possesses exceptionally potent trypsin-inhibitory activity, and has promise as a stable peptide-based drug template. Within its compact structure, SFTI-1 combines a head-to-tail cyclized backbone and a disulfide bond. In this study, we synthesized a range of acyclic and disulfide-deficient analogs of SFTI-1 to investigate enzyme-assisted cyclization of the peptide backbone and proteolytic degradation that occurs as a result of incubation with trypsin. Electrospray and matrix-assisted laser desorption ionization mass spectrometry allowed the characterization of a range of novel degradation products and elucidation of the time-course for cyclization and/or proteolysis. Trypsin displayed the ability to resynthesize the scissile bond(s) and hence cyclize two of the linear permutants, whereas irreversible degradation was observed for another two permutants. An interesting ring contraction mediated by trypsin was observed, supporting a role for protease catalyzed splicing as a way of increasing the combinatorial ersity of cyclic peptides in nature. Disulfide-deficient mutants were degraded within minutes, emphasizing the critical role of the cysteine bridge in maintaining proteolytic stability of SFTI-1. Overall, the study provides additional support for the proposal that naturally occurring cyclic peptides like SFTI-1 are biosynthesized by proteolytic enzymes effectively catalyzing the reverse of their normal reaction to make, rather than break peptide bonds.

Stabilization of α-Conotoxin AuIB: Influences of Disulfide Connectivity and Backbone Cyclization

Publisher: Mary Ann Liebert Inc

Date: 2011

DOI: 10.1089/ARS.2009.3068

Abstract: α-Conotoxins are peptides isolated from the venom ducts of cone snails that target nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential applications for treating a range of conditions in humans, including pain. However, like all peptides, conotoxins are susceptible to degradation, and to enhance their therapeutic potential it is important to elucidate the factors contributing to instability and to develop approaches for improving stability. AuIB is a unique member of the α-conotoxin family because the nonnative "ribbon" disulfide isomer exhibits enhanced activity at the nAChR in rat parasympathetic neurons compared with the native "globular" isomer. Here we show that the ribbon isomer of AuIB is also more resistant to disulfide scrambling, despite having a nonnative connectivity and flexible structure. This resistance to disulfide scrambling does not correlate with overall stability in serum because the ribbon isomer is degraded in human serum more rapidly than the globular isomer. Cyclization via the joining of the N- and C-termini with peptide linkers of four to seven amino acids prevented degradation of the ribbon isomer in serum and stabilized the globular isomers to disulfide scrambling. The linker length used for cyclization strongly affected the relative proportions of the disulfide isomers produced by oxidative folding. Overall, the results of this study provide important insights into factors influencing the stability and oxidative folding of α-conotoxin AuIB and might be valuable in the design of more stable antagonists of nAChRs.

Effects of microstructure and growth conditions on quantum emitters in gallium nitride

Publisher: AIP Publishing

Date: 08-2019

DOI: 10.1063/1.5098794

Abstract: Single-photon emitters in gallium nitride (GaN) are gaining interest as attractive quantum systems due to the well-established techniques for growth and nanofabrication of the host material, as well as its remarkable chemical stability and optoelectronic properties. We investigate the nature of such single-photon emitters in GaN with a systematic analysis of various s les produced under different growth conditions. We explore the effect that intrinsic structural defects (dislocations and stacking faults), doping, and crystal orientation in GaN have on the formation of quantum emitters. We investigate the relationship between the position of the emitters—determined via spectroscopy and photoluminescence measurements—and the location of threading dislocations—characterized both via atomic force microscopy and cathodoluminescence. We find that quantum emitters do not correlate with stacking faults or dislocations instead, they are more likely to originate from point defects or impurities whose density is modulated by the local extended defect density.

The Three-dimensional Solution Structure of NaD1, a New Floral Defensin from Nicotiana alata and its Application to a Homology Model of the Crop Defense Protein alfAFP

Publisher: Elsevier BV

Date: 2003

DOI: 10.1016/S0022-2836(02)01103-8

Abstract: NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of NaD1, a novel antifungal and insecticidal protein isolated from the flowers of Nicotiana alata. NaD1 is a basic, cysteine-rich protein of 47 residues and is the first ex le of a plant defensin from flowers to be characterized structurally. Its three-dimensional structure consists of an alpha-helix and a triple-stranded antiparallel beta-sheet that are stabilized by four intramolecular disulfide bonds. NaD1 features all the characteristics of the cysteine-stabilized alphabeta motif that has been described for a variety of proteins of differing functions ranging from antibacterial insect defensins and ion channel-perturbing scorpion toxins to an elicitor of the sweet taste response. The protein is biologically active against insect pests, which makes it a potential candidate for use in crop protection. NaD1 shares 31% sequence identity with alfAFP, an antifungal protein from alfalfa that confers resistance to a fungal pathogen in transgenic potatoes. The structure of NaD1 was used to obtain a homology model of alfAFP, since NaD1 has the highest level of sequence identity with alfAFP of any structurally characterized antifungal defensin. The structures of NaD1 and alfAFP were used in conjunction with structure-activity data for the radish defensin Rs-AFP2 to provide an insight into structure-function relationships. In particular, a putative effector site was identified in the structure of NaD1 and in the corresponding homology model of alfAFP.

Disulfide-rich macrocyclic peptides as templates in drug design

Publisher: Elsevier BV

Date: 04-2014

DOI: 10.1016/J.EJMECH.2014.03.011

Abstract: Recently disulfide-rich head-to-tail cyclic peptides have attracted the interest of medicinal chemists owing to their exceptional thermal, chemical and enzymatic stability brought about by their constrained structures. Here we review current trends in the field of peptide-based pharmaceuticals and describe naturally occurring cyclic disulfide-rich peptide scaffolds, discussing their pharmaceutically attractive properties and benefits. We describe how we can utilise these stable frameworks to graft and/or engineer pharmaceutically interesting epitopes to increase their selectivity and bioactivity, opening up new possibilities for addressing 'difficult' pharmaceutical targets.

Chemical synthesis and folding pathways of large cyclic polypeptides: Studies of the cystine knot polypeptide kalata B1

Publisher: American Chemical Society (ACS)

Date: 21-07-1999

DOI: 10.1021/BI990605B

Structure and Activity of α-Conotoxin PeIA at Nicotinic Acetylcholine Receptor Subtypes and GABAB Receptor-coupled N-type Calcium Channels

Publisher: Elsevier BV

Date: 03-2011

DOI: 10.1074/JBC.M110.196170

Evaluation of toxicity and antitumor activity of cycloviolacin O2 in mice

Publisher: Wiley

Date: 2010

DOI: 10.1002/BIP.21408

Abstract: Cycloviolacin O2 is a small cyclic cysteine-rich protein belonging to the group of plant proteins called cyclotides. This cyclotide has been previously shown to exert cytotoxic activity against a variety of human tumor cell lines as well as primary cultures of human tumor cells in vitro. This study is the first evaluation of its tolerability and antitumor activity in vivo. Maximal-tolerated doses were estimated to 1.5 mg/kg for single intravenous (i.v.) dosing and 0.5 mg/kg for daily repeated dosing, respectively. Two different in vivo methods were used: the hollow fiber method with single dosing (i.v., 1.0 mg/kg) and traditional xenografts with repeated dosing over 2 weeks (i.v., 0.5 mg/kg daily, 5 days a week). The human tumor cell lines used displayed dose-dependent in vitro sensitivity (including growth in hollow fibers to confirm passage of cycloviolacin O2 through the polyvinylidene fluoride fibers), with IC5o values in the micromolar range. Despite this sensitivity in vitro, no significant antitumor effects were detected in vivo, neither with single dosing in the hollow fiber method nor with repeated dosing in xenografts. In summary, the results indicate that antitumor effects are minor or absent at tolerable (sublethal) doses, and cycloviolacin O2 has a very abrupt in vivo toxicity profile, with lethality after single injection at 2 mg/kg, but no signs of discomfort to the animals at 1.5 mg/kg. Repeated dosing of 1 mg/kg gave a local-inflammatory reaction at the site of injection after 2-3 days lower doses were without complications.

The Cyclic Cystine Ladder in θ-Defensins Is Important for Structure and Stability, but Not Antibacterial Activity

Publisher: Elsevier BV

Date: 04-2013

DOI: 10.1074/JBC.M113.451047

Rational domestication of a plant-based recombinant expression system expands its biosynthetic range

Publisher: Oxford University Press (OUP)

Date: 20-06-2022

DOI: 10.1093/JXB/ERAC273

Abstract: Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead, and an orally active analgesic is demonstrated.

A new era for cyclotide sequencing.

Publisher: Wiley

Date: 2010

DOI: 10.1002/BIP.21400

Abstract: In recent years, the discovery of a large family of macrocyclic peptides, the cyclotides, has revealed Natures ingenuity in molecular drug design. The incorporation of a cyclic peptide backbone and a knotted arrangement of disulfide bridges into their structures confers extraordinary chemical, thermal, and enzymatic stability on these biologically active peptides. However, these structural attributes present challenges in the identification of cyclotides. Until now, the sequencing of cyclotides has been slow and inefficient owing to inherent difficulties in the separation of these hydrophobic peptides from plants, the multiple chemical and enzymatic derivatization steps required to make them amenable to mass spectrometric sequencing, and the lack of software tools to efficiently deal with these circular permutants. The current bottleneck slowing the speed of cyclotide sequencing is the requirement for multiple HPLC purification steps before analysis. Here, we have applied proteomic strategies to fast-track the discovery of known, modified and novel sequences. Using four fractions from a previously well-characterized cyclotide-containing plant species, Viola odorata, 11 new sequences, as well as a plethora of known and modified cyclotides, were uncovered. In addition, the methodology was validated through analysis of crude leaf extracts ofOldenlandia affinis and Arabidopsis thaliana. The unambiguous identification of a suite of cyclotides in the Oldenlandia affinis extract provided the ultimate proof-of-concept for this application. Major advances in methodology include the use of optimized LC-MS/MS conditions and design of a custom-built cyclotide database, in which mature cyclotide sequences are excised, replicated and appended, marking a new "era" for cyclotide sequencing.

Solution structures by 1 H NMR of the novel cyclic trypsin inhibitor SFTI-1 from sunflower seeds and an acyclic permutant 1 1Edited by M. F. Summers

Publisher: Elsevier BV

Date: 08-2001

DOI: 10.1006/JMBI.2001.4887

Tuning the Anti-Angiogenic Effect of the P15 Peptide Using Cyclic Trypsin Inhibitor Scaffolds

Publisher: American Chemical Society (ACS)

Date: 21-04-2021

DOI: 10.1021/ACSCHEMBIO.0C00907

The N‐terminal pro‐domain of the kalata B1 cyclotide precursor is intrinsically unstructured

Publisher: Wiley

Date: 11-2016

DOI: 10.1002/BIP.22977

Abstract: Cyclotides are plant-derived, gene-encoded, circular peptides with a range of host-defense functions, including insecticidal activity. They also have potential as pharmaceutical scaffolds and understanding their biosynthesis is important to facilitate their large-scale production. Insights into the biosynthesis of cyclotides are emerging but there are still open questions, particularly regarding the influence of the structure of the precursor proteins on processing/biosynthetic pathways. The precursor protein of kalata B1, encoded by the plant Oldenlandia affinis, contains N- and C-terminal propeptides that flank the mature cyclotide domain. The C-terminal region (ctr) is important for the cyclization process, whereas the N-terminal repeat (ntr) has been implicated in vacuolar targeting. In this study we examined the structure and folding of various truncated constructs of the ntr coupled to the mature domain of kalata B1. Despite the ntr having a well-defined helical structure in isolation, once coupled to the natively folded mature domain there is no evidence of an ordered structure. Surprisingly, the ntr appears to be highly disordered and induces self-association of the precursor. This self-association might be associated with the role of the ntr as a vacuolar-targeting signal, as previously shown for unrelated storage proteins.

The Antimicrobial Activity of Sub3 is Dependent on Membrane Binding and Cell‐Penetrating Ability

Publisher: Wiley

Date: 26-08-2013

DOI: 10.1002/CBIC.201300274

Abstract: Because of their high activity against microorganisms and low cytotoxicity, cationic antimicrobial peptides (AMPs) have been explored as the next generation of antibiotics. Although they have common structural features, the modes of action of AMPs are extensively debated, and a single mechanism does not explain the activity of all AMPs reported so far. Here we investigated the mechanism of action of Sub3, an AMP previously designed and optimised from high-throughput screening with bactenecin as the template. Sub3 has potent activity against Gram-negative and Gram-positive bacteria as well as against fungi, but its mechanism of action has remained elusive. By using AFM imaging, ζ potential, flow cytometry and fluorescence methodologies with model membranes and bacterial cells, we found that, although the mechanism of action involves membrane targeting, Sub3 internalises inside bacteria at lethal concentrations without permeabilising the membrane, thus suggesting that its antimicrobial activity might involve both the membrane and intracellular targets. In addition, we found that Sub3 can be internalised into human cells without being toxic. As some bacteria are able to survive intracellularly and consequently evade host defences and antibiotic treatment, our findings suggest that Sub3 could be useful as an intracellular antimicrobial agent for infections that are notoriously difficult to treat.

Understanding the Diversity and Distribution of Cyclotides from Plants of Varied Genetic Origin

Publisher: American Chemical Society (ACS)

Date: 04-05-2017

DOI: 10.1021/ACS.JNATPROD.7B00061

Abstract: Cyclotides are a large family of naturally occurring plant-derived macrocyclic cystine-knot peptides, with more than 400 having been identified in species from the Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae, and Solanaceae families. Nevertheless, their specialized distribution within the plant kingdom remains poorly understood. In this study, the ersity of cyclotides was explored through the screening of 197 plants belonging to 43 different families. In total, 28 cyclotides were sequenced from 15 plant species, one of which belonged to the Rubiaceae and 14 to the Violaceae. Every Violaceae species screened contained cyclotides, but they were only sparsely represented in Rubiaceae and nonexistent in other families. The study thus supports the hypothesis that cyclotides are ubiquitous in the Violaceae, and it adds to the list of plants found to express kalata S and cycloviolacin O12. Finally, previous studies suggested the existence of cyclotide isoforms with either an Asn or an Asp at the C-terminal processing site of the cyclotide domain within the precursor proteins. Here we found that despite the discovery of a few cyclotides genuinely containing an Asp in loop 6 as evidenced by gene sequencing, deamidation of Asn during enzymatic digestion resulted in the artifactual presence of Asp isoforms. This result is consistent with studies suggesting that peptides can undergo deamidation after being subjected to external factors, including pH, temperature, and enzymatic digestion.

Cellular Uptake and Cytosolic Delivery of a Cyclic Cystine Knot Scaffold

Publisher: American Chemical Society (ACS)

Date: 19-02-2020

DOI: 10.26434/CHEMRXIV.11860155

Abstract: Cyclotides are macrocyclic peptides that have exceptionally stable structures and been reported to penetrate cells, making them promising scaffolds for the delivery of peptide inhibitory sequences to target intracellular proteins. However, their cellular uptake and cytosolic localization have been poorly understood until now, which has limited their therapeutic potential. In this study, the recently developed chloroalkane penetration assay was combined with established assays to characterize the cellular uptake and cytosolic delivery of the prototypic cyclotide, kalata B1. We show that kalata B1 enters the cytosol at low efficiency, but introducing various epitopes, including a single hydrophobic amino acid, into its loop 6 significantly improved its cytosolic delivery. Our results provide a foundation for the further development of a structurally unique class of scaffolds for the delivery of therapeutic cargoes into cells. br

Cyclization of the Antimicrobial Peptide Gomesin with Native Chemical Ligation: Influences on Stability and Bioactivity

Publisher: Wiley

Date: 20-02-2013

DOI: 10.1002/CBIC.201300034

Abstract: Gomesin is an 18-residue peptide originally isolated from the hemocytes of the Brazilian spider Acanthoscurria gomesiana. A broad spectrum of bioactivities have been attributed to gomesin, including in vivo and in vitro cytotoxicity against tumour cells, antimicrobial, antifungal, anti-Leishmania and antimalarial effects. Given the potential therapeutic applications of gomesin, it was of interest to determine if an engineered version with a cyclic backbone has improved stability and bioactivity. Cyclization has been shown to confer enhanced stability and activity to a range of bioactive peptides and, in the case of a cone snail venom peptide, confer oral activity in a pain model. The current study demonstrates that cyclization improves the in vitro stability of gomesin over a 24 hour time period and enhances cytotoxicity against a cancer cell line without being toxic to a noncancerous cell line. In addition, antimalarial activity is enhanced upon cyclization. These findings provide additional insight into the influences of backbone cyclization on the therapeutic potential of peptides.

Synthesis of cyclic penta- and hexapeptides: A general synthetic strategy on DAS resin

Publisher: Springer Science and Business Media LLC

Date: 04-1994

DOI: 10.1007/BF01901567

NMR solution structure of the RNA-binding peptide from human immunodeficiency virus (type 1) Rev

Publisher: American Chemical Society (ACS)

Date: 04-07-1995

DOI: 10.1021/BI00026A005

Abstract: NMR spectroscopy has been used to solve the three-dimensional solution structure of a minimal RNA-binding domain of the Rev protein from the human immunodeficiency virus (type 1), an essential regulatory protein for viral replication. The presence of 10 arginine residues in the 17-residue peptide Rev34-50 caused significant problems in assignment of the NMR spectra. To improve spectral resolution, the peptide was synthesized with an alanine replacing a nonessential arginine and with selectively 15N-labeled residues. Contrary to Chou-Fasman modeling predictions an alpha-helix was detected in both water and 20% trifluoroethanol (TFE) and was found to span residues that constitute the RNA-binding and nuclear-localizing domains of Rev. The sequence-specific information provided by the NMR data gives a full description of the solution conformation of Rev34-50 which serves as a template for investigating binding of the peptide to RNA from the Rev response element (RRE). Preliminary modeling suggests that the helix can fit neatly into the expanded major groove of the RRE where interactions between the peptide side chains and the RNA can be identified. These data may aid the construction of a suitable pharmacophore model for the rational design of molecules that block Rev-RNA binding and inhibit HIV replication.

Bioactive Cyclization Optimizes the Affinity of a Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Peptide Inhibitor

Publisher: American Chemical Society (ACS)

Date: 23-12-2020

DOI: 10.1021/ACS.JMEDCHEM.0C01766

A peptide corresponding to the N‐terminal 13 residues of T4 lysozyme forms an α‐helix

Publisher: Wiley

Date: 11-01-1993

DOI: 10.1016/0014-5793(93)81187-5

Abstract: Solid-phase methods have been used to synthesize LYS(1-13), a peptide corresponding to the first 13 residues of T4 lysozyme. 2D 1H NMR techniques were used to investigate its solution structure in the presence of SDS micelles. The identification of numerous medium-range NOESY crosspeaks and several slowly exchanging NH protons indicated the presence of an alpha-helical structure. This was confirmed by simulated annealing calculations performed using XPLOR.

Spider peptide toxin HwTx-IV engineered to bind to lipid membranes has an increased inhibitory potency at human voltage-gated sodium channel hNa V 1.7

Publisher: Elsevier BV

Date: 05-2017

DOI: 10.1016/J.BBAMEM.2017.01.020

Abstract: The human voltage-gated sodium channel sub-type 1.7 (hNa

Effects of linker sequence modifications on the structure, stability, and biological activity of a cyclic α‐conotoxin

Publisher: Wiley

Date: 11-2016

DOI: 10.1002/BIP.22848

Abstract: The cyclic conotoxin analogue cVc1.1 is a promising lead molecule for the development of new treatments for neuropathic and chronic pain. The design of this peptide includes a linker sequence that joins the N and C termini together, improving peptide stability while maintaining the structure and activity of the original linear Vc1.1. The effect of linker length on the structure, activity and stability of cyclised conotoxins has been studied previously but the effect of altering the composition of the linker sequence has not been investigated. In this study, we designed three analogues of cVc1.1 with linker sequences that varied in charge, hydrophobicity and hydrogen bonding capacity and examined the effect on structure, stability, membrane permeability and biological activity. The three designed peptides were successfully synthesized using solid phase peptide synthesis approaches and had similar structures and stability compared with cVc1.1. Despite modifications in charge, hydrophobicity and hydrogen bonding potential, which are all factors that can affect membrane permeability, no changes in the ability of the peptides to pass through membranes in either PAMPA or Caco-2 cell assay were observed. Surprisingly, modification of the linker sequence was deleterious to biological activity. These results suggest the linker sequence might be a useful part of the molecule for optimization of bioactivity and not just the physiochemical properties of cVc1.1. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 864-875, 2016.

Iterative Optimization of the Cyclic Peptide SFTI-1 Yields Potent Inhibitors of Neutrophil Proteinase 3

Publisher: American Chemical Society (ACS)

Date: 19-07-2019

DOI: 10.1021/ACSMEDCHEMLETT.9B00253

Mutagenesis of cyclotide Cter 27 exemplifies a robust folding strategy for bracelet cyclotides

Publisher: Wiley

Date: 27-05-2022

DOI: 10.1002/PEP2.24284

Abstract: In contrast to Möbius and trypsin inhibitor cyclotides, members of the bracelet subfamily are typically intractable to chemical synthesis and folding. In a significant advance in the field, the bracelet cyclotides ribe 33 and Cter 27 were successfully produced synthetically in moderate yield in a recent study. That synthetic method was a breakthrough as members of the bracelet subfamily of cyclotides had hitherto eluded attempts to be synthetically produced, apart from one report of cyO2 production in which a complicated folding strategy was used. In the current study the successful in vitro folding of three mutants of bracelet cyclotide Cter 27 is reported. This study broadens our understanding of the folding of bracelet cyclotides and elucidates the three dimensional structure of synthetic Cter 27, providing a new class of cyclotide molecular grafting scaffold for drug design applications.

Solution structure of χ‐conopeptide MrIA, a modulator of the human norepinephrine transporter

Publisher: Wiley

Date: 2005

DOI: 10.1002/BIP.20302

Abstract: The chi-conopeptides MrIA and MrIB are 13-residue peptides with two disulfide bonds that inhibit human and rat norepinephrine transporter systems and are of significant interest for the design of novel drugs involved in pain treatment. In the current study we have determined the solution structure of MrIA using NMR spectroscopy. The major element of secondary structure is a beta-hairpin with the two strands connected by an inverse gamma-turn. The residues primarily involved in activity have previously been shown to be located in the turn region (Sharpe, I. A. Palant, E. Schroder, C. I. Kaye, D. M. Adams, D. J. Alewood, P. F. Lewis, R. J. J Biol Chem 2003, 278, 40317-40323), which appears to be more flexible than the beta-strands based on disorder in the ensemble of calculated structures. Analogues of MrIA with N-terminal truncations indicate that the N-terminal residues play a role in defining a stable conformation and the native disulfide connectivity. In particular, noncovalent interactions between Val3 and Hyp12 are likely to be involved in maintaining a stable conformation. The N-terminus also affects activity, as a single N-terminal deletion introduced additional pharmacology at rat vas deferens, while deleting the first two amino acids reduced chi-conopeptide potency.

Engineering Cyclic Peptide Toxins

Publisher: Elsevier

Date: 2012

DOI: 10.1016/B978-0-12-396962-0.00003-3

Cyclotides as templates in drug design

Publisher: Elsevier BV

Date: 2010

DOI: 10.1016/J.DRUDIS.2009.10.007

Abstract: Cyclotides are remarkably stable proteins from plants that have a range of pharmaceutical and agricultural applications based on both their various bioactivities and their potential for use as stable protein-engineering templates. This article discusses literature on pharmaceutically relevant activities of cyclotides, including anti-HIV, antimicrobial and cytotoxic activities, and evaluates their potential therapeutic applications. Their applications as templates for the design of antiangiogenic agents for the treatment of cancer and as anti-infective agents are also described. Toxic effects of cyclotides, whose native function is as insecticidal agents, can be removed by simple mutagenesis, thus rationalizing the apparent conundrum of proposing insecticidal agents as leads for human therapeutics.

Cell Membrane Composition Drives Selectivity and Toxicity of Designed Cyclic Helix–Loop–Helix Peptides with Cell Penetrating and Tumor Suppressor Properties

Publisher: American Chemical Society (ACS)

Date: 07-08-2019

DOI: 10.1021/ACSCHEMBIO.9B00593

Abstract: The tumor suppressor protein p53 is inactive in a large number of cancers, including some forms of sarcoma, breast cancer, and leukemia, due to overexpression of its intrinsic inhibitors MDM2 and MDMX. Reactivation of p53 tumor suppressor activity, via disruption of interactions between MDM2/X and p53 in the cytosol, is a promising strategy to treat cancer. Peptides able to bind MDM2 and/or MDMX were shown to prevent MDM2/X:p53 interactions, but most possess low cell penetrability, low stability, and/or high toxicity to healthy cells. Recently, the designed peptide cHLH-p53-R was reported to possess high affinity for MDM2, resistance toward proteases, cell-penetrating properties, and toxicity toward cancer cells. This peptide uses a stable cyclic helix-loop-helix (cHLH) scaffold, which includes two helices connected with a Gly loop and cyclized to improve stability. In the current study, we were interested in examining the cell selectivity of cHLH-p53-R, its cellular internalization, and ability to reactivate the p53 pathway. We designed analogues of cHLH-p53-R and employed biochemical and biophysical methodologies using

A designed cyclic analogue of gomesin has potent activity against Staphylococcus aureus biofilms

Publisher: Oxford University Press (OUP)

Date: 29-09-2022

DOI: 10.1093/JAC/DKAC309

Abstract: Infections caused by bacterial biofilms are very difficult to treat. The use of currently approved antibiotics even at high dosages often fails, making the treatment of these infections very challenging. Novel antimicrobial agents that use distinct mechanisms of action are urgently needed. To explore the use of [G1K,K8R]cGm, a designed cyclic analogue of the antimicrobial peptide gomesin, as an alternative approach to treat biofilm infections. We studied the activity of [G1K,K8R]cGm against biofilms of Staphylococcus aureus, a pathogen associated with several biofilm-related infections. A combination of atomic force and real-time confocal laser scanning microscopies was used to study the mechanism of action of the peptide. The peptide demonstrated potent activity against 24 h-preformed biofilms through a concentration-dependent ability to kill biofilm-embedded cells. Mechanistic studies showed that [G1K,K8R]cGm causes morphological changes on bacterial cells and permeabilizes their membranes across the biofilm with a half-time of 65 min. We also tested an analogue of [G1K,K8R]cGm without disulphide bonds, and a linear unfolded analogue, and found both to be inactive. The results suggest that the 3D structure of [G1K,K8R]cGm and its stabilization by disulphide bonds are essential for its antibacterial and antibiofilm activities. Moreover, our findings support the potential application of this stable cyclic antimicrobial peptide to fight bacterial biofilms.

Chemical Synthesis of Naturally-Occurring Cyclic Mini-Proteins from Plants and Animals

Publisher: Wiley

Date: 07-09-2011

DOI: 10.1002/IJCH.201100067

Abstract: Cyclization of a peptide backbone is a relatively minor modification in one sense, in that it involves the addition of just one extra peptide bond to a protein sequence, but the consequences of this addition can be profound in terms of stability of the protein. Until recently, most studies of head‐to‐tail cyclic peptides were limited to rather small peptides, typically less than 12 amino acids in size, but over the last decade it has become clear that cyclization is readily applicable to a wide range of peptides and proteins, including those that contain one or more disulfide bonds. This article describes studies involving the solid phase peptide synthesis of two classes of disulfide‐rich peptides that contain a cystine knot and a cystine ladder motif, respectively. The cyclotides comprise around 30 amino acids, with their six conserved cysteine residues arranged in a cystine knot motif, whereas the θ‐defensins comprise 18 amino acids with the cysteine residues forming a cystine ladder.

Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the α-conotoxin MII

Publisher: Proceedings of the National Academy of Sciences

Date: 14-09-2005

DOI: 10.1073/PNAS.0504613102

Abstract: Conotoxins (CTXs), with their exquisite specificity and potency, have recently created much excitement as drug leads. However, like most peptides, their beneficial activities may potentially be undermined by susceptibility to proteolysis in vivo . By cyclizing the α-CTX MII by using a range of linkers, we have engineered peptides that preserve their full activity but have greatly improved resistance to proteolytic degradation. The cyclic MII analogue containing a seven-residue linker joining the N and C termini was as active and selective as the native peptide for native and recombinant neuronal nicotinic acetylcholine receptor subtypes present in bovine chromaffin cells and expressed in Xenopus oocytes, respectively. Furthermore, its resistance to proteolysis against a specific protease and in human plasma was significantly improved. More generally, to our knowledge, this report is the first on the cyclization of disulfide-rich toxins. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine.

Conopeptide characterization and classifications: An analysis using ConoServer

Publisher: Elsevier BV

Date: 07-2010

DOI: 10.1016/J.TOXICON.2010.03.002

Abstract: Cone snails are carnivorous marine gastropods that have evolved potent venoms to capture their prey. These venoms comprise a rich and erse cocktail of peptide toxins, or conopeptides, whose high ersity has arisen from an efficient hypermutation mechanism, combined with a high frequency of post-translational modifications. Conopeptides bind with high specificity to distinct membrane receptors, ion channels, and transporters of the central and muscular nervous system. As well as serving their natural function in prey capture, conopeptides have been utilized as versatile tools in neuroscience and have proven valuable as drug leads that target the nervous system in humans. This paper examines current knowledge on conopeptide sequences based on an analysis of gene and peptide sequences in ConoServer (www.conoserver.org), a specialized database of conopeptide sequences and three-dimensional structures. We describe updates to the content and organization of ConoServer and discuss correlations between gene superfamilies, cysteine frameworks, pharmacological families targeted by conopeptides, and the phylogeny, habitat, and diet of cone snails. The study identifies gaps in current knowledge of conopeptides and points to potential directions for future research.

The Role of Conserved Glu Residue on Cyclotide Stability and Activity: A Structural and Functional Study of Kalata B12, a Naturally Occurring Glu to Asp Mutant

Publisher: American Chemical Society (ACS)

Date: 25-04-2011

DOI: 10.1021/BI2004153

Abstract: Cyclotides are a family of plant defense proteins with a unique cyclic backbone and cystine knot. Their remarkable stability under harsh thermal, enzymatic, and chemical conditions, combined with their range of bioactivities, including anti-HIV activity, underpins their potential as protein drug scaffolds. The vast majority of cyclotides possess a conserved glutamate residue in loop 1 of the sequence that is involved in a structurally important network of hydrogen bonds to an adjacent loop (loop 3). A single native cyclotide sequence, kalata B12, has been discovered that has an aspartic acid in this otherwise conserved position. Previous studies have determined that methylation of the glutamate or substitution with alanine abolishes the membrane disrupting activity that is characteristic of the family. To further understand the role of this conserved structural feature, we studied the folding, structure, stability, and activity of the natural aspartic acid variant kalata B12 and compared it to the prototypical cyclotide kalata B1, along with its glutamate to alanine or aspartate mutants. We show that the overall fold of kalata B12 is similar to the structure of other cyclotides, confirming that the cyclotide framework is robust and tolerant to substitution, although the structure appears to be more flexible than other cyclotides. Modification of the glutamate in kalata B1 or replacing the aspartate in kalata B12 with a glutamate reduces the efficiency of oxidative folding relative to the native peptides. The bioactivity of all modified glutamate cyclotides is abolished, suggesting an important functional role of this conserved residue. Overall, this study shows that the presence of a glutamic acid in loop 1 of the cyclotides improves stability and is essential for the membrane disrupting activity of cyclotides.

Modified defence peptides from horseshoe crab target and kill bacteria inside host cells

Publisher: Cold Spring Harbor Laboratory

Date: 28-06-2021

DOI: 10.1101/2021.06.27.450110

Abstract: Bacteria that occupy an intracellular niche can evade extracellular host immune responses and antimicrobial molecules. In addition to classic intracellular pathogens, other bacteria including uropathogenic Escherichia coli (UPEC) can adopt both extracellular and intracellular lifestyles. UPEC intracellular survival and replication complicates treatment, as many therapeutic molecules do not effectively reach all components of the infection cycle. In this study, we explored cell penetrating antimicrobial peptides from distinct structural classes as alternative molecules for targeting bacteria. We identified two β-hairpin peptides from the horseshoe crab, tachyplesin I and polyphemusin I, with broad antimicrobial activity toward a panel of pathogenic and non-pathogenic bacteria in planktonic form. Peptide analogues [I11A]tachyplesin I and [I11S]tachyplesin I maintained activity toward bacteria, but were less toxic to mammalian cells than native tachyplesin I. This important increase in therapeutic window allowed treatment with higher concentrations of [I11A]tachyplesin I and [I11S]tachyplesin I, to significantly reduce intramacrophage survival of UPEC in an in vitro infection model. Mechanistic studies using bacterial cells, model membranes and cell membrane extracts, suggest that tachyplesin I and polyphemusin I peptides kill UPEC by selectively binding and disrupting bacterial cell membranes. Moreover, treatment of UPEC with sublethal peptide concentrations increased zinc toxicity and enhanced innate macrophage antimicrobial pathways. In summary, our combined data show that cell penetrating peptides are attractive alternatives to traditional small molecule antibiotics for treating UPEC infection, and that optimization of native peptide sequences can deliver effective antimicrobials for targeting bacteria in extracellular and intracellular environments.

The Chemistry and Biology of Theta Defensins

Publisher: Wiley

Date: 30-07-2014

DOI: 10.1002/ANIE.201402167

Determination of the α-Conotoxin Vc1.1 Binding Site on the α9α10 Nicotinic Acetylcholine Receptor

Publisher: American Chemical Society (ACS)

Date: 29-04-2013

DOI: 10.1021/JM400041H

Abstract: α-Conotoxin Vc1.1 specifically and potently inhibits the nicotinic acetylcholine receptor subtype α9α10 (α9α10 nAChR) and is a potential novel treatment for neuropathic pain. Here, we used a combination of computational modeling and electrophysiology experiments to determine the Vc1.1 binding site on the α9α10 nAChR. Interactions of Vc1.1 with two probable binding sites, α9α10 and α10α9, were modeled. Mutational energies calculated by assuming specific interactions in the α10α9 binding site correlated better with electrophysiological recordings than those assuming interactions with the α9α10 binding site. Two novel Vc1.1 analogues, [N9F]Vc1.1 and [N9W]Vc1.1, were predicted to have large differences in affinity between the two binding sites. Data from functional studies were consistent with computational predictions that assumed preferred binding of Vc1.1 to the α10α9 pocket. Moreover, our modeling study suggested that a single hydrogen bond formed between Vc1.1 and position 59 of the α10α9 pocket confers specificity to rat versus human α9α10 nAChRs.

Designed β-Hairpins Inhibit LDH5 Oligomerization and Enzymatic Activity

Publisher: American Chemical Society (ACS)

Date: 25-03-2021

DOI: 10.1021/ACS.JMEDCHEM.0C01898

Cyclotide Isolation and Characterization

Publisher: Elsevier

Date: 2012

DOI: 10.1016/B978-0-12-394291-3.00024-1

Chemical modification of conotoxins to improve stability and activity

Publisher: American Chemical Society (ACS)

Date: 07-2007

DOI: 10.1021/CB700091J

Abstract: Conotoxins are small disulfide-rich peptides from the venom of cone snails. Along with other conopeptides, they target a wide range of membrane receptors, ion channels, and transporters, and because of their high potency and selectivity for defined subtypes of these receptors, they have attracted a great deal of attention recently as leads in drug development. However, like most peptides, conopeptides potentially suffer from the disadvantages of poor absorption, poor stability, or short biological half-lives. Recently, various chemical approaches, including residue substitutions, backbone cyclization, and disulfide-bridge modification, have been reported to increase the stability of conopeptides. These manufactured interventions add to the array of post-translational modifications that occur naturally in conopeptides. They enhance the versatility of these peptides as tools in neuroscience and as drug leads.

Structural Characterization of the Cyclic Cystine Ladder Motif of θ-Defensins

Publisher: American Chemical Society (ACS)

Date: 20-11-2012

DOI: 10.1021/BI301363A

Abstract: The θ-defensins are, to date, the only known ribosomally synthesized cyclic peptides in mammals, and they have promising antimicrobial bioactivities. The characteristic structural motif of the θ-defensins is the cyclic cystine ladder, comprising a cyclic peptide backbone and three parallel disulfide bonds. In contrast to the cyclic cystine knot, which characterizes the plant cyclotides, the cyclic cystine ladder has not been as well described as a structural motif. Here we report the solution structures and nuclear magnetic resonance relaxation properties in aqueous solution of three representative θ-defensins from different species. Our data suggest that the θ-defensins are more rigid and structurally defined than previously thought. In addition, all three θ-defensins were found to self-associate in aqueous solution in a concentration-dependent and reversible manner, a property that might have a role in their mechanism of action. The structural definition of the θ-defensins and the cyclic cystine ladder will help to guide exploitation of these molecules as structural frameworks for the design of peptide drugs.

The Anthelmintic Activity of the Cyclotides: Natural Variants with Enhanced Activity

Publisher: Wiley

Date: 08-2008

DOI: 10.1002/CBIC.200800174

Abstract: The cyclotides are a family of backbone-cyclised cystine-knot-containing peptides from plants that possess anthelmintic activity against Haemonchus contortus and Trichostrongylus colubriformis, two important gastrointestinal nematode parasites of sheep. In the current study, we investigated the in vitro effects of newly discovered natural cyclotides on the viability of larval and adult life stages of these pests. The natural variants cycloviolacin O2, cycloviolacin O3, cycloviolacin O8, cycloviolacin O13, cycloviolacin O14, cycloviolacin O15, and cycloviolacin O16 extracted from Viola odorata showed up to 18-fold greater potency than the prototypic cyclotide kalata B1 in nematode larval development assays. Cycloviolacin O2 and cycloviolacin O14 were significantly more potent than kalata B1 in adult H. contortus motility assays. The lysine and glutamic acid residues of cycloviolacin O2, the most potent anthelmintic cyclotide, were chemically modified to investigate the role of these charged residues in modulating the biological activity. The single glutamic acid residue, which is conserved across all known cyclotides, was shown to be essential for activity, with a sixfold decrease in potency of cycloviolacin O2 following methylation. The three lysine residues present in cycloviolacin O2 were acetylated to effectively mask the positive charge, resulting in a 18-fold decrease in anthelmintic activity. The relative anthelmintic activities of the natural variants assayed against nematode larvae correlated with the number of charged residues present in their sequence.

Conformational Flexibility Is a Determinant of Permeability for Cyclosporin

Publisher: American Chemical Society (ACS)

Date: 19-02-2018

DOI: 10.1021/ACS.JPCB.7B12419

Abstract: Several cyclic peptides have been reported to have unexpectedly high membrane permeability. Of these, cyclosporin A is perhaps the most well-known ex le, particularly in light of its relatively high molecular weight. Observations that cyclosporin A changes conformation depending on its solvent environment led to the hypothesis that conformational dynamics is a prerequisite for its permeability however, this hypothesis has been difficult to validate experimentally. Here, we use molecular dynamics simulations to explicitly determine the conformational behavior of cyclosporin A and other related cyclic peptides as they spontaneously transition between different environments, including through a lipid bilayer. These simulations are referenced against simulations in explicit water, chloroform, and cyclohexane and further validated against NMR experiments, measuring conformational exchange, nuclear spin relaxation, and three-dimensional structures in membrane-mimicking environments, such as in dodecylphosphocholine micelles, to build a comprehensive understanding of the role of dynamics. We find that conformational flexibility is a key determinant of the membrane permeability of cyclosporin A and similar membrane-permeable cyclic peptides, as conformationally constrained variants have limited movement into, then through, and finally out of the membrane in silico. We envisage that a better understanding of dynamics might thus provide new opportunities to modulate peptide function and enhance their delivery.

NMR of plant proteins

Publisher: Elsevier BV

Date: 05-2013

DOI: 10.1016/J.PNMRS.2013.01.003

Discovery of an unusual biosynthetic origin for circular proteins in legumes

Publisher: Proceedings of the National Academy of Sciences

Date: 18-05-2011

DOI: 10.1073/PNAS.1103660108

Abstract: Cyclotides are plant-derived proteins that have a unique cyclic cystine knot topology and are remarkably stable. Their natural function is host defense, but they have a erse range of pharmaceutically important activities, including uterotonic activity and anti-HIV activity, and have also attracted recent interest as templates in drug design. Here we report an unusual biosynthetic origin of a precursor protein of a cyclotide from the butterfly pea, Clitoria ternatea , a representative member of the Fabaceae plant family. Unlike all previously reported cyclotides, the domain corresponding to the mature cyclotide from this Fabaceae plant is embedded within an albumin precursor protein. We confirmed the expression and correct processing of the cyclotide encoded by the Cter M precursor gene transcript following extraction from C. ternatea leaf and sequencing by tandem mass spectrometry. The sequence was verified by direct chemical synthesis and the peptide was found to adopt a classic knotted cyclotide fold as determined by NMR spectroscopy. Seven additional cyclotide sequences were also identified from C. ternatea leaf and flower, five of which were unique. Cter M displayed insecticidal activity against the cotton budworm Helicoverpa armigera and bound to phospholipid membranes, suggesting its activity is modulated by membrane disruption. The Fabaceae is the third largest family of flowering plants and many Fabaceous plants are of huge significance for human nutrition. Knowledge of Fabaceae cyclotide gene transcripts should enable the production of modified cyclotides in crop plants for a variety of agricultural or pharmaceutical applications, including plant-produced designer peptide drugs.

Cloning, synthesis, and characterization of αO-conotoxin GeXIVA, a potent α9α10 nicotinic acetylcholine receptor antagonist

Publisher: Proceedings of the National Academy of Sciences

Date: 13-07-2015

DOI: 10.1073/PNAS.1503617112

Abstract: The α9α10 nicotinic AChR (nAChR) subtype is a recently identified target for the development of breast cancer chemotherapeutics and analgesics, particularly to treat neuropathic pain. Structure/function analyses of antagonists of this subtype are therefore essential for the development of specific therapeutic compounds. The Conus genus is a rich source of pharmacologically active peptides, and we report here that the αO-conotoxin GeXIVA is a potent and selective antagonist of the α9α10 nAChR subtype. GeXIVA displays unique structural properties among other Conus peptides and represents a previously unidentified template for molecules active against neuropathic pain.

Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa. 2. Three-dimensional solution structure

Publisher: Wiley

Date: 08-2000

DOI: 10.1046/J.1432-1327.2000.01507.X

Abstract: The three-dimensional solution structure of conotoxin TVIIA, a 30-residue polypeptide from the venom of the piscivorous cone snail Conus tulipa, has been determined using 2D 1H NMR spectroscopy. TVIIA contains six cysteine residues which form a 'four-loop' structural framework common to many peptides from Conus venoms including the omega-, delta-, kappa-, and muO-conotoxins. However, TVIIA does not belong to these well-characterized pharmacological classes of conotoxins, but displays high sequence identity with conotoxin GS, a muscle sodium channel blocker from Conus geographus. Structure calculations were based on 562 interproton distance restraints inferred from NOE data, together with 18 backbone and nine side-chain torsion angle restraints derived from spin-spin coupling constants. The final family of 20 structures had mean pairwise rms differences over residues 2-27 of 0.18+/-0.05 A for the backbone atoms and 1.39+/-0.33 A for all heavy atoms. The structure consists of a triple-stranded, antiparallel beta sheet with +2x, -1 topology (residues 7-9, 16-20 and 23-27) and several beta turns. The core of the molecule is formed by three disulfide bonds which form a cystine knot motif common to many toxic and inhibitory polypeptides. The global fold, molecular shape and distribution of amino-acid sidechains in TVIIA is similar to that previously reported for conotoxin GS, and comparison with other four-loop conotoxin structures provides further indication that TVIIA and GS represent a new and distinct subgroup of this structural family. The structure of TVIIA determined in this study provides the basis for determining a structure-activity relationship for these molecules and their interaction with target receptors.

Insights into the Molecular Flexibility of θ-Defensins by NMR Relaxation Analysis

Publisher: American Chemical Society (ACS)

Date: 25-11-2014

DOI: 10.1021/JP507754C

Cyclotides as a basis for drug design

Publisher: Informa Healthcare

Date: 21-02-2012

DOI: 10.1517/17460441.2012.661554

Abstract: Cyclotides are plant-made defence proteins with a head-to-tail cyclic backbone combined with a conserved, six cystine knot. They have a range of biological activities, including uterotonic and anti-HIV activity, which have attracted attention to their potential pharmaceutical applications. Furthermore, their unique structures and high stability make them appealing as peptide-based templates for drug design applications. Methods have been developed for their production, including solid phase peptide synthesis as well as recombinant methods. This article reviews the recent literature associated with therapeutic applications of naturally occurring and synthetically modified cyclotides. It includes applications of cyclotides and cyclotide-like molecules as peptide-based drug leads and diagnostic agents. The ultra-stable cyclotides are promising templates for drug development applications and are currently being assessed for the potential breadth of their applications. For synthetic versions of cyclotides to enter human clinical trials further studies to examine their biopharmaceutical properties and toxicities are required. However, several promising proof-of-concept studies have established that pharmaceutically relevant bioactive peptide sequences can be grafted into cyclotide frameworks and thereby stabilised, while maintaining biological activity. These studies include ex les directed at cancer, cardiovascular disease and infectious diseases. Solid phase peptide synthesis has been the preferred approach for making pharmaceutically modified cyclotides so far, but promising progress is being made in biological approaches to cyclotide production.

Anticancer and Toxic Properties of Cyclotides are Dependent on Phosphatidylethanolamine Phospholipid Targeting

Publisher: Wiley

Date: 05-08-2014

DOI: 10.1002/CBIC.201402144

Abstract: Cyclotides, ultrastable disulfide-rich cyclic peptides, can be engineered to bind and inhibit specific cancer targets. In addition, some cyclotides are toxic to cancer cells, though not much is known about their mechanisms of action. Here we delineated the potential mode of action of cyclotides towards cancer cells. A novel set of analogues of kalata B1 (the prototypic cyclotide) and kalata B2 and cycloviolacin O2 were examined for their membrane-binding affinity and selectivity towards cancer cells. By using solution-state NMR, surface plasmon resonance, flow cytometry and bioassays we show that cyclotides are toxic against cancer and non-cancerous cells and their toxicity correlates with their ability to target and disrupt lipid bilayers that contain phosphatidylethanolamine phospholipids. Our results suggest that the potential of cyclotides as anticancer therapeutics might best be realised by combining their amenability to epitope engineering with their ability to bind cancer cell membranes.

Identification, Characterization, and Three-Dimensional Structure of the Novel Circular Bacteriocin, Enterocin NKR-5-3B, from Enterococcus faecium

Publisher: American Chemical Society (ACS)

Date: 31-07-2015

DOI: 10.1021/ACS.BIOCHEM.5B00196

Abstract: Enterocin NKR-5-3B, one of the multiple bacteriocins produced by Enterococcus faecium NKR-5-3, is a 64-amino acid novel circular bacteriocin that displays broad-spectrum antimicrobial activity. Here we report the identification, characterization, and three-dimensional nuclear magnetic resonance solution structure determination of enterocin NKR-5-3B. Enterocin NKR-5-3B is characterized by four helical segments that enclose a compact hydrophobic core, which together with its circular backbone impart high stability and structural integrity. We also report the corresponding structural gene, enkB, that encodes an 87-amino acid precursor peptide that undergoes a yet to be described enzymatic processing that involves adjacent cleavage and ligation of Leu(24) and Trp(87) to yield the mature (circular) enterocin NKR-5-3B.

The Yariv reagent: Behaviour in different solvents and interaction with a gum arabic arabinogalactanprotein

Publisher: Elsevier BV

Date: 06-2014

DOI: 10.1016/J.CARBPOL.2014.01.009

Abstract: The β-D-Glc Yariv reagent is frequently used to isolate and to study the structure of arabinogalactan-proteins with the arabinogalactan type II structure. The present paper describes the aggregation features of the Yariv reagent in water, salt solutions and in organic solvents as determined by NMR, absorption spectroscopy and light scattering experiments. The results indicate that in water the Yariv reagent forms aggregates of up to 300 units and in 1% aqueous NaCl the degree of aggregation is approx. 150. The aggregates are formed both by H-bonds and hydrophobic interactions, the former appearing to be of most importance in water. The interaction between the Yariv reagent and an AGP fraction from gum arabic, showed a degree of aggregation of the Yariv reagent when using 1% NaCl to be of approx. 150 units, whereas disruption of the aggregate took place in 10% NaCl with an aggregation number of approx. 100. Partial acid hydrolysis of an AGP from gum Arabic (Acacia Senegal) and analyses of the linkage types remaining indicated that a certain length of (1→3)-β-linked galactose units was necessary for binding between the Yariv reagent and the AGP. This is in accordance to what also was recently observed by Kitazawa et al. (2013).

The Three-dimensional Solution Structure by 1H NMR of a 6-Kda Proteinase Inhibitor Isolated from the Stigma of Nicotiana alata

Publisher: Elsevier BV

Date: 09-1994

DOI: 10.1006/JMBI.1994.1575

Abstract: The three-dimensional structure and disulfide connectivities of a 6-kDa protein isolated from the stigma of the ornamental tobacco Nicotiana alata has been determined by 1H NMR spectroscopy combined with simulated annealing calculations. The protein, termed C1, is a chymotrypsin inhibitor and is one of five homologous proteinase inhibitors that are proteolytically cleaved from a 40.3-kDa precursor protein. The other four proteinase inhibitors (T1 to T4) contain reactive sites for trypsin. The three-dimensional structure of C1 is generally well defined and contains a triple stranded beta-sheet as the dominant secondary structural feature. Several turns and a short region of 3(10) helix are also present. The putative chymotrypsin reactive site is present on an exposed loop which is less defined than the rest of the protein. The overall shape of C1 is disc-like and the N and C termini are exposed, supporting the proposal that this protein results from post-translational processing of the 40.3-kDa precursor protein.

Translational diffusion of cyclic peptides measured using pulsed-field gradient NMR

Publisher: American Chemical Society (ACS)

Date: 15-09-2014

DOI: 10.1021/JP506678F

Abstract: Cyclic peptides are increasingly being recognized as valuable templates for drug discovery or design. To facilitate efforts in the structural characterization of cyclic peptides, we explore the use of pulse-field gradient experiments as a convenient and noninvasive approach for characterizing their diffusion properties in solution. We present diffusion coefficient measurements of five cyclic peptides, including dichC, SFTI-1, cVc1.1, kB1, and kB2. These peptides range in size from six to 29 amino acids and have various therapeutically interesting activities. We explore the use of internal standards, such as dioxane and acetonitrile, to evaluate the hydrodynamic radius from the diffusion coefficient, and show that 2,2-dimethyl-2-silapentane-5-sulfonic acid, a commonly used chemical shift reference, can be used as an internal standard to avoid spectral overlap issues and simplify data analysis. The experimentally measured hydrodynamic radii correlate with increasing molecular weight and in silico predictions. We further applied diffusion measurements to characterize the self-association of kB2 and showed that it forms oligomers in a concentration-dependent manner, which may be relevant to its mechanism of action. Diffusion coefficient measurements appear to have broad utility in cyclic peptide structural biology, allowing for the rapid characterization of their molecular shape in solution.

Isolation, Solution Structure, and Insecticidal Activity of Kalata B2, a Circular Protein with a Twist:  Do Möbius Strips Exist in Nature?^,

Publisher: American Chemical Society (ACS)

Date: 24-12-2004

DOI: 10.1021/BI047837H

Abstract: A large number of macrocyclic miniproteins with erse biological activities have been isolated from the Rubiaceae, Violaceae, and Cucurbitaceae plant families in recent years. Here we report the three-dimensional structure determined using (1)H NMR spectroscopy and demonstrate potent insecticidal activity for one of these peptides, kalata B2. This peptide is one of the major components of an extract from the leaves of the plant Oldenlandia affinis. The structure consists of a distorted triple-stranded beta-sheet and a cystine knot arrangement of the disulfide bonds and is similar to those described for other members of the cyclotide family. The unique cyclic and knotted nature of these molecules makes them a fascinating ex le of topologically complex proteins. Examination of the sequences reveals that they can be separated into two subfamilies, one of which contains a larger number of positively charged residues and has a bracelet-like circularization of the backbone. The second subfamily contains a backbone twist due to a cis-peptidyl-proline bond and may conceptually be regarded as a molecular Mobius strip. Kalata B2 is the second putative member of the Mobius cyclotide family to be structurally characterized and has a cis-peptidyl-proline bond, thus validating the suggested name for this subfamily of cyclotides. The observation that kalata B2 inhibits the growth and development of Helicoverpa armigera larvae suggests a role for the cyclotides in plant defense. A comparison of the sequences and structures of kalata B1 and B2 provides insight into the biological activity of these peptides.

Prediction and characterization of cyclic proteins from sequences in three domains of life

Publisher: Elsevier BV

Date: 2014

DOI: 10.1016/J.BBAPAP.2013.05.002

Abstract: Cyclic proteins (CPs) have circular chains with a continuous cycle of peptide bonds. Their unique structural traits result in greater stability and resistance to degradation when compared to their acyclic counterparts. They are also promising targets for pharmaceutical/therapeutic applications. To date, only a few hundred CPs are known, although recent studies suggest that their numbers might be substantially higher. Here we developed a first-of-its-kind, accurate and high-throughput method called CyPred that predicts whether a given protein chain is cyclic. CyPred considers currently well-represented CP families: cyclotides, cyclic defensins, bacteriocins, and trypsin inhibitors. Empirical tests demonstrate that CyPred outperforms commonly used alignment methods. We used CyPred to estimate the incidence of CPs and found ~3500 putative CPs among 5.7+ million chains from 642 fully sequenced proteomes from archaea, bacteria, and eukaryotes. The median number of putative CPs per species ranges from three for archaea proteomes to two for eukaryotes/bacteria, with 7% of archaea, 11% of bacterial, and 16% of eukaryotic proteomes having 10+ CPs. The differences in the estimated fractions of CPs per proteome are as large as three orders of magnitude. Among eukaryotes, animals have higher ratios of CPs compared to fungi, while plants have the largest spread of the ratios. We also show that proteomes enriched in cyclic proteins evolve more slowly than proteomes with fewer cyclic chains. Our results suggest that further research is needed to fully uncover the scope and potential of cyclic proteins. A list of putative CPs and the CyPred method are available at biomine.ece.ualberta.ca/CyPred/. This article is part of a Special Issue entitled: Computational Proteomics, Systems Biology & Clinical Implications. Guest Editor: Yudong Cai.

Three-dimensional solution structure of α-conotoxin MII by NMR spectroscopy effects of solution environment on helicity

Publisher: American Chemical Society (ACS)

Date: 23-10-1998

DOI: 10.1021/BI981535W

Membrane-active peptides escape drug-resistance in cancer

Publisher: Cold Spring Harbor Laboratory

Date: 28-10-2022

DOI: 10.1101/2022.10.27.513961

Abstract: Acquired drug-resistance is a recurring problem in cancer treatment, and this is particularly true for patients with metastatic melanoma that carry a BRAF V600E mutation. In the current study, we explored the use of membrane-active peptides as an alternative therapeutic modality to target drug-resistant melanoma cells. We produced slow-cycling and drug-resistant melanoma cells using dabrafenib, a small molecule drug that targets tumor cells with BRAF V600E mutation, and characterised their lipidome and proteome to investigate the role of membrane lipids in acquired drug-resistance. Despite some changes in the lipid composition, tested anti-melanoma membrane-active cyclic peptides (cTI and cGm) killed melanoma cells that are sensitive, tolerant, or resistant to dabrafenib. Importantly, melanoma cells did not develop resistance to cTI or cGm, nor changed their lipid composition with long-term peptide treatment. Therefore, these peptides are well suited as templates to design therapeutic leads to target drug-resistant metastatic melanoma cells and/or as co-treatment with small molecule drugs.

Importance of the Cyclic Cystine Knot Structural Motif for Immunosuppressive Effects of Cyclotides

Publisher: American Chemical Society (ACS)

Date: 30-09-2021

DOI: 10.1021/ACSCHEMBIO.1C00524

Late-Stage Functionalization with Cysteine Staples Generates Potent and Selective Melanocortin Receptor-1 Agonists

Publisher: American Chemical Society (ACS)

Date: 27-09-2022

DOI: 10.1021/ACS.JMEDCHEM.2C00793

Abstract: In this work, cysteine staples were used as a late-stage functionalization strategy to ersify peptides and build conjugates targeting the melanocortin G-protein-coupled receptors [melanocortin receptor-1 (MC1R) and MC3R-MC5R]. Monocyclic and bicyclic agonists based on sunflower trypsin inhibitor-1 were used to generate a selection of stapled peptides that were evaluated for binding (p

Potent, Selective, and Cell-Penetrating Inhibitors of Kallikrein-Related Peptidase 4 Based on the Cyclic Peptide MCoTI-II

Publisher: American Chemical Society (ACS)

Date: 21-11-2018

DOI: 10.1021/ACSMEDCHEMLETT.8B00422

Unlocking the Potential of the Antimicrobial Peptide Gomesin: From Discovery and Structure–Activity Relationships to Therapeutic Applications

Publisher: MDPI AG

Date: 20-03-2023

DOI: 10.3390/IJMS24065893

Abstract: Gomesin is a cationic antimicrobial peptide which is isolated from the haemocytes of the Brazilian tarantula Acanthoscurria gomesiana and can be produced chemically by Fmoc solid-phase peptide synthesis. Gomesin exhibits a range of biological activities, as demonstrated by its toxicity against therapeutically relevant pathogens such as Gram-positive or Gram-negative bacteria, fungi, cancer cells, and parasites. In recent years, a cyclic version of gomesin has been used for drug design and development as it is more stable than native gomesin in human serum and can penetrate and enter cancer cells. It can therefore interact with intracellular targets and has the potential to be developed as a drug lead for to treat cancer, infectious diseases, and other human diseases. This review provides a perspective on the discovery, structure–activity relationships, mechanism of action, biological activity, and potential clinical applications of gomesin.

Discovery and optimization of peptide macrocycles

Publisher: Informa UK Limited

Date: 16-10-2016

DOI: 10.1080/17460441.2016.1245720

Abstract: Macrocyclic peptides are generally more resistant to proteolysis and often have higher potency than linear peptides and so they are excellent leads in drug design. Their study is significant because they offer potential as a new generation of drugs that are potent and specific, and thus might have fewer side effects than traditional small molecule drugs. Areas covered: This article covers macrocyclic drug leads based on nature-derived cyclic peptides as well as synthetic cyclic peptides and close derivatives. The natural peptides include cyclotides, sunflower-derived peptides, theta-defensins and orbitides. Technologies to make engineered cyclic peptides covered here include cyclization via amino acid linkers, CLIPS, templates, and stapled peptides. Expert opinion: Macrocyclic peptides are promising drug leads and several are in clinical trials. The authors believe they offer key advantages over traditional small molecule drugs, as well as some advantages over protein-based 'biologics' such as antibodies or growth factors. These include the ability to penetrate cells and attack intracellular targets such as protein-protein interactions as well as to hit extracellular targets. Some macrocyclic peptides such as cyclotides offer the potential for production in plants, thus reducing manufacture costs and potentially increasing opportunities for their distribution to developing countries at low cost.

Characterization of a Novel α-Conotoxin TxID from Conus textile That Potently Blocks Rat α3β4 Nicotinic Acetylcholine Receptors

Publisher: American Chemical Society (ACS)

Date: 22-11-2013

DOI: 10.1021/JM401254C

A Computationally Designed Peptide Derived from Escherichia coli as a Potential Drug Template for Antibacterial and Antibiofilm Therapies

Publisher: American Chemical Society (ACS)

Date: 22-10-2018

DOI: 10.1021/ACSINFECDIS.8B00219

Abstract: Computer-aided screening of antimicrobial peptides (AMPs) is a promising approach for discovering novel therapies against multidrug-resistant bacterial infections. Here, we functionally and structurally characterized an Escherichia coli-derived AMP (EcDBS1R5) previously designed through pattern identification [α-helical set (KK[ILV]

Structural properties of relaxin chimeras: NMR characterization of the R3/I5 relaxin peptide

Publisher: Wiley

Date: 04-2009

DOI: 10.1111/J.1749-6632.2008.03805.X

Abstract: Relaxin-3 interacts with high potency with three relaxin family peptide receptors (RXFP1, RXFP3, and RXFP4). Therefore, the development of selective agonist and antagonist analogs is important for in vivo studies characterizing the biological significance of the different receptor-ligand systems and for future pharmaceutical applications. Recent reports demonstrated that a peptide selective for RXFP3 and RXFP4 over RXFP1 can be generated by the combination of the relaxin-3 B chain with the A chain from insulin-like peptide 5 (INSL5), creating an R3/I5 chimera. We have used NMR spectroscopy to determine the three-dimensional structure of this peptide to gain structural insights into the consequences of combining chains from two different relaxins. The R3/I5 structure reveals a similar backbone conformation for the relaxin-3 B chain compared to native relaxin-3, and the INSL5 A chain displays a relaxin/insulin-like fold with two parallel helices. The findings indicate that binding and activation of RXFP3 and RXFP4 mainly require the B chain and that the A chain functions as structural support. RXFP1, however, demonstrates a more complex binding mechanism, involving both the A chain and the B chain. The creation of chimeras is a promising strategy for generating new structure-activity data on relaxins.

Plant-based production of an orally active cyclotide for the treatment of multiple sclerosis

Publisher: Springer Science and Business Media LLC

Date: 17-03-2023

DOI: 10.1007/S11248-023-00341-1

Abstract: Multiple sclerosis (MS) is a debilitating disease that requires prolonged treatment with often severe side effects. One experimental MS therapeutic currently under development is a single amino acid mutant of a plant peptide termed kalata B1, of the cyclotide family. Like all cyclotides, the therapeutic candidate [T20K]kB1 is highly stable as it contains a cyclic backbone that is cross-linked by three disulfide bonds in a knot-like structure. This stability is much sought after for peptide drugs, which despite exquisite selectivity for their targets, are prone to rapid degradation in human serum. In preliminary investigations, it was found that [T20K]kB1 retains oral activity in experimental autoimmune encephalomyelitis, a model of MS in mice, thus opening up opportunities for oral dosing of the peptide. Although [T20K]kB1 can be synthetically produced, a recombinant production system provides advantages, specifically for reduced scale-up costs and reductions in chemical waste. In this study, we demonstrate the capacity of the Australian native Nicotiana benthamiana plant to produce a structurally identical [T20K]kB1 to that of the synthetic peptide. By optimizing the co-expressed cyclizing enzyme, precursor peptide arrangements, and transgene regulatory regions, we demonstrate a [T20K]kB1 yield in crude peptide extracts of ~ 0.3 mg/g dry mass) in whole plants and close to 1.0 mg/g dry mass in isolated infiltrated leaves. With large-scale plant production facilities coming on-line across the world, the sustainable and cost-effective production of cyclotide-based therapeutics is now within reach.

Structure of α-conotoxin BuIA: influences of disulfide connectivity on structural dynamics

Publisher: Springer Science and Business Media LLC

Date: 20-04-2007

DOI: 10.1186/1472-6807-7-28

Abstract: α-Conotoxins have exciting therapeutic potential based on their high selectivity and affinity for nicotinic acetylcholine receptors. The spacing between the cysteine residues in α-conotoxins is variable, leading to the classification of sub-families. BuIA is the only α-conotoxin containing a 4/4 cysteine spacing and thus it is of significant interest to examine the structure of this conotoxin. In the current study we show the native globular disulfide connectivity of BuIA displays multiple conformations in solution whereas the non-native ribbon isomer has a single well-defined conformation. Despite having multiple conformations in solution the globular form of BuIA displays activity at the nicotinic acetylcholine receptor, contrasting with the lack of activity of the structurally well-defined ribbon isomer. These findings are opposite to the general trends observed for α-conotoxins where the native isomers have well-defined structures and the ribbon isomers are generally disordered. This study thus highlights the influence of the disulfide connectivity of BuIA on the dynamics of the three-dimensional structure.

Proteome analysis of the hyaluronic acid-producing bacterium, Streptococcus zooepidemicus

Publisher: Springer Science and Business Media LLC

Date: 28-03-2009

DOI: 10.1186/1477-5956-7-13

Abstract: Streptococcus equi subsp. zooepidemicus ( S. zooepidemicus ) is a commensal of horses and an opportunistic pathogen in many animals and humans. Some strains produce copious amounts of hyaluronic acid, making S. zooepidemicus an important industrial microorganism for the production of this valuable biopolymer used in the pharmaceutical and cosmetic industry. Encapsulation by hyaluronic acid is considered an important virulence factor in other streptococci, though the importance in S. zooepidemicus remains poorly understood. Proteomics may provide a better understanding of virulence factors in S. zooepidemicus , facilitate the design of better diagnostics and treatments, and guide engineering of superior production strains. Using hyaluronidase to remove the capsule and by optimising cellular lysis, a reference map for S. zooepidemicus was completed. This protocol significantly increased protein recovery, allowing for visualisation of 682 spots and the identification of 86 proteins using mass spectrometry (LC-ESI-MS/MS and MALDI-TOF/TOF) of which 16 were membrane proteins. The data presented constitute the first reference map for S. zooepidemicus and provide new information on the identity and characteristics of the more abundantly expressed proteins.

Combined x-ray and NMR analysis of the stability of the cyclotide cystine knot fold that underpins its insecticidal activity and potential use as a drug scaffold

Publisher: Elsevier BV

Date: 04-2009

DOI: 10.1074/JBC.M900021200

Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa: 1. Isolation, characterization and chemical synthesis

Publisher: Wiley

Date: 08-2000

DOI: 10.1046/J.1432-1327.2000.01508.X

Abstract: A novel conotoxin belonging to the 'four-loop' structural class has been isolated from the venom of the piscivorous cone snail Conus tulipa. It was identified using a chemical-directed strategy based largely on mass spectrometric techniques. The new toxin, conotoxin TVIIA, consists of 30 amino-acid residues and contains three disulfide bonds. The amino-acid sequence was determined by Edman analysis as SCSGRDSRCOOVCCMGLMCSRGKCVSIYGE where O = 4-transL-hydroxyproline. Two under-hydroxylated analogues, [Pro10]TVIIA and [Pro10,11]TVIIA, were also identified in the venom of C. tulipa. The sequences of TVIIA and [Pro10]TVIIA were further verified by chemical synthesis and coelution studies with native material. Conotoxin TVIIA has a six cysteine/four-loop structural framework common to many peptides from Conus venoms including the omega-, delta- and kappa-conotoxins. However, TVIIA displays little sequence homology with these well-characterized pharmacological classes of peptides, but displays striking sequence homology with conotoxin GS, a peptide from Conus geographus that blocks skeletal muscle sodium channels. These new toxins and GS share several biochemical features and represent a distinct subgroup of the four-loop conotoxins.

Disulfide Bond Mutagenesis and the Structure and Function of the Head-to-Tail Macrocyclic Trypsin Inhibitor SFTI-1^,

Publisher: American Chemical Society (ACS)

Date: 04-01-2005

DOI: 10.1021/BI048297R

Abstract: SFTI-1 is a novel 14 amino acid peptide comprised of a circular backbone constrained by three proline residues, a hydrogen-bond network, and a single disulfide bond. It is the smallest and most potent known Bowman-Birk trypsin inhibitor and the only one with a cyclic peptidic backbone. The solution structure of [ABA(3,11)]SFTI-1, a disulfide-deficient analogue of SFTI-1, has been determined by (1)H NMR spectroscopy. The lowest energy structures of native SFTI-1 and [ABA(3,11)]SFTI-1 are similar and superimpose with a root-mean-square deviation over the backbone and heavy atoms of 0.26 +/- 0.09 and 1.10 +/- 0.22 A, respectively. The disulfide bridge in SFTI-1 was found to be a minor determinant for the overall structure, but its removal resulted in a slightly weakened hydrogen-bonding network. To further investigate the role of the disulfide bridge, NMR chemical shifts for the backbone H(alpha) protons of two disulfide-deficient linear analogues of SFTI-1, [ABA(3,11)]SFTI-1[6,5] and [ABA(3,11)]SFTI-1[1,14] were measured. These correspond to analogues of the cleavage product of SFTI-1 and a putative biosynthetic precursor, respectively. In contrast with the cyclic peptide, it was found that the disulfide bridge is essential for maintaining the structure of these open-chain analogues. Overall, the hydrogen-bond network appears to be a crucial determinant of the structure of SFTI-1 analogues.

Biosynthesis of circular proteins in plants

Publisher: Wiley

Date: 30-10-2007

DOI: 10.1111/J.1365-313X.2007.03357.X

The Plant Decapeptide OSIP108 Can Alleviate Mitochondrial Dysfunction Induced by Cisplatin in Human Cells

Publisher: MDPI AG

Date: 19-09-2014

DOI: 10.3390/MOLECULES190915088

Peptide-Membrane Interactions Affect the Inhibitory Potency and Selectivity of Spider Toxins ProTx-II and GpTx-1.

Publisher: American Chemical Society (ACS)

Date: 03-12-2019

DOI: 10.1021/ACSCHEMBIO.8B00989

Abstract: Gating modifier toxins (GMTs) from spider venom can inhibit voltage gated sodium channels (Na

A novel suite of cyclotides from Viola odorata: sequence variation and the implications for structure, function and stability

Publisher: Portland Press Ltd.

Date: 27-10-2006

DOI: 10.1042/BJ20060627

Abstract: Cyclotides are a fascinating family of plant-derived peptides characterized by their head-to-tail cyclized backbone and knotted arrangement of three disulfide bonds. This conserved structural architecture, termed the CCK (cyclic cystine knot), is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. Cyclotides have a variety of biological activities, but their insecticidal activities suggest that their primary function is in plant defence. In the present study, we determined the cyclotide content of the sweet violet Viola odorata, a member of the Violaceae family. We identified 30 cyclotides from the aerial parts and roots of this plant, 13 of which are novel sequences. The new sequences provide information about the natural ersity of cyclotides and the role of particular residues in defining structure and function. As many of the biological activities of cyclotides appear to be associated with membrane interactions, we used haemolytic activity as a marker of bioactivity for a selection of the new cyclotides. The new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. The results show that while biological activity varies with the sequence, the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. The structure of one of the new cyclotides, cycloviolacin O14, was determined and shown to contain the CCK motif. This study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens.

Less is More: Design of a Highly Stable Disulfide-Deleted Mutant of Analgesic Cyclic α-Conotoxin Vc1.1

Publisher: Springer Science and Business Media LLC

Date: 20-08-2015

DOI: 10.1038/SREP13264

Abstract: Cyclic α-conotoxin Vc1.1 (cVc1.1) is an orally active peptide with analgesic activity in rat models of neuropathic pain. It has two disulfide bonds, which can have three different connectivities, one of which is the native and active form. In this study we used computational modeling and nuclear magnetic resonance to design a disulfide-deleted mutant of cVc1.1, [C2H,C8F]cVc1.1, which has a larger hydrophobic core than cVc1.1 and, potentially, additional surface salt bridge interactions. The new variant, hcVc1.1, has similar structure and serum stability to cVc1.1 and is highly stable at a wide range of pH and temperatures. Remarkably, hcVc1.1 also has similar selectivity to cVc1.1, as it inhibited recombinant human α9α10 nicotinic acetylcholine receptor-mediated currents with an IC 50 of 13 μM and rat N-type (Ca v 2.2) and recombinant human Ca v 2.3 calcium channels via GABA B receptor activation, with an IC 50 of ~900 pM. Compared to cVc1.1, the potency of hcVc1.1 is reduced three-fold at both analgesic targets, whereas previous attempts to replace Vc1.1 disulfide bonds by non-reducible dicarba linkages resulted in at least 30-fold decreased activity. Because it has only one disulfide bond, hcVc1.1 is not subject to disulfide bond shuffling and does not form multiple isomers during peptide synthesis.

Single amino acid substitutions in α-conotoxin PnIA shift selectivity for subtypes of the mammalian neuronal nicotinic acetylcholine receptor

Publisher: Elsevier BV

Date: 12-1999

DOI: 10.1074/JBC.274.51.36559

Molecular basis for the production of cyclic peptides by plant asparaginyl endopeptidases

Publisher: Springer Science and Business Media LLC

Date: 20-06-2018

DOI: 10.1038/S41467-018-04669-9

Abstract: Asparaginyl endopeptidases (AEPs) are proteases that have crucial roles in plant defense and seed storage protein maturation. Select plant AEPs, however, do not function as proteases but as transpeptidases (ligases) catalyzing the intra-molecular ligation of peptide termini, which leads to peptide cyclization. These ligase-type AEPs have potential biotechnological applications ranging from in vitro peptide engineering to plant molecular farming, but the structural features enabling these enzymes to catalyze peptide ligation/cyclization rather than proteolysis are currently unknown. Here, we compare the sequences, structures, and functions of erse plant AEPs by combining molecular modeling, sequence space analysis, and functional testing in planta. We find that changes within the substrate-binding pocket and an adjacent loop, here named the “marker of ligase activity”, together play a key role for AEP ligase efficiency. Identification of these structural determinants may facilitate the discovery of more ligase-type AEPs and the engineering of AEPs with tailored catalytic properties.

Models for the binding of amiodarone to the thyroid hormone receptor

Publisher: Springer Science and Business Media LLC

Date: 02-1992

DOI: 10.1007/BF00124384

Structural and functional characterization of chimeric cyclotides from the Möbius and trypsin inhibitor subfamilies

Publisher: Wiley

Date: 2017

DOI: 10.1002/BIP.22927

Abstract: Cyclotides are plant-derived host defense peptides displaying exceptional stability due to their cyclic cystine knot comprising three intertwined disulfide bonds and a cyclic backbone. Their six conserved cysteine residues are separated by backbone loops with erse sequences. Prototypical cyclotides from the Möbius (kalata B1) and trypsin inhibitor (MCoTI-II) subfamilies lack sequence homology with one another, but both are able to penetrate cells, apparently via different mechanisms. To delineate the influence of the sequences of the loops on the structure and cell internalization of these two cyclotide subfamilies, a series of Möbius/trypsin inhibitor loop-chimeras of kalata B1 and MCoTI-II were synthesized, and structurally and functionally characterized. NMR analysis showed that the structural fold of the majority of chimeric peptides was minimally affected by the loop substitutions. Substituting loops 3, 5, or 6 of MCoTI-II into the corresponding loops of kalata B1 attenuated its hemolytic and cytotoxic activities, and greatly reduced its cell-penetrating properties. On the other hand, replacing loops of MCoTI-II with the corresponding loops of kalata B1 did not introduce cytotoxicity into the chimeras. Loops 2, 3, and 4 of MCoTI-II were found to contribute little to cell-penetrating properties. Overall, this study provides valuable insights into the structural basis for the hemolytic, cytotoxic, and cell-penetrating properties of kalata B1 and MCoTI-II, which could be useful for future engineering of cyclotides to carry bioactive epitopes to intracellular targets.

Ribosomally-synthesised cyclic peptides from plants as drug leads and pharmaceutical scaffolds

Publisher: Elsevier BV

Date: 06-2018

DOI: 10.1016/J.BMC.2017.08.005

Abstract: Owing to their exceptional stability and favourable pharmacokinetic properties, plant-derived cyclic peptides have recently attracted significant attention in the field of peptide-based drug design. This article describes the three major classes of ribosomally-synthesised plant peptides - the cyclotides, the PawS-derived peptides and the orbitides - and reviews their applications as leads or scaffolds in drug design. These ribosomally-produced peptides have a range of biological activities, including anti-HIV, cytotoxic and immunomodulatory activity. In addition, recent interest has focused on their use as scaffolds to stabilise bioactive peptide sequences, thereby enhancing their biopharmaceutical properties. There are now more than 30 published papers on such 'grafting' applications, most of which have been reported only in the last few years, and several such studies have reported in vivo activity of orally delivered cyclic peptides. In this article, we describe approaches to the synthesis of cyclic peptides and their pharmaceutically-grafted derivatives as well as outlining their biosynthetic routes. Finally, we describe possible bioproduction routes for pharmaceutically active cyclic peptides, involving plants and plant suspension cultures.

Discovery, Characterization, and Engineering of LvIC, an α4/4-Conotoxin That Selectively Blocks Rat α6/α3β4 Nicotinic Acetylcholine Receptors

Publisher: American Chemical Society (ACS)

Date: 22-01-2023

DOI: 10.1021/ACS.JMEDCHEM.2C01786

Beta-arrestin 2 is required for complement C1q expression in macrophages and constrains factor-independent survival

Publisher: Elsevier BV

Date: 12-2009

DOI: 10.1016/J.MOLIMM.2009.09.012

Abstract: The beta-arrestins (ARRB1 and ARRB2) regulate G-protein coupled receptor (GPCR) dependent- and independent-signaling pathways and are ubiquitously expressed. Here we show that ARRB2 mRNA and protein expression is enriched in macrophages, and that it regulates complement C1q expression and cell survival. Basal and Toll-like receptor (TLR) inducible expression of mRNAs encoding the complement subcomponents C1qa, C1qb and C1qc was greatly reduced in bone marrow-derived macrophages (BMM) from ARRB2-deficient, but not ARRB1-deficient mice, while factor-independent survival of ARRB2(-/-) BMM was enhanced compared to wildtype BMM. TatARRB2(23), a cell-permeable peptide that contains the MAPK JNK-binding motif from within the ARRB2 C-domain, impaired ARRB2 interaction with JNK3, down-regulated C1q expression and permitted factor-independent survival in BMM, thus suggesting that this peptide antagonises ARRB2 function in macrophages. In addition, TatARRB2(23) transiently activated the phosphorylation of JNK and ERK, but not p38 in BMM. These data imply that ARRB2 acts to limit JNK/ERK activation and survival in macrophages, but is required for basal and TLR-inducible complement C1q expression. Given that loss of C1q function is strongly associated with the development of systemic lupus erythematosus, ARRB2 may act to limit the development of autoimmune disease.

Engineering pro-angiogenic peptides using stable, disulfide-rich cyclic scaffolds

Publisher: American Society of Hematology

Date: 15-12-2011

DOI: 10.1182/BLOOD-2011-06-359141

Abstract: Fragments from the extracellular matrix proteins laminin and osteopontin and a sequence from VEGF have potent proangiogenic activity despite their small size ( 10 residues). However, these linear peptides have limited potential as drug candidates for therapeutic angiogenesis because of their poor stability. In the present study, we show that the therapeutic potential of these peptides can be significantly improved by “grafting” them into cyclic peptide scaffolds. Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) and sunflower trypsin inhibitor-1 (SFTI-1), naturally occurring, plant-derived cyclic peptides of 34 and 14 residues, respectively, were used as scaffolds in this study. Using this approach, we have designed a peptide that, in contrast to the small peptide fragments, is stable in human serum and at nanomolar concentration induces angiogenesis in vivo. This is the first report of using these scaffolds to improve the activity and stability of angiogenic peptide sequences and is a promising approach for promoting angiogenesis for therapeutic uses.

Cyclisation of Disulfide‐Rich Conotoxins in Drug Design Applications

Publisher: Wiley

Date: 07-2016

DOI: 10.1002/EJOC.201600402

Abstract: Conotoxins are disulfide‐rich peptides found in the venoms of marine snails of the genus Conus . They have attracted great attention from the pharmaceutical industry because of their potential uses as drug leads, but like most peptides, conotoxins are susceptible to proteolysis and typically are not orally bioavailable. Here we discuss approaches that have been used to stabilise conotoxins to improve their potential pharmaceutical use. Specifically, we focus on the use of backbone cyclisation to improve their stability in biological fluids. The Microreview provides an introduction to the various classes of conotoxins, including their frameworks (cysteine patterns) and a background on the receptors that they interact with, as well as an analysis of the binding interactions between conotoxins and their receptors.

Solution structure of amyloid β-peptide(1-40) in a water-micelle environment. Is the membrane-spanning domain where we think it is?

Publisher: American Chemical Society (ACS)

Date: 16-07-1998

DOI: 10.1021/BI972979F

Antimicrobial and Anticancer Properties of Synthetic Peptides Derived from the Wasp Parachartergus fraternus

Publisher: Wiley

Date: 24-02-2021

DOI: 10.1002/CBIC.202000716

Abstract: Agelaia‐MPI and protonectin are antimicrobial peptides isolated from the wasp Parachartergus fraternus that show antimicrobial and neuroactive activities. Previously, two analogues of these peptides, neuroVAL and protonectin‐F, were designed to reduce nonspecific toxicity and improve potency. Here, the three‐dimensional structures of neuroVAL, protonectin and protonectin‐F were determined by using circular dichroism and NMR spectroscopy. Antibacterial, antifungal, cytotoxic and hemolytic activities were tested for the parent peptides and analogues. All peptides showed moderate antimicrobial activity against Gram‐positive bacteria, with agelaia‐MPI being the most active. Protonectin and protonectin‐F were found to be toxic to cancerous and noncancerous cell lines. Internalization experiments revealed that these peptides accumulate inside both cell types. By contrast, neuroVAL was nontoxic to all tested cells and was able to enter cells without accumulating. In summary, neuroVAL has potential as a nontoxic cell‐penetrating peptide, while protonectin‐F needs further modification to realize its potential as an antitumor peptide.

Kalata B1 and Kalata B2 Have a Surfactant-Like Activity in Phosphatidylethanolomine-Containing Lipid Membranes

Publisher: American Chemical Society (ACS)

Date: 21-06-2017

DOI: 10.1021/ACS.LANGMUIR.7B01642

Abstract: Cyclotides are cyclic disulfide-rich peptides that are chemically and thermally stable and possess pharmaceutical and insecticidal properties. The activities reported for cyclotides correlate with their ability to target phosphatidylethanolamine (PE)-phospholipids and disrupt cell membranes. However, the mechanism by which this disruption occurs remains unclear. In the current study we examine the effect of the prototypic cyclotides, kalata B1 (kB1) and kalata B2 (kB2), on tethered lipid bilayer membranes (tBLMs) using swept frequency electrical impedance spectroscopy. We confirmed that kB1 and kB2 bind to bilayers only if they contain PE-phospholipids. We hypothesize that the increase in membrane conduction and capacitance observed upon addition of kB1 or kB2 is unlikely to result from ion channel like pores but is consistent with the formation of lipidic toroidal pores. This hypothesis is supported by the concentration dependence of effects of kB1 and kB2 being suggestive of a critical micelle concentration event rather than a progressive increase in conduction arising from increased channel insertion. Additionally, conduction behavior is readily reversible when the peptide is rinsed from the bilayer. Our results support a mechanism by which kB1 and kB2 bind to and disrupt PE-containing membranes by decreasing the overall membrane critical packing parameter, as would a surfactant, which then opens or increases the size of existing membrane defects. The cyclotides need not participate directly in the conductive pore but might exert their effect indirectly through altering membrane packing constraints and inducing purely lipidic conductive pores.

The Cyclic Cystine Ladder of Theta‐Defensins as a Stable, Bifunctional Scaffold: A Proof‐of‐Concept Study Using the Integrin‐Binding RGD Motif.

Publisher: Wiley

Date: 02-01-2014

DOI: 10.1002/CBIC.201300568

Abstract: Peptides have the specificity and size required to target the protein-protein interactions involved in many diseases. Some cyclic peptides have been utilised as scaffolds for peptide drugs because of their stability however, other cyclic peptide scaffolds remain to be explored. θ-Defensins are cyclic peptides from mammals they are characterised by a cyclic cystine ladder motif and have low haemolytic and cytotoxic activity. Here we demonstrate the potential of the cyclic cystine ladder as a scaffold for peptide drug design by introducing the integrin-binding Arg-Gly-Asp (RGD) motif into the θ-defensin RTD-1. The most active analogue had an IC50 of 18 nM for the αv β3 integrin as well as high serum stability, thus demonstrating that a desired bioactivity can be imparted to the cyclic cystine ladder. This study highlights how θ-defensins can provide a stable and conformationally restrained scaffold for bioactive epitopes in a β-strand or turn conformation. Furthermore, the symmetry of the cyclic cystine ladder presents the opportunity to design peptides with dual bioactive epitopes to increase activity and specificity.

Potential therapeutic applications of the cyclotides and related cystine knot mini-proteins

Publisher: Informa Healthcare

Date: 26-04-2007

DOI: 10.1517/13543784.16.5.595

Abstract: Cyclotides are naturally occurring mini-proteins that have a cyclic peptide backbone and a knotted arrangement of three disulfide bonds. They are remarkably stable and have a erse range of therapeutically useful biological activities, including antimicrobial and anti-HIV activity, although their natural function appears to be as plant defence agents. Cyclotides are amenable to chemical synthesis and the potential exists to graft new bioactivities onto their cyclic cystine knot framework as a way of stabilising peptide drugs. Over the last few years, proof-of-concept that bioactive peptide epitopes can be grafted onto cyclotides and related cystine knot mini-proteins has been obtained. The cystine knot framework is tolerant to a wide range of residue substitutions and is showing great promise as a scaffold in drug design and protein engineering.

α-Conotoxin Vc1.1 inhibits human dorsal root ganglion neuroexcitability and mouse colonic nociception via GABAB receptors.

Publisher: BMJ

Date: 17-02-2016

DOI: 10.1136/GUTJNL-2015-310971

Discovery, isolation, and structural characterization of cyclotides from Viola sumatrana Miq

Publisher: Wiley

Date: 11-2016

DOI: 10.1002/BIP.22914

Abstract: Cyclotides are cyclic peptides from plants in the Violaceae, Rubiaceae, Fabaceae, Cucurbitaceae, and Solanaceae families. They are sparsely distributed in most of these families, but appear to be ubiquitous in the Violaceae, having been found in every plant so far screened from this family. However, not all geographic regions have been examined and here we report the discovery of cyclotides from a Viola species from South-East Asia. Two novel cyclotides (Visu 1 and Visu 2) and two known cyclotides (kalata S and kalata B1) were identified in V. sumatrana. NMR studies revealed that kalata S and kalata B1 had similar secondary structures. Their biological activities were determined in cytotoxicity assays both had similar cytotoxic activity and were more toxic to U87 cells compared with other cell lines. Overall, the study strongly supports the ubiquity of cyclotides in the Violaceae and adds to our understanding of their distribution and cytotoxic activity.

Solution structure and characterization of the LGR8 receptor binding surface of insulin-like peptide 3.

Publisher: Elsevier BV

Date: 09-2006

DOI: 10.1074/JBC.M603829200

Photodegradation of irinotecan (CPT-11) in aqueous solutions: Identification of fluorescent products and influence of solution composition

Publisher: American Geophysical Union (AGU)

Date: 12-1997

DOI: 10.1021/JS970110C

A suite of kinetically superior AEP ligases can cyclise an intrinsically disordered protein

Publisher: Springer Science and Business Media LLC

Date: 25-07-2019

DOI: 10.1038/S41598-019-47273-7

Abstract: Asparaginyl endopeptidases (AEPs) are a class of enzymes commonly associated with proteolysis in the maturation of seed storage proteins. However, a subset of AEPs work preferentially as peptide ligases, coupling release of a leaving group to formation of a new peptide bond. These “ligase-type” AEPs require only short recognition motifs to ligate a range of targets, making them useful tools in peptide and protein engineering for cyclisation of peptides or ligation of separate peptides into larger products. Here we report the recombinant expression, ligase activity and cyclisation kinetics of three new AEPs from the cyclotide producing plant Oldenlandia affinis with superior kinetics to the prototypical recombinant AEP ligase OaAEP1 b . These AEPs work preferentially as ligases at both acidic and neutral pH and we term them “canonical AEP ligases” to distinguish them from other AEPs where activity preferences shift according to pH. We show that these ligases intrinsically favour ligation over hydrolysis, are highly efficient at cyclising two unrelated peptides and are compatible with organic co-solvents. Finally, we demonstrate the broad scope of recombinant AEPs in biotechnology by the backbone cyclisation of an intrinsically disordered protein, the 25 kDa malarial vaccine candidate Plasmodium falciparum merozoite surface protein 2 (MSP2).

An Ultrapotent and Selective Cyclic Peptide Inhibitor of Human β-Factor XIIa in a Cyclotide Scaffold

Publisher: American Chemical Society (ACS)

Date: 11-2021

DOI: 10.1021/JACS.1C07574

Abstract: Cyclotides are plant-derived peptides with complex structures shaped by their head-to-tail cyclic backbone and cystine knot core. These structural features underpin the native bioactivities of cyclotides, as well as their beneficial properties as pharmaceutical leads, including high proteolytic stability and cell permeability. However, their inherent structural complexity presents a challenge for cyclotide engineering, particularly for accessing libraries of sufficient chemical ersity to design potent and selective cyclotide variants. Here, we report a strategy using mRNA display enabling us to select potent cyclotide-based FXIIa inhibitors from a library comprising more than 10

Conformational dynamics of thyroid hormones by variable temperature nuclear magnetic resonance: The role of side chain rotations and cisoid/transoid interconversions

Publisher: American Chemical Society (ACS)

Date: 07-1997

DOI: 10.1021/JM9606610

Bioinformatics-Aided Venomics.

Publisher: MDPI AG

Date: 11-06-2015

DOI: 10.3390/TOXINS7062159

Invited reviewnative chemical ligation applied to the synthesis and bioengineering of circular peptides and proteins

Publisher: Wiley

Date: 2010

DOI: 10.1002/BIP.21372

Abstract: Native chemical ligation methodology developed in the laboratory of Stephen Kent is a versatile approach to the linkage of peptide fragments using a native peptide bond. It is readily adaptable to the task of joining the N- and C-termini of peptides to produce cyclic molecules and we have used it for the cyclization of a range of disulfide-rich peptides. Specifically, it has been valuable for the synthesis of cyclotides, naturally occurring peptides characterized by a head-to-tail cyclized backbone and a knotted arrangement of three conserved disulfide bonds. Cyclotides have a erse range of biological activities, including anti-HIV, antimicrobial, and insecticidal activities. They are ultrastable owing to their cyclic cystine knot motif, and native chemical ligation methodology has been invaluable in the synthesis of a range of native and modified cyclotides to explore their structure-activity relationships and applications in drug design. Similar studies have also been applied to a smaller cyclic peptide produced in sunflower seeds, sunflower trypsin inhibitor-1, which also shows promise as a template in drug design applications. We have also found native chemical ligation to be a valuable methodology for the cyclization of conotoxins, small disulfide-rich peptides from the venoms of marine cone snails. Conotoxins target a range of ions channels and receptors and are exciting leads in drug design applications. The synthetic cyclization of conotoxins with peptide linkers stabilizes them and improves their biopharmaceutical properties. In summary, this article illustrates the use of native chemical ligation technology in the cyclization of cyclotides, sunflower trypsin inhibitor-1, and conotoxins in our laboratory.

High-affinity Cyclic Peptide Matriptase Inhibitors

Publisher: Elsevier BV

Date: 05-2013

DOI: 10.1074/JBC.M113.460030

¹⁵N cyclotides by whole plant labeling

Publisher: Wiley

Date: 2008

DOI: 10.1002/BIP.21012

Abstract: Cyclotides are 28-37 amino acid peptides incorporating three disulfide bonds and a cyclic backbone. Their cyclic and knotted topology renders them immune to denaturation by heat or organic solvents and highly resistant to proteolysis. They have a range of interesting and potentially useful pharmaceutical properties and have been proposed as scaffolds within which peptides with drug activities can be stabilized for delivery. Some members of the family also have agricultural applications deriving from their potent insecticidal activity. Labeling peptides with the NMR-active and stable 15N isotope facilitates a range of studies by NMR, including structural and dynamics studies and their use as tracers. However, owing to their head-to-tail cyclized peptide backbone labeled cyclotides are not amenable to conventional recombinant labeling strategies. We have developed an approach to overcome this limitation by growing the cyclotide-bearing plant Oldenlandia affinis on nitrogen-free agar media supplemented with 15N salts and obtaining complete labeling at no detriment to plant biomass. We purified the insecticidal cyclotides kalata B1 and kalata B2 as ex les and provide heteronuclear single quantum coherence (HSQC) NMR spectra for each. This method of labeling cyclotides involves only a fraction of the cost of uniform labeling by solid-phase peptide synthesis.

A determination of the solution conformation of the nonmammalian tachykinin eledoisin by NMR and CD spectroscopy

Publisher: American Chemical Society (ACS)

Date: 07-06-1994

DOI: 10.1021/BI00188A008

Abstract: The nonmammalian tachykinin eledoisin was investigated by use of CD and two-dimensional NMR techniques. In aqueous solution the peptide is conformationally averaged, but on addition of 50% trifluoroethanol (TFE) or sodium dodecyl sulfate (SDS) it adopts an alpha-helical structure. In TFE/H2O and SDS, residues 6-10 of eledoisin show more conformational order than the terminal regions, which undergo dynamic fraying. A possible turn in the N-terminal "address" region, the putative receptor recognition site of the peptide, is detected by NMR spectroscopy but appears to undergo substantial conformational averaging. The NMR data indicate that the helical central core of eledoisin is better defined in the micellar environment than in TFE however, partial unfolding via 3(10) intermediates occurs in both cases. The conformational preference for SDS-bound eledoisin was examined by three-dimensional structure calculations using NMR-derived distance information in simulated annealing calculations.

Thyroid hormone uptake by hepatocytes: structure-activity relationships of phenylanthranilic acids with inhibitory activity

Publisher: American Chemical Society (ACS)

Date: 04-1993

DOI: 10.1021/JM00061A019

Abstract: The synthesis of a series of mono- and disubstituted N-phenylanthranilic acids is described. Substituents on the phenyl ring include Cl, CN, OH, CF3, Br, I, CH3, OCH3, and OCF2CF2H. These compounds have been tested for their inhibitory effect on triiodothyronine (T3) uptake by H4 hepatocytes. The nonsteroidal antiinflammatory drugs flufenamic acid, mefenamic acid, and meclofenamic acid and the structurally related compounds 2,3-dimethyldiphenylamine and diclofenac were also tested. The most potent compounds were found to be, in order of decreasing activity, meclofenamic acid (2,6-Cl2,3-CH3), flufenamic acid (3-CF3), mefenamic acid (2,3-(CH3)2), and the compounds with 3,5-Cl2 and 3-OCF2CF2H substituents. The least potent compounds had 3-CN and 3-OH substituents. An analysis of quantitative structure-activity relationships (QSAR) for the series of phenylanthranilic acids showed that the inhibition of T3 uptake is highly dependent on the hydrophobicity of the compound. The relationship between uptake inhibition and the calculated octanol-water partition coefficient (clogP) was found to be parabolic, with optimum inhibitory activity found when the clogP of the phenylanthranilic acid was 5.7. It was also found that the 1-carboxylic acid group of the phenylanthranilic acids was not a prerequisite for uptake inhibition to occur, but its removal or alteration resulted in reduced inhibition.

Substrate-Guided Design of Selective FXIIa Inhibitors Based on the Plant-Derived Momordica cochinchinensis Trypsin Inhibitor-II (MCoTI-II) Scaffold

Publisher: American Chemical Society (ACS)

Date: 08-2016

DOI: 10.1021/ACS.JMEDCHEM.6B00557

Abstract: Thrombosis is a leading cause of morbidity and mortality associated with cardiovascular diseases. Inhibition of factor XIIa (FXIIa) provides thrombus protection without bleeding complications. Here, we defined the extended substrate specificity of FXIIa and its close homologue factor Xa and used these data, together with inhibitor-based and structure-guided methods, to engineer selective FXIIa inhibitors based on Momordica cochinchinensis trypsin inhibitor-II.

Structure-Activity Relationship Study of the Plant-Derived Decapeptide OSIP108 Inhibiting Candida albicans Biofilm Formation

Publisher: American Society for Microbiology

Date: 08-2014

DOI: 10.1128/AAC.03336-14

Abstract: We performed a structure-activity relationship study of the antibiofilm plant-derived decapeptide OSIP108. Introduction of positively charged amino acids R, H, and K resulted in an up-to-5-fold-increased antibiofilm activity against Candida albicans compared to native OSIP108, whereas replacement of R9 resulted in complete abolishment of its antibiofilm activity. By combining the most promising amino acid substitutions, we found that the double-substituted OSIP108 analogue Q6R/G7K had an 8-fold-increased antibiofilm activity.

Structural Analysis of the Complex of Human Transthyretin with 3′,5′-Dichlorophenylanthranilic Acid at 1.5 Å Resolution

Publisher: MDPI AG

Date: 25-10-2022

DOI: 10.3390/MOLECULES27217206

Abstract: Human transthyretin (hTTR) can form amyloid deposits that accumulate in nerves and organs, disrupting cellular function. Molecules such as tafamidis that bind to and stabilize the TTR tetramer can reduce such amyloid formation. Here, we studied the interaction of VCP-6 (2-((3,5-dichlorophenyl)amino)benzoic acid) with hTTR. VCP-6 binds to hTTR with 5 times the affinity of the cognate ligand, thyroxine (T4). The structure of the hTTR:VCP-6 complex was determined by X-ray crystallography at 1.52 Å resolution. VCP-6 binds deeper in the binding channel than T4 with the 3′,5′-dichlorophenyl ring binding in the ‘forward’ mode towards the channel centre. The dichlorophenyl ring lies along the 2-fold axis coincident with the channel centre, while the 2-carboxylatephenylamine ring of VCP-6 is symmetrically displaced from the 2-fold axis, allowing the 2-carboxylate group to form a tight intermolecular hydrogen bond with Nζ of Lys15 and an intramolecular hydrogen bond with the amine of VCP-6, stabilizing its conformation and explaining the greater affinity of VCP-6 compared to T4. This arrangement maintains optimal halogen bonding interactions in the binding sites, via chlorine atoms rather than iodine of the thyroid hormone, thereby explaining why the dichloro substitution pattern is a stronger binder than either the diiodo or dibromo analogues.

Enabling Efficient Folding and High-Resolution Crystallographic Analysis of Bracelet Cyclotides

Publisher: MDPI AG

Date: 13-09-2021

DOI: 10.3390/MOLECULES26185554

Abstract: Cyclotides have attracted great interest as drug design scaffolds because of their unique cyclic cystine knotted topology. They are classified into three subfamilies, among which the bracelet subfamily represents the majority and comprises the most bioactive cyclotides, but are the most poorly utilized in drug design applications. A long-standing challenge has been the very low in vitro folding yields of bracelets, h ering efforts to characterize their structures and activities. Herein, we report substantial increases in bracelet folding yields enabled by a single point mutation of residue Ile-11 to Leu or Gly. We applied this discovery to synthesize mirror image enantiomers and used quasi-racemic crystallography to elucidate the first crystal structures of bracelet cyclotides. This study provides a facile strategy to produce bracelet cyclotides, leading to a general method to easily access their atomic resolution structures and providing a basis for development of biotechnological applications.

Cyclization of conotoxins to improve their biopharmaceutical properties

Publisher: Elsevier BV

Date: 03-2012

DOI: 10.1016/J.TOXICON.2010.12.003

Abstract: Conotoxins are disulfide-rich peptides from the venoms of marine cone snails that are used in prey capture. Due to their exquisite potency and selectivity for different ion channels, receptors and transporters they have attracted much interest as leads in drug design. This article gives a brief background on conotoxins, describes their structures and highlights methods for synthetic cyclization to improve their biopharmaceutical properties. The proximity of the N and C termini of many conotoxins makes them particularly suitable for cyclization with linkers of on average five to seven amino acids. By linking the ends of conotoxins it is possible to significantly decrease their susceptibility to proteolysis without loss of their intrinsic biological activity. Here, the principles of conotoxin cyclization are illustrated with applications to the α- and χ- conotoxin classes, which have been implicated as leads for the treatment of pain and a range of other disorders including neuroprotection, schizophrenia, depression and cancer.

A Consensus Structure for ω-Conotoxins with Different Selectivities for Voltage-sensitive Calcium Channel Subtypes: Comparison of MVIIA, SVIB and SNX-202

Publisher: Elsevier BV

Date: 10-1996

DOI: 10.1006/JMBI.1996.0576

Abstract: The omega-conotoxins are a set of structurally related peptides that have a wide range of specificities for different subtypes of the voltage-sensitive calcium channel (VSCC). To understand their VSCC subtype differentiation we studied the structure of two naturally occurring omega-conotoxins, MVIIA (specific to N-type) and SVIB (specific to P/Q-type) and a synthetic hybrid, SNX-202, which has altered specificities to both VSCC subtypes. The secondary structures of the three peptides are almost identical, consisting of a triple-stranded beta-sheet and several turns. A comparison of NMR data emphasizes the structural similarities between the peptides and highlights some minor structural differences. In the three-dimensional structures of SVIB and MVIIA these are manifested as orientational differences between two key loops. The structural rigidity of MVIIA was also examined. H alpha shifts are similar in a range of solvents, indicating that there are no solvent-induced changes in structure. The omega-conotoxins form a consensus structure despite differences in sequence and VSCC subtype specificity. This indicates that the omega-conotoxin macrosites for the N/P/Q-subfamily of VSCCs are related, with specificity for receptor targets being conferred by the positions of functional side-chains on the surface of the peptides.

Anthelminthic activity of the cyclotides (kalata B1 and B2) against schistosome parasites

Publisher: Wiley

Date: 09-2013

DOI: 10.1002/BIP.22229

Abstract: The risk of reduced sensitivity of the human schistosomes to praziquantel has led to efforts to find new therapies. Here, the cyclotides kalata B1 (kB1), kalata B2 (kB2), MCoCC‐1, and MCoTI‐II, cyclic peptides extracted from plants and shown to be potent against nematodes and insects, were tested for antischistosome activity. In vitro assays showed that high concentrations (500–1000 μ g/mL) of either kB1 or kB2 killed Schistosoma japonicum and Schistosoma mansoni adults within 5 min, whereas MCoTI‐II and MCoCC‐1 had no effect. Lethal concentrations to kill 50% of the population for kB2 was 15.5 ± 7.4 μg/mL at 1 h for male S. japonicum (Philippine strain). Males were more susceptible than females. kB2 showed higher antischistosome activity than kB1 and killing time was concentration‐dependent. Mode of action studies revealed that kB1 and 2 lysed the tegument of adult worms. Lysis of myofibrils was not demonstrated, but longitudinal and radial muscle fibers were distorted, an observation consistent with strong coiling of the parasites after drug exposure. A single dose of kB2 administered either orally or intravenously, reduced worm burdens in S. japonicum‐infected mice from 15% to 60%. However, treatment of S. mansoni‐infected mice did not result in reduction in worm burdens. Our studies show that kB2 acts as a promising antischistosomal against Philippine S. japonicum, and it or other cyclotides may be developed further as general anthelminthics. With thousands of cyclotides predicted to occur in plants, and the amenability of these peptides to combinatorial variation, there is potential for their exploitation as wide‐spectrum anthelminthics. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 461–470, 2013.

Neurotoxic peptides from the venom of the giant Australian stinging tree

Publisher: American Association for the Advancement of Science (AAAS)

Date: 18-09-2020

DOI: 10.1126/SCIADV.ABB8828

Abstract: The pain-inducing components of Australian stinging tree venom are miniproteins that modulate voltage-gated sodium channels.

Novel Inhibitor Cystine Knot Peptides from Momordica charantia

Publisher: Public Library of Science (PLoS)

Date: 08-10-2013

DOI: 10.1371/JOURNAL.PONE.0075334

The Prototypic Cyclotide Kalata B1 Has a Unique Mechanism of Entering Cells

Publisher: Elsevier BV

Date: 08-2015

DOI: 10.1016/J.CHEMBIOL.2015.07.012

Abstract: Cyclotides combine the stability of disulfide-rich peptides with the intracellular accessibility of cell-penetrating peptides, giving them outstanding potential as drug scaffolds with an ability to inhibit intracellular protein-protein interactions. To realize and optimize the application of cyclotides as a drug framework and delivery system, we studied the ability of the prototypic cyclotide, kalata B1, to enter mammalian cells. We show that kalata B1 can enter cells via both endocytosis and direct membrane translocation. Both pathways are initiated by targeting phosphatidylethanolamine phospholipids at the cell surface and inducing membrane curvature. This unusual approach to initiate internalization might be harnessed to deliver drugs into cells and, in particular, cancer cells, which present a higher proportion of surface-exposed phosphatidylethanolamine phospholipids. Our findings highlight the potential of these peptides as drug leads for the modulation of traditionally "undruggable" targets, such as intracellular protein-protein interactions.

Cyclotides: Disulfide-rich peptide toxins in plants.

Publisher: Elsevier BV

Date: 10-2019

DOI: 10.1016/J.TOXICON.2019.10.244

Abstract: Cyclotides are a plant-derived family of peptides that comprise approximately 30 amino acid residues, a cyclic backbone and a cystine knot. Due to their unique structure, cyclotides are exceptionally stable to heat or proteolytic degradation and are tolerant to amino acid substitutions in their backbone loops between conserved cysteine residues. Their toxicity to insect pests and their make-up of natural amino acids has led to their applications in eco-friendly crop protection. Furthermore, their stability and cell penetrating properties make cyclotides ideal scaffolds for bioactive epitope grafting. This article gives a brief overview of cyclotide discovery, characterization, distribution, synthesis and mode of action mechanisms. We focus on their toxicities to insect pests and their medical and agricultural applications.

Comparison of a Short Linear Antimicrobial Peptide with Its Disulfide-Cyclized and Cyclotide-Grafted Variants against Clinically Relevant Pathogens

Publisher: MDPI AG

Date: 08-06-2021

DOI: 10.3390/MICROORGANISMS9061249

Abstract: According to the World Health Organization (WHO) the development of resistance against antibiotics by microbes is one of the most pressing health concerns. The situation will intensify since only a few pharmacological companies are currently developing novel antimicrobial compounds. Discovery and development of novel antimicrobial compounds with new modes of action are urgently needed. Antimicrobial peptides (AMPs) are known to be able to kill multidrug-resistant bacteria and, therefore, of interest to be developed into antimicrobial drugs. Proteolytic stability and toxicities of these peptides are challenges to overcome, and one strategy frequently used to address stability is cyclization. Here we introduced a disulfide-bond to cyclize a potent and nontoxic 9mer peptide and, in addition, as a proof-of-concept study, grafted this peptide into loop 6 of the cyclotide MCoTI-II. This is the first time an antimicrobial peptide has been successfully grafted onto the cyclotide scaffold. The disulfide-cyclized and grafted cyclotide showed moderate activity in broth and strong activity in 1/5 broth against clinically relevant resistant pathogens. The linear peptide showed superior activity in both conditions. The half-life time in 100% human serum was determined, for the linear peptide, to be 13 min, for the simple disulfide-cyclized peptide, 9 min, and, for the grafted cyclotide 7 h 15 min. The addition of 10% human serum led to a loss of antimicrobial activity for the different organisms, ranging from 1 to -fold for the cyclotide. For the disulfide-cyclized version and the linear version, activity also dropped to different degrees, 2 to 18-fold, and 1 to 30-fold respectively. Despite the massive difference in stability, the linear peptide still showed superior antimicrobial activity. The cyclotide and the disulfide-cyclized version demonstrated a slower bactericidal effect than the linear version. All three peptides were stable at high and low pH, and had very low hemolytic and cytotoxic activity.

Isolation, Sequencing, and Structure−Activity Relationships of Cyclotides

Publisher: American Chemical Society (ACS)

Date: 18-08-2010

DOI: 10.1021/NP1000413

Abstract: Cyclotides are a topologically fascinating family of miniproteins discovered over the past decade that have expanded the ersity of plant-derived natural products. They are approximately 30 amino acids in size and occur in plants of the Violaceae, Rubiaceae, and Cucurbitaceae families. Despite their proteinaceous composition, cyclotides behave in much the same way as many nonpeptidic natural products in that they are resistant to degradation by enzymes or heat and can be extracted from plants using methanol. Their stability arises, in large part, due to their characteristic cyclic cystine knot (CCK) structural motif. Cystine knots are present in a variety of proteins of insect, plant, and animal origin, comprising a ring formed by two disulfide bonds and their connecting backbone segments that is threaded by a third disulfide bond. In cyclotides, the cystine knot is uniquely embedded within a head-to-tail cyclized peptide backbone, leading to the ultrastable CCK structural motif. Apart from the six absolutely conserved cysteine residues, the majority of amino acids in the six backbone loops of cyclotides are tolerant to variation. It has been predicted that the family might include up to 50,000 members although, so far, sequences for only 140 have been reported. Cyclotides exhibit a variety of biological activities, including insecticidal, nematocidal, molluscicidal, antimicrobial, antibarnacle, anti-HIV, and antitumor activities. Due to their erse activities and common structural core from which variable loops protrude, cyclotides can be thought of as combinatorial peptide templates capable of displaying a variety of amino acid sequences. They have thus attracted interest in drug design as well as in crop protection applications.

Development of novel frog‐skin peptide scaffolds with selectivity towards melanocortin receptor subtypes

Publisher: Wiley

Date: 26-10-2020

DOI: 10.1002/PEP2.24209

Abstract: Melanocortin receptors are pharmaceutically important receptors that are involved in complex physiological functions. They have been associated with various diseases including obesity, erectile dysfunction, acne, and skin cancer. It has been challenging to transform nonselective endogenous agonist and antagonist ligands into selective and potent ligands. In this study, we investigated naturally occurring peptides derived from frog skin secretions for selectivity and activity toward melanocortin receptors. Three peptides (ORB, ORB2K and ranacyclin‐T) were found to have selectivity towards the melanocortin receptor 5 (MC5R). ORB and ORB2K had partial binding affinity at nanomolar concentrations, whereas ranacyclin‐T had 57% binding efficiency at 1.6 μM. Backbone cyclization of ORB and ORB2K altered the binding efficiency to melanocortin receptors. Our results suggest that these frog‐skin peptides could be modified for developing melanocortin‐specific ligands and potentially future therapeutics.

In Vivo Efficacy of Anuran Trypsin Inhibitory Peptides against Staphylococcal Skin Infection and the Impact of Peptide Cyclization

Publisher: American Society for Microbiology

Date: 04-2015

DOI: 10.1128/AAC.04324-14

Abstract: Staphylococcus aureus is a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weak in vitro inhibitory activities against S. aureus , but several had strong antibacterial activities against S. aureus in an in vivo murine wound infection model. pYR, an immunomodulatory peptide from Rana sevosa , was the most potent, with complete bacterial clearance at 3 mg · kg −1 . Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen.

The biological activity of the prototypic cyclotide Kalata B1 is modulated by the formation of multimeric pores

Publisher: Elsevier BV

Date: 07-2009

DOI: 10.1074/JBC.M109.003384

Decoding the Membrane Activity of the Cyclotide Kalata B1

Publisher: Elsevier BV

Date: 07-2011

DOI: 10.1074/JBC.M111.253393

Engineered EGF-A Peptides with Improved Affinity for Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9)

Publisher: American Chemical Society (ACS)

Date: 29-01-2021

DOI: 10.1021/ACSCHEMBIO.0C00991

Angler Peptides: Macrocyclic Conjugates Inhibit p53:MDM2/X Interactions and Activate Apoptosis in Cancer Cells

Publisher: American Chemical Society (ACS)

Date: 03-02-2021

DOI: 10.1021/ACSCHEMBIO.0C00988

Bibliometric Review of the Literature on Cone Snail Peptide Toxins from 2000 to 2022

Publisher: MDPI AG

Date: 25-02-2023

DOI: 10.3390/MD21030154

Abstract: The venom of marine cone snails is mainly composed of peptide toxins called conopeptides, among which conotoxins represent those that are disulfide-rich. Publications on conopeptides frequently state that conopeptides attract considerable interest for their potent and selective activity, but there has been no analysis yet that formally quantifies the popularity of the field. We fill this gap here by providing a bibliometric analysis of the literature on cone snail toxins from 2000 to 2022. Our analysis of 3028 research articles and 393 reviews revealed that research in the conopeptide field is indeed prolific, with an average of 130 research articles per year. The data show that the research is typically carried out collaboratively and worldwide, and that discoveries are truly a community-based effort. An analysis of the keywords provided with each article revealed research trends, their evolution over the studied period, and important milestones. The most employed keywords are related to pharmacology and medicinal chemistry. In 2004, the trend in keywords changed, with the pivotal event of that year being the approval by the FDA of the first peptide toxin drug, ziconotide, a conopeptide, for the treatment of intractable pain. The corresponding research article is among the top ten most cited articles in the conopeptide literature. From the time of that article, medicinal chemistry aiming at engineering conopeptides to treat neuropathic pain r ed up, as seen by an increased focus on topological modifications (e.g., cyclization), electrophysiology, and structural biology.

Discovery and structures of the cyclotides: novel macrocyclic peptides from plants

Publisher: Springer Science and Business Media LLC

Date: 2001

DOI: 10.1023/A:1016262020925

Phosphatidylethanolamine binding is a conserved feature of cyclotide-membrane interactions

Publisher: Elsevier BV

Date: 09-2012

DOI: 10.1074/JBC.M112.372011

Production of a structurally validated cyclotide in rice suspension cells is enabled by a supporting biosynthetic enzyme.

Publisher: Springer Science and Business Media LLC

Date: 05-11-2020

DOI: 10.1007/S00425-020-03505-Z

Abstract: We demonstrate the production of a structurally correct cyclotide in rice suspension cells with co-expression of a ligase-type AEP, which unlocks monocotyledons as production platforms to produce cyclotides. Cyclotides are a class of backbone-cyclic plant peptides that harbor a cystine knot composed of three disulfide bonds. These structural features make cyclotides particularly stable, and thus they have attracted significant attention for their use in biotechnological applications such as drug design. Currently, chemical synthesis is the predominant strategy to produce cyclotides for research purposes. However, synthetic production becomes costly both economically and environmentally at large scale. Plants offer an attractive alternative to chemical synthesis because of their lower cost and environmental footprint. In this study, rice suspension cells were engineered to produce the prototypical cyclotide, kalata B1 (kB1), a cyclotide with insecticidal properties from the African plant Oldenlandia affinis. Engineered rice cells produced structurally validated kB1 at yields of 64.21 µg/g (DW), which was dependent on the co-expression of a peptide ligase-competent asparaginyl endopeptidase OaAEP1

Binding Mode of &#945;-Conotoxins to an Acetylcholine Binding Protein Determined by Saturation Transfer Difference NMR

Publisher: Bentham Science Publishers Ltd.

Date: 09-2008

DOI: 10.2174/092986608785849335

Abstract: The saturation transfer difference (STD) NMR technique was employed to study the complex of the alpha-conotoxins Vc1.1 and MII bound to the acetylcholine binding protein (AChBP) from Lymnea stagnalis, a model system of the alpha7 subunit of the nicotinic acetylcholine receptor. MII was found to be the more potent ligand for AChBP, consistent with data from electrophysiology measurements for the nicotinic acetylcholine receptor. Both peptides displayed strong interactions on aromatic residues in the alpha-helical part of their sequences, i.e., Tyr10 in Vc1.1 and His9 in MII respectively. From the STD NMR spectra it was determined that the peptides are buried in the nicotinic binding site of ACBP as has been previously shown for the conotoxins PnIA[A10L, D14K], ImI and TxIA[A10L] by X-ray crystallography. This study demonstrates the value of STD NMR in the study of conotoxin binding to receptor proteins.

Transforming conotoxins into cyclotides: Backbone cyclization of P‐superfamily conotoxins

Publisher: Wiley

Date: 11-2015

DOI: 10.1002/BIP.22699

Abstract: Peptide backbone cyclization is a widely used approach to improve the activity and stability of small peptides but until recently it had not been applied to peptides with multiple disulfide bonds. Conotoxins are disulfide-rich conopeptides derived from the venoms of cone snails that have applications in drug design and development. However, because of their peptidic nature, they can suffer from poor bioavailability and poor stability in vivo. In this study two P-superfamily conotoxins, gm9a and bru9a, were backbone cyclized by joining the N- and C-termini with short peptide linkers using intramolecular native chemical ligation chemistry. The cyclized derivatives had conformations similar to the native peptides showing that backbone cyclization can be applied to three disulfide-bonded peptides with cystine knot motifs. Cyclic gm9a was more potent at high voltage-activated (HVA) calcium channels than its acyclic counterpart, highlighting the value of this approach in developing active and stable conotoxins containing cyclic cystine knot motifs.

Small Molecular Probes for G-Protein-Coupled C5a Receptors:  Conformationally Constrained Antagonists Derived from the C Terminus of the Human Plasma Protein C5a

Publisher: American Chemical Society (ACS)

Date: 08-1998

DOI: 10.1021/JM9800651

The evolution of Momordica cyclic peptides.

Publisher: Oxford University Press (OUP)

Date: 06-11-2015

DOI: 10.1093/MOLBEV/MSU307

Abstract: Cyclic proteins have evolved for millions of years across all kingdoms of life to confer structural stability over their acyclic counterparts while maintaining intrinsic functional properties. Here, we show that cyclic miniproteins (or peptides) from Momordica (Cucurbitaceae) seeds evolved in species that erged from an African ancestor around 19 Ma. The ability to achieve head-to-tail cyclization of Momordica cyclic peptides appears to have been acquired through a series of mutations in their acyclic precursor coding sequences following recent and independent gene expansion event(s). Evolutionary analysis of Momordica cyclic peptides reveals sites that are under selection, highlighting residues that are presumably constrained for maintaining their function as potent trypsin inhibitors. Molecular dynamics of Momordica cyclic peptides in complex with trypsin reveals site-specific residues involved in target binding. In a broader context, this study provides a basis for selecting Momordica species to further investigate the biosynthesis of the cyclic peptides and for constructing libraries that may be screened against evolutionarily related serine proteases implicated in human diseases.

Analgesic α-Conotoxins Vc1.1 and Rg1A Inhibit N-Type Calcium Channels in Rat Sensory Neurons via GABA_BReceptor Activation

Publisher: Society for Neuroscience

Date: 22-10-2008

DOI: 10.1523/JNEUROSCI.3594-08.2008

Abstract: α-Conotoxins Vc1.1 and Rg1A are peptides from the venom of marine Conus snails that are currently in development as a treatment for neuropathic pain. Here we report that the α9α10 nicotinic acetylcholine receptor-selective conotoxins Vc1.1 and Rg1A potently and selectively inhibit high-voltage-activated (HVA) calcium channel currents in dissociated DRG neurons in a concentration-dependent manner. The post-translationally modified peptides vc1a and [P6O]Vc1.1 were inactive, as were all other α-conotoxins tested. Vc1.1 inhibited the ω-conotoxin-sensitive HVA currents in DRG neurons but not those recorded from Xenopus oocytes expressing Ca V 2.2, Ca V 2.1, Ca V 2.3, or Ca V 1.2 channels. Inhibition of HVA currents by Vc1.1 was not reversed by depolarizing prepulses but was abolished by pertussis toxin (PTX), intracellular GDPβS, or a selective inhibitor of pp60c-src tyrosine kinase. These data indicate that Vc1.1 does not interact with N-type calcium channels directly but inhibits them via a voltage-independent mechanism involving a PTX-sensitive, G-protein-coupled receptor. Preincubation with a variety of selective receptor antagonists demonstrated that only the GABA B receptor antagonists, [ S -( R *, R *)][-3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxy propyl]([3,4]-cyclohexylmethyl) phosphinic acid hydrochloride (2 S )-3[[(1 S )-1-(3,4-dichlorophenyl)-ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid and phaclofen, blocked the effect of Vc1.1 and Rg1A on Ca 2+ channel currents. Together, the results identify Ca V 2.2 as a target of Vc1.1 and Rg1A, potentially mediating their analgesic actions. We propose a novel mechanism by which α-conotoxins Vc1.1 and Rg1A modulate native N-type (Ca V 2.2) Ca 2+ channel currents, namely acting as agonists via G-protein-coupled GABA B receptors.

Ultra‐Stable Peptide Scaffolds for Protein Engineering—Synthesis and Folding of the Circular Cystine Knotted Cyclotide Cycloviolacin O2

Publisher: Wiley

Date: 19-12-2007

DOI: 10.1002/CBIC.200700357

Abstract: The cyclic cystine knot motif, as defined by the cyclotide peptide family, is an attractive scaffold for protein engineering. To date, however, the utilisation of this scaffold has been limited by the inability to synthesise members of the most erse and biologically active subfamily, the bracelet cyclotides. This study describes the synthesis and first direct oxidative folding of a bracelet cyclotide-cycloviolacin O2-and thus provides an efficient method for exploring the most potent cyclic cystine knot peptides. The linear chain of cycloviolacin O2 was assembled by solid-phase Fmoc peptide synthesis and cyclised by thioester-mediated native chemical ligation, and the inherent difficulties of folding bracelet cyclotides were successfully overcome in a single-step reaction. The folding pathway was characterised and was found to include predominating fully oxidised intermediates that slowly converted to the native peptide structure.

Design of Potent and Selective Cathepsin G Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold

Publisher: American Chemical Society (ACS)

Date: 18-01-2017

DOI: 10.1021/ACS.JMEDCHEM.6B01509

Abstract: Neutrophils are directly responsible for destroying invading pathogens via reactive oxygen species, antimicrobial peptides, and neutrophil serine proteases (NSPs). Imbalance between NSP activity and endogenous protease inhibitors is associated with chronic inflammatory disorders, and engineered inhibitors of NSPs are a potential therapeutic pathway. In this study we characterized the extended substrate specificity (P4-P1) of the NSP cathepsin G using a peptide substrate library. Substituting preferred cathepsin G substrate sequences into sunflower trypsin inhibitor-1 (SFTI-1) produced a potent cathepsin G inhibitor (K

The E15R point mutation in scorpion toxin Cn2 uncouples its depressant and excitatory activities on human NaV1. 6

Publisher: American Chemical Society (ACS)

Date: 03-02-2018

DOI: 10.1021/ACS.JMEDCHEM.7B01609

Abstract: We report the chemical synthesis of scorpion toxin Cn2, a potent and highly selective activator of the human voltage-gated sodium channel Na

New indole alkaloids from the roots of Ochrosia acuminata

Publisher: American Chemical Society (ACS)

Date: 19-08-2004

DOI: 10.1021/NP049812P

Abstract: Two new indole alkaloids, polyneuridine-N-oxide (1) and 17-hydroxy-10-methoxy-yohimbane (2), together with seven known alkaloids were isolated from the roots of Ochrosia acuminata collected in Savu, Indonesia. 9-Methoxyellipticine (3) and ellipticine (4) were responsible for the antitumor activities of the extract. The structures of all compounds were elucidated using MS and NMR methods.

Structural Refinement of Insecticidal Plant Proteinase Inhibitors from Nicotiana alata

Publisher: Bentham Science Publishers Ltd.

Date: 09-2008

DOI: 10.2174/092986608785849326

Abstract: Ornamental tobacco (Nicotiana alata) produces a series of 6 kDa proteinase inhibitors belonging to the potato type II inhibitor family. These proteins inhibit trypsin and chymotrypsin, the main digestive enzymes of predatory insects, thus leading to starvation, impaired larval development or death. In this context, the three-dimensional structures of these inhibitors are important for developing novel strategies for pest control. The solution structures of C1 and T1, the two main prototypes of the N. alata inhibitors, were originally determined more than a decade ago (J. Mol. Biol. 242, 231-243 (1994) and Biochemistry 34, 14304-14311 (1995)). Since then methods for NMR structure calculations have evolved considerably. Here we report the refinement of the structures of C1 and T1 with state-of-the-art protocols for NMR structure calculations. This refinement leads to an improved quality of the structures, making them a more reliable basis for the development of novel pesticides and modeling applications.

Single Amino Acid Substitution in α-Conotoxin TxID Reveals a Specific α3β4 Nicotinic Acetylcholine Receptor Antagonist

Publisher: American Chemical Society (ACS)

Date: 25-09-2018

DOI: 10.1021/ACS.JMEDCHEM.8B00967

Abstract: The α3β4 nicotinic acetylcholine receptor (nAChR) is an important target implicated in various disease states. α-Conotoxin TxID (1) is the most potent antagonist of α3β4 nAChR, but it also exhibits inhibition of α6/α3β4 nAChR. The results of alanine scanning of 1 suggested a vital role for Ser9 in the selectivity of the peptide. In this study, Ser9 was substituted with a series of 14 amino acids, including some non-natural amino acids, displaying different physicochemical characteristics to further improve the selectivity of 1 toward α3β4 nAChR. The pharmacological activities of the mutants were evaluated using an electrophysiological approach. The best selectivity was obtained with [S9K]TxID, 12, which inhibited α3β4 nAChR with an IC

Biochemical and biophysical characterization of a novel plant protein disulfide isomerase.

Publisher: Wiley

Date: 04-11-2009

DOI: 10.1002/BIP.21113

Abstract: We recently isolated a protein disulfide isomerase (PDI) from the Rubiaceae (coffee family) plant Oldenlandia affinis (OaPDI) and demonstrated that it facilitates the production of disulfide-knotted defense proteins called cyclotides. PDIs are major folding catalysts in the eukaryotic ER where they are responsible for formation, breakage, or shuffling of disulfide bonds in substrate polypeptides and are important chaperones in the secretory pathway. Here, we report the first detailed analysis of the oligomerization behavior of a plant PDI, based on characterization of OaPDI using various biochemical and biophysical techniques, including size-exclusion chromatography, NMR spectroscopy, surface plasmon resonance and atomic force microscopy. In solution at low concentration OaPDI comprises mainly monomers, but fractions of dimers and/or higher-order oligomers were observed at increased conditions, raising the possibility that dimerization and/or oligomerization could be a mechanism to adapt to the various-sized polypeptide substrates of PDI. Unlike mammalian PDIs, oligomerization of the plant PDI is not driven by the formation of intermolecular disulfide bonds, but by noncovalent interactions. The information derived in this study advances our understanding of the oligomerization behavior of OaPDI in particular but is potentially of broader interest for understanding the mechanism and role of oligomerization, and hence the catalytic and physiological mechanism, of the ubiquitous folding catalyst PDI.

Circular Permutation of the Native Enzyme-Mediated Cyclization Position in Cyclotides

Publisher: American Chemical Society (ACS)

Date: 23-03-2020

DOI: 10.1021/ACSCHEMBIO.9B00996

The acyclotide ribe 31 from Rinorea bengalensis has selective cytotoxicity and potent insecticidal properties in Drosophila

Publisher: Elsevier BV

Date: 10-2022

DOI: 10.1016/J.JBC.2022.102413

New Alkaloids from Pandanus amaryllifolius

Publisher: American Chemical Society (ACS)

Date: 24-12-2004

DOI: 10.1021/NP0303310

Abstract: Three new alkaloids, the two pyrrolidine type alkaloids (1 and 2) and 6E-pandanamine (3), together with five known alkaloids (4-8), were isolated from the leaves of Pandanus amaryllifolius collected in West Java, Indonesia. All the new alkaloids have two alpha-methyl alpha,beta-unsaturated gamma-lactone moieties, while compound 2 also has an additional seven-membered ring, which has not been encountered before in Pandanus alkaloids. Two different extraction methods, namely, a solvent partitioning extraction and acid-base treatment, were tested, giving secondary and tertiary amines, respectively. Spectroscopic and chemical studies showed that the tertiary amines isolated from the acid-base treatment were artifacts formed during the extraction process. This finding suggests that the use of conventional acid-base treatment in isolating Pandanus alkaloids should be reviewed since it can introduce artifacts.

Anti-HIV Cyclotides from the Chinese Medicinal Herb Viola yedoensis

Publisher: American Chemical Society (ACS)

Date: 15-12-2007

DOI: 10.1021/NP070393G

Targeted Delivery of Cyclotides via Conjugation to a Nanobody

Publisher: American Chemical Society (ACS)

Date: 24-09-2018

DOI: 10.1021/ACSCHEMBIO.8B00653

Abstract: Many naturally occurring peptides have poor proteolytic stability, which limits their therapeutic applications. Cyclotides are plant-derived cyclic peptides that resist proteolysis due to their highly constrained structure, comprising a head-to-tail cyclic backbone and three disulfide bonds that form a cystine-knotted core. This structure makes them useful as scaffolds onto which peptide sequences (epitopes) can be grafted. In this study, VHH7, an alpaca-derived nanobody that targets murine class II MHC molecules, was used for the targeted delivery of cyclotides to antigen-presenting cells (APCs). The cyclotides MCoTI-I, and MCoTI-I with a HA-tag (YPYDVPDYA) grafted into loop 6 (MCoTI-HA), were tested for immunogenic properties. To produce the requisite VHH7-peptide conjugates, a site-specific sortase A-catalyzed reaction in combination with a copper-free strain-promoted cycloaddition reaction was used. MCoTI-I alone did not display any obvious antibody response, thus showing the capacity of cyclotides as immunologically silent scaffolds. By contrast, MCoTI-I conjugated to VHH7 elicited antibodies against cyclic or linear MCoTI-I, thus suggesting a simple and robust approach for targeting cyclotides to APCs, and potentially to other cell types. A similar antibody response was observed when MCoTI-HA was conjugated to VHH7, but there was no reactivity toward a linear HA-tag itself, suggesting differences in conformational constraint between cyclotide-presented and linear epitopes. Studies of commercially available HA antibodies applied to MCoTI-HA confirmed that the conformation of peptide immunogens affects their reactivity. Thus, the production of antibodies that recognize constrained epitopes may benefit from engraftment onto scaffolds such as cyclotides. More broadly, this study validates that a prototypic cyclotide, a member of a peptide family that has proven to be useful as drug design scaffolds in many other studies, can efficiently reach a specific target in vivo.

Design and characterization of novel antimicrobial peptides, R-BP100 and RW-BP100, with activity against Gram-negative and Gram-positive bacteria

Publisher: Elsevier BV

Date: 03-2013

DOI: 10.1016/J.BBAMEM.2012.12.002

Abstract: BP100 is a short cationic antimicrobial peptide with a mechanism of action dependent on peptide-lipid interactions and microbial surface charge neutralization. Although active against Gram-negative bacteria, BP100 is inactive against Gram-positive bacteria. In this study we report two newly designed BP100 analogues, RW-BP100 and R-BP100 that have the Tyr residue replaced with a Trp and/or the Lys residues replaced with an Arg. The new analogues in addition to being active against Gram-negative bacteria, possess activity against all tested Gram-positive bacteria. Mechanistic studies using atomic force microscopy, surface plasmon resonance and fluorescence methodologies reveal that the antibacterial efficiency follows the affinity for bacterial membrane. The studies suggest that the activity of BP100 and its analogues against Gram-negative bacteria is mainly driven by electrostatic interactions with the lipopolysaccharide layer and is followed by binding to and disruption of the inner membrane, whereas activity against Gram-positive bacteria, in addition to electrostatic attraction to the exposed lipoteichoic acids, requires an ability to more deeply insert in the membrane environment, which is favoured with Arg residues and is facilitated in the presence of a Trp residue. Knowledge on the mechanism of action of these antimicrobial peptides provides information that assists in the design of antimicrobials with higher efficacy and broader spectra of action, but also on the design of peptides with higher specificity if required.

Thermal, Chemical, and Enzymatic Stability of the Cyclotide Kalata B1:  The Importance of the Cyclic Cystine Knot

Publisher: American Chemical Society (ACS)

Date: 05-2004

DOI: 10.1021/BI049711Q

Abstract: The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata B2, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC-MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5-Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.

A new family of cystine knot peptides from the seeds of Momordica cochinchinensis

Publisher: Elsevier BV

Date: 2013

DOI: 10.1016/J.PEPTIDES.2012.09.018

Abstract: Momordica cochinchinensis, a Cucurbitaceae plant commonly found in Southeast Asia, has the unusual property of containing both acyclic and backbone-cyclized trypsin inhibitors with inhibitor cystine knot (ICK) motifs. In the current study we have shown that M. cochinchinensis also contains another family of acyclic ICK peptides. We recently reported two novel peptides from M. cochinchinensis but have now discovered four additional peptides (MCo-3-MCo-6) with related sequences. Together these peptides form a novel family of M. cochinchinensis ICK peptides (MCo-ICK) that do not have sequence homology with other known peptides and are not potent trypsin inhibitors. Otherwise these new peptides MCo-3 to MCo-6 were evaluated for antimalarial activity against Plasmodium falciparum, and cytotoxic activity against the cancer cell line MDA-MB-231. But these peptides were not active.

Solution structure of μ-conotoxin PIIIA, a preferential inhibitor of persistent tetrodotoxin-sensitive sodium channels

Publisher: Elsevier BV

Date: 07-2002

DOI: 10.1074/JBC.M201611200

Conformation of a Peptide Corresponding to T4 Lysozyme Residues 59-81 by NMR and CD Spectroscopy

Publisher: American Chemical Society (ACS)

Date: 09-1994

DOI: 10.1021/BI00203A013

Abstract: The conformation, in solution, of a peptide corresponding to residues 59-81 from T4 lysozyme [LYS(59-81)] has been determined by 1H NMR and CD spectroscopy. This peptide spans the region corresponding to helix C in the crystal structure of T4 lysozyme. Secondary structure predictions indicated that the peptide would possibly be helical in an aqueous environment, but in a more hydrophobic environment the peptide would certainly adopt a helical conformation. This prediction was confirmed by the far-UV CD and NMR studies, which showed the peptide to be relatively unstructured in aqueous solution and significantly helical in the presence of either TFE or SDS micelles, although the 1H NMR results did give some indication of the presence of nascent helix in aqueous solution. For LYS(59-81), in TFE, the three-dimensional structure derived from the NMR data showed that the helix had a more pronounced curvature than the gradual bend observed in the crystal structure.

Small cyclic sodium channel inhibitors

Publisher: Elsevier BV

Date: 2021

DOI: 10.1016/J.BCP.2020.114291

Innentitelbild: Application and Structural Analysis of Triazole‐Bridged Disulfide Mimetics in Cyclic Peptides (Angew. Chem. 28/2020)

Publisher: Wiley

Date: 26-05-2020

DOI: 10.1002/ANGE.202006713

Development of Antiplasmodial Peptide–Drug Conjugates Using a Human Protein-Derived Cell-Penetrating Peptide with Selectivity for Infected Cells

Publisher: American Chemical Society (ACS)

Date: 26-05-2023

DOI: 10.1021/ACS.BIOCONJCHEM.3C00147

Conopressin-T from Conus tulipa reveals an antagonist switch in vasopressin-like peptides

Publisher: Elsevier BV

Date: 03-2008

DOI: 10.1074/JBC.M706477200

Lysine-scanning mutagenesis reveals an amendable face of the cyclotide kalata B1 for the optimization of nematocidal activity

Publisher: Elsevier BV

Date: 04-2010

DOI: 10.1074/JBC.M109.089854

Structure–Activity Studies of Cysteine‐Rich α‐Conotoxins that Inhibit High‐Voltage‐Activated Calcium Channels via GABA_B Receptor Activation Reveal a Minimal Functional Motif

Publisher: Wiley

Date: 07-03-2016

DOI: 10.1002/ANIE.201600297

Abstract: α-Conotoxins are disulfide-rich peptides that target nicotinic acetylcholine receptors. Recently we identified several α-conotoxins that also modulate voltage-gated calcium channels by acting as G protein-coupled GABA(B) receptor (GABA(B)R) agonists. These α-conotoxins are promising drug leads for the treatment of chronic pain. To elucidate the ersity of α-conotoxins that act through this mechanism, we synthesized and characterized a set of peptides with homology to α-conotoxins known to inhibit high voltage-activated calcium channels via GABA(B)R activation. Remarkably, all disulfide isomers of the active α-conotoxins Pu1.2 and Pn1.2, and the previously studied Vc1.1 showed similar levels of biological activity. Structure determination by NMR spectroscopy helped us identify a simplified biologically active eight residue peptide motif containing a single disulfide bond that is an excellent lead molecule for developing a new generation of analgesic peptide drugs.

Design of a Stable Cyclic Peptide Analgesic Derived from Sunflower Seeds that Targets the κ-Opioid Receptor for the Treatment of Chronic Abdominal Pain

Publisher: American Chemical Society (ACS)

Date: 24-06-2021

DOI: 10.1021/ACS.JMEDCHEM.1C00158

Inhibition of tau aggregation using a naturally-occurring cyclic peptide scaffold

Publisher: Elsevier BV

Date: 02-2016

DOI: 10.1016/J.EJMECH.2016.01.006

Abstract: Disulfide-rich macrocyclic peptides are emerging as versatile scaffolds for the development of stable biochemical tools. This potential is due to the combination of their structural stability and range of bioactivities. Here, we explored the activity of these peptides on fibril growth of the hexapeptide Ac-VQIVYK-NH2 (AcPHF6), which is a tau-derived peptide that has been widely used to understand the pathological mechanism of numerous tauopathies, including Alzheimer's disease. Of the cyclic peptides tested, SFTI-1 and kB1 showed an inherent ability to inhibit AcPHF6 fibril formation. Using an end-capping strategy and combining it with a molecular grafting approach, we demonstrated that SFTI-1 could be used as a starting point to design more potent fibril inhibitors. We further identified chemical and structural features of SFTI-1 and its analogues that underpin their inhibitory activity. The ability to inhibit fibril growth using the strategy employed herein supports the 'steric zipper' model of AcPHF6 fibril formation and shows that naturally-occurring cyclic peptides have potential as drug leads or molecular probes for understanding fibril formation.

Lysine-rich Cyclotides: A New Subclass of Circular Knotted Proteins from Violaceae

Publisher: American Chemical Society (ACS)

Date: 15-09-2015

DOI: 10.1021/ACSCHEMBIO.5B00454

Abstract: Cyclotides are macrocyclic proteins produced by plants for host defense. Although they occur sparsely in other plant families, cyclotides have been detected in every Violaceae plant species so far screened. Many of the Violaceae species examined until now have been from closely related geographical regions or habitats. To test the hypothesis that cyclotides are ubiquitous in this family, two geographically isolated (and critically endangered) species of Australasian Violaceae, namely Melicytus chathamicus and M. latifolius, were examined. Surprisingly, we discovered a suite of cyclotides possessing novel sequence features, including a lysine-rich nature, distinguishing them from "conventional" cyclotides and suggesting that they might have different physiological activities in plants to those reported to date. The newly discovered cyclotides were found to bind to lipid membranes and were cytotoxic against cancer cell lines but had low toxicity against red blood cells, which is advantageous for potential therapeutic applications. This suite of novel Lys-rich cyclotides emphasizes the broad ersity of cyclotides in Violaceae species.

Effects of arginine 10 to lysine substitution on ω-conotoxin CVIE and CVIF block of Cav2.2 channels

Publisher: Wiley

Date: 10-06-2014

DOI: 10.1111/BPH.12686

Effects of Chirality at Tyr13 on the Structure−Activity Relationships of ω-Conotoxins from Conus magus

Publisher: American Chemical Society (ACS)

Date: 05-1999

DOI: 10.1021/BI982980U

Structures of muO-conotoxins from Conus marmoreus. I nhibitors of tetrodotoxin (TTX)-sensitive and TTX-resistant sodium channels in mammalian sensory neurons.

Publisher: Elsevier BV

Date: 06-2004

DOI: 10.1074/JBC.M313002200

A bifunctional asparaginyl endopeptidase efficiently catalyzes both cleavage and cyclization of cyclic trypsin inhibitors.

Publisher: Springer Science and Business Media LLC

Date: 27-03-2020

DOI: 10.1038/S41467-020-15418-2

Abstract: Asparaginyl endopeptidases (AEPs) catalyze the key backbone cyclization step during the biosynthesis of plant-derived cyclic peptides. Here, we report the identification of two AEPs from Momordica cochinchinensis and biochemically characterize MCoAEP2 that catalyzes the maturation of trypsin inhibitor cyclotides. Recombinantly produced MCoAEP2 catalyzes the backbone cyclization of a linear cyclotide precursor (MCoTI-II-NAL) with a k cat /K m of 620 mM −1 s −1 , making it one of the fastest cyclases reported to date. We show that MCoAEP2 can mediate both the N-terminal excision and C-terminal cyclization of cyclotide precursors in vitro. The rate of cyclization/hydrolysis is primarily influenced by varying pH, which could potentially control the succession of AEP-mediated processing events in vivo. Furthermore, MCoAEP2 efficiently catalyzes the backbone cyclization of an engineered MCoTI-II analog with anti-angiogenic activity. MCoAEP2 provides enhanced synthetic access to structures previously inaccessible by direct chemistry approaches and enables the wider application of trypsin inhibitor cyclotides in biotechnology applications.

Improving on Nature: Making a Cyclic Heptapeptide Orally Bioavailable

Publisher: Wiley

Date: 12-09-2014

DOI: 10.1002/ANIE.201405364

Abstract: The use of peptides in medicine is limited by low membrane permeability, metabolic instability, high clearance, and negligible oral bioavailability. The prediction of oral bioavailability of drugs relies on physicochemical properties that favor passive permeability and oxidative metabolic stability, but these may not be useful for peptides. Here we investigate effects of heterocyclic constraints, intramolecular hydrogen bonds, and side chains on the oral bioavailability of cyclic heptapeptides. NMR-derived structures, amide H-D exchange rates, and temperature-dependent chemical shifts showed that the combination of rigidification, stronger hydrogen bonds, and solvent shielding by branched side chains enhances the oral bioavailability of cyclic heptapeptides in rats without the need for N-methylation.

Mirror Images of Antimicrobial Peptides Provide Reflections on Their Functions and Amyloidogenic Properties

Publisher: American Chemical Society (ACS)

Date: 26-04-2016

DOI: 10.1021/JACS.6B02575

Abstract: Enantiomeric forms of BTD-2, PG-1, and PM-1 were synthesized to delineate the structure and function of these β-sheet antimicrobial peptides. Activity and lipid-binding assays confirm that these peptides act via a receptor-independent mechanism involving membrane interaction. The racemic crystal structure of BTD-2 solved at 1.45 Å revealed a novel oligomeric form of β-sheet antimicrobial peptides within the unit cell: an antiparallel trimer, which we suggest might be related to its membrane-active form. The BTD-2 oligomer extends into a larger supramolecular state that spans the crystal lattice, featuring a steric-zipper motif that is common in structures of amyloid-forming peptides. The supramolecular structure of BTD-2 thus represents a new mode of fibril-like assembly not previously observed for antimicrobial peptides, providing structural evidence linking antimicrobial and amyloid peptides.

Cyclic alpha-conotoxin peptidomimetic chimeras as potent GLP-1R agonists

Publisher: Elsevier BV

Date: 10-2015

DOI: 10.1016/J.EJMECH.2015.08.046

Abstract: Type 2 diabetes mellitus (T2DM) results from compromised pancreatic β-cell function, reduced insulin production, and lowered insulin sensitivity in target organs resulting in hyperglycemia. The GLP-1 hormone has two biologically active forms, GLP-1-(7-37) and GLP-1-(7-36)amide, which are equipotent at the glucagon-like peptide-1 receptor (GLP-1R). These peptides are central both to normal glucose metabolism and dysregulation in T2DM. Several structurally modified GLP-1 analogues are now approved drugs, and a number of other analogues are in clinical trials. None of these compounds is orally bioavailable and all require parenteral delivery. Recently, a number of smaller peptidomimetics containing 11-12 natural and unnatural amino acids have been identified that have similar insulin regulating profiles as GLP-1. The α-conotoxins are a class of disulfide rich peptide venoms isolated from cone snails, and are known for their highly constrained structures and resistance to enzymatic degradation. In this study, we examined whether 11-residue peptidomimetics incorporated into α-conotoxin scaffolds, forming monocyclic or bicyclic compounds constrained by disulfide bonds and/or backbone cyclization, could activate the GLP-1 receptor (GLP-1R). Several compounds showed potent (nanomolar) agonist activity at GLP-1R, as evaluated via cAMP signaling. In addition, HPLC retention times and in silico calculations suggested that mono- and bicyclic compounds had more favorable n-octanol/water partition coefficients according to the virtual partition coefficient model (vLogP), while maintaining a smaller radius of gyration compared to corresponding uncyclized peptidomimetics. Our findings suggest that cyclic peptidomimetics provide a potential avenue for future design of potent, compact ligands targeting GLP-1R and possessing improved physicochemical properties.