ORCID Profile
0000-0002-9961-0006
Current Organisations
Friedrich-Alexander-Universität Erlangen-Nürnberg
,
University of Melbourne
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Medicinal and Biomolecular Chemistry | Proteins and Peptides | Medical Biochemistry: Proteins and Peptides (incl. Medical Proteomics) | Industrial Chemistry | Synthesis of Materials | Medicinal and Biomolecular Chemistry not elsewhere classified | Medical Microbiology not elsewhere classified | Biocatalysis and Enzyme Technology |
ANZSRC Socio-Economic Objective (SEO)
Human Pharmaceutical Treatments (e.g. Antibiotics) | Human Pharmaceutical Products not elsewhere classified | Diabetes | Digestive System Disorders | Expanding Knowledge in the Biological Sciences | Environmentally Sustainable Manufacturing not elsewhere classified | Expanding Knowledge in the Medical and Health Sciences | Health Protection and/or Disaster Response
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Publications
Simultaneous Post-cysteine(S-Acm) Group Removal Quenching of Iodine and Isolation of Peptide by One Step Ether Precipitation
Publisher: Springer Science and Business Media LLC
Date: 16-09-2008
Novel peptides bearing pyrene and coumarin units with or without β-cyclodextrin in their side chains exhibit intramolecular fluorescence resonance energy transfer
Publisher: American Chemical Society (ACS)
Date: 22-08-2003
DOI: 10.1021/JA036427Y
Abstract: Novel peptides bearing the pyrene/coumarin FRET pair in their side chains have been designed and synthesized. Peptide 1 having endogenous beta-cyclodextrin (beta-CD) in the side chain exhibits FRET in aqueous solution, indicating that coumarin, being accommodated into the CD cavity, is separated from pyrene. Guest-induced quenching of the fluorophores in 1 indicates that coumarin, being excluded from the CD cavity, comes into close contact with pyrene. Peptide 2 shows FRET only after addition of external beta-CD that again reflects the idea that beta-CD surely caps the coumarin unit in its hydrophobic cavity, and, therefore, quenching of the fluorophores can be prevented in FRET peptide probes. With this strategy, various peptide-based FRET probes can be developed that would be useful for studying biological phenomena in living cells.
Direct Measurement of Pore Dynamics and Leakage Induced by a Model Antimicrobial Peptide in Single Vesicles and Cells
Publisher: American Chemical Society (ACS)
Date: 17-06-2016
DOI: 10.1021/ACS.LANGMUIR.6B00596
Abstract: Antimicrobial peptides are promising therapeutic alternatives to counter growing antimicrobial resistance. Their precise mechanism of action remains elusive, however, particularly with respect to live bacterial cells. We investigated the interaction of a fluorescent melittin analogue with single giant unilamellar vesicles, giant multilamellar vesicles, and bilamellar Gram-negative Escherichia coli (E. coli) bacteria. Time-lapse fluorescence lifetime imaging microscopy was employed to determine the population distribution of the fluorescent melittin analogue between pore state and membrane surface state, and simultaneously measure the leakage of entrapped fluorescent species from the vesicle (or bacterium) interior. In giant unilamellar vesicles, leakage from vesicle interior was correlated with an increase in level of pore states, consistent with a stable pore formation mechanism. In giant multilamellar vesicles, vesicle leakage occurred more gradually and did not appear to correlate with increased pore states. Instead pore levels remained at a low steady-state level, which is more in line with coupled equilibria. Finally, in single bacterial cells, significant increases in pore levels were observed over time, which were correlated with only partial loss of cytosolic contents. These observations suggested that pore formation, as opposed to complete dissolution of membrane, was responsible for the leakage of contents in these systems, and that the bacterial membrane has an adaptive capacity that resists peptide attack. We interpret the three distinct pore dynamics regimes in the context of the increasing physical and biological complexity of the membranes.
Fluorescence resonance energy transfer in a novel cyclodextrin–Peptide conjugate for detecting steroid molecules
Publisher: Elsevier BV
Date: 12-2003
DOI: 10.1016/J.BMCL.2003.09.051
Abstract: A novel cyclodextrin conjugated peptide, 1, having two different fluorophores, coumarin and pyrene, in the side chains has been designed and synthesized. The circular dichroism study reveals that 1 shows typical alpha-helix pattern, and forms intramolecular inclusion complex with coumarin. The fluorescence emission study shows that the peptide exhibits intramolecular fluorescence resonance energy transfer (FRET) without quenching of two fluorophores. We have determined the binding constants of 1 for various biologically important steroid molecules as guests using the guest-responsive variation in the fluorescence emission intensity of coumarin.
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.
The effect of selective D- or na-methyl arginine substitution on the activity of the proline-rich antimicrobial peptide, Chex1-Arg20
Publisher: Frontiers Media SA
Date: 19-01-2017
Structure and function relationship of murine insulin-like peptide 5 (INSL5): Free C-terminus is essential for RXFP4 receptor binding and activation
Publisher: American Chemical Society (ACS)
Date: 08-09-2011
DOI: 10.1021/BI201093M
Abstract: Insulin-like peptide 5 (INSL5) is a member of insulin/relaxin superfamily of peptides. It has recently been identified as the cognate ligand for the G-protein-coupled receptor, RXFP4. Although the complete physiological role of this naturally occurring peptide is still under investigation, there is evidence that it acts to both stimulate appetite and activate colon motility. This suggests that both agonists and antagonists of the peptide may have potential therapeutic applications. To further investigate the physiological role of this peptide and because of the ready availability of the mouse as an experimental animal, the preparation of mouse INSL5 was undertaken. Because of its complex structure and the intractable nature of the two constituent chains, different solid phase synthesis strategies were investigated, including the use of a temporary B-chain solubilizing tag. Unfortunately, none provided significantly improved yield of purified mouse INSL5 which reflects the complexity of this peptide. In addition to the native peptide, two mouse INSL5 analogues were also prepared. One had its two chains as C-terminal amides, and the other contained a europium chelate monolabel for use in RXFP4 receptor assays. It was found that the INSL5 amide was substantially less potent than the native acid form. A similar observation was made for the human peptide acid and amide, highlighting the necessity for free C-terminal carboxylates for function. Two additional human INSL5 analogues were prepared to further investigate the necessity of a free C-terminal. The results together provide a first insight into the mechanism whereby INSL5 binds to and activates RXFP4.
Association and guest-induced dissociation of a novel alpha-helix peptide bearing pyrene and gamma-cyclodextrin in the side chains
Publisher: The Chemical Society of Japan
Date: 03-2000
DOI: 10.1246/CL.2000.252
The Chemical Synthesis of Insulin: An Enduring Challenge
Publisher: American Chemical Society (ACS)
Date: 09-03-2021
Exploring the Use of Helicogenic Amino Acids for Optimising Single Chain Relaxin-3 Peptide Agonists
Publisher: MDPI AG
Date: 14-10-2020
DOI: 10.3390/BIOMEDICINES8100415
Abstract: Relaxin-3 is a highly conserved two-chain neuropeptide that acts through its endogenous receptor the Relaxin Family Peptide-3 (RXFP3) receptor. The ligand/receptor system is known to modulate several physiological processes, with changes in food intake and anxiety-levels the most well studied in rodent models. Agonist and antagonist analogues based on the native two-chain peptide are costly to synthesise and not ideal drug leads. Since RXFP3 interacting residues are found in the relaxin B-chain only, this has been the focus of analogue development. The B-chain is unstructured without the A-chain support, but in single-chain variants structure can be induced by dicarba-based helical stapling strategies. Here we investigated whether alternative helical inducing strategies also can enhance structure and activity at RXFP3. Combinations of the helix inducing α-aminoisobutyric acid (Aib) were incorporated into the sequence of the relaxin-3 B-chain. Aib residues at positions 13, 17 and 18 partially reintroduce helicity and activity of the relaxin-3 B-chain, but other positions are generally not suited for modifications. We identify Thr21 as a putative new receptor contact residue important for RXFP3 binding. Cysteine residues were also incorporated into the sequence and cross-linked with dichloroacetone or α, α’-dibromo-m-xylene. However, in contrast to previously reported dicarba variants, neither were found to promote structure and RXFP3 activity.
Slow Insertion Kinetics during Interaction of a Model Antimicrobial Peptide with Unilamellar Phospholipid Vesicles
Publisher: American Chemical Society (ACS)
Date: 22-12-2011
DOI: 10.1021/LA203770J
Abstract: The mechanism of interaction between a model antimicrobial peptide and phospholipid unilamellar vesicle membranes was studied using fluorescence spectroscopy, fluorescence lifetime measurements, and light scattering. The peptide, a mellitin mutant, was labeled at position K14 with the polarity-sensitive probe AlexaFluor 430. The kinetics of the interaction of this derivative with various concentrations of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) vesicles was examined. Our work unveiled two novel aspects of peptide-lipid interactions. First, the AB plot or phasor analysis of the fluorescence lifetime studies revealed at least three different peptide states, the population of which depended on the lipid to peptide (L:P) concentration ratio. Second, complex fluorescence kinetics were observed over extended time-scales from 30 s to 2 h. The extended kinetics was only observed at particular lipid concentrations (L:P ratios 20:1 and 10:1) and not at others (30, 40, 50 and 100:1 L:P ratio). Analysis of the complex kinetics revealed several intermediates. We assign these to distinct states of the peptide formed during helix insertion into the vesicle membrane that are intermediate to lytic pore formation.
Phosphorylation of a full length amyloid-β peptide modulates its amyloid aggregation, cell binding and neurotoxic properties
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C7MB00249A
Abstract: Phosphorylation of Aβ42 promotes the formation of amyloid plaques in the brain, which lack the neurotoxic properties associated with oligomeric species causing pathogenesis in Alzheimer's disease.
H3 Relaxin Demonstrates Antifibrotic Properties via the RXFP1 Receptor
Publisher: American Chemical Society (ACS)
Date: 31-01-2011
DOI: 10.1021/BI1013968
Abstract: Human gene 3 (H3) relaxin is the most recently discovered member of the relaxin peptide family and can potentially bind all of the defined relaxin family peptide receptors (RXFP1-4). While its effects as a neuromodulator are being increasingly studied through its primary receptor, RXFP3, its actions via other RXFPs are poorly understood. Hence, we specifically determined the antifibrotic effects and mechanisms of action of H3 relaxin via the RXFP1 receptor using primary rat ventricular fibroblasts in vitro, which naturally express RXFP1, but not RXFP3, and a mouse model of fibrotic cardiomyopathy in vivo. Transforming growth factor β1 (TGF-β1) administration to ventricular fibroblasts significantly increased Smad2 phosphorylation, myofibroblast differentiation, and collagen deposition (all p < 0.05 vs untreated controls), while having no marked effect on matrix metalloproteinase (MMP) 9, MMP-13, tissue inhibitor of metalloproteinase (TIMP) 1, or TIMP-2 expression over 72 h. H3 relaxin (at 100 and 250 ng/mL) almost completely abrogated the TGF-β1-stimulated collagen deposition over 72 h, and its effects at 100 ng/mL were equivalent to that of the same dose of H2 relaxin. Furthermore, H3 relaxin (100 ng/mL) significantly inhibited TGF-β1-stimulated cardiac myofibroblast differentiation and TIMP-1 and TIMP-2 expression to an equivalent extent as H2 relaxin (100 ng/mL), while also inhibiting Smad2 phosphorylation to approximately half the extent of H2 relaxin (all p < 0.05 vs TGF-β1). Lower doses of H3 (50 ng/mL) and H2 (50 ng/mL) relaxin additively inhibited TGF-β1-stimulated collagen deposition in vitro, while H3 relaxin was also found to reverse left ventricular collagen overexpression in the model of fibrotic cardiomyopathy in vivo. These combined findings demonstrate that H3 relaxin exerts antifibrotic actions via RXFP1 and may enhance the collagen-inhibitory effects of H2 relaxin.
Enhancing proline-rich antimicrobial peptide action by homodimerization: influence of bifunctional linker
Publisher: Royal Society of Chemistry (RSC)
Date: 2022
DOI: 10.1039/D1SC05662J
Abstract: Homodimerization of a proline-rich antimicrobial peptide via bioconjugation to perfluoroaromatic linkers confers increased antimicrobial, antibiofilm and immunomodulatory activity. The dimers are promising new therapeutic leads against WHO priority multidrug resistant bacteria.
Synthetic relaxins
Publisher: Elsevier BV
Date: 10-2014
DOI: 10.1016/J.CBPA.2014.09.014
Abstract: The relaxin subfamily of peptides within the human insulin superfamily consists of seven members including relaxin-2 and relaxin-3. The former is a pleiotropic hormone that is a vasodilator and cardiac stimulant in the cardiovascular system and an antifibrotic agent whereas the latter is primarily a neuropeptide involved in stress and metabolic control. Both possess the unique three-disulfide heterodimeric peptide structure of insulin. Consequently, the synthesis, both chemical and biological, of relaxin-2 and relaxin-3 has long represented a special challenge to further understanding their structural and functional relationships. This review highlights past and recent developments in the use of chemical and recombinant DNA methods of synthesis of these peptides and current resulting knowledge of their biology.
Rapid Photolysis‐Mediated Folding of Disulfide‐Rich Peptides
Publisher: Wiley
Date: 22-05-2019
Abstract: Structure-activity relationship studies are a highly time-consuming aspect of peptide-based drug development, particularly in the assembly of disulfide-rich peptides, which often requires multiple synthetic steps and purifications. Therefore, it is vital to develop rapid and efficient chemical methods to readily access the desired peptides. We have developed a photolysis-mediated "one-pot" strategy for regioselective disulfide bond formation. The new pairing system utilises two ortho-nitroveratryl protected cysteines to generate two disulfide bridges in less than one hour in good yield. This strategy was applied to the synthesis of complex disulfide-rich peptides such as Rho-conotoxin ρ-TIA and native human insulin.
Solid phase synthesis and structural analysis of novel A-chain dicarba analogs of human relaxin-3 (INSL7) that exhibit full biological activity
Publisher: Royal Society of Chemistry (RSC)
Date: 2009
DOI: 10.1039/B821882J
Abstract: Replacement of disulfide bonds with non-reducible isosteres can be a useful means of increasing the in vivo stability of a protein. We describe the replacement of the A-chain intramolecular disulfide bond of human relaxin-3 (H3 relaxin, INSL7), an insulin-like peptide that has potential applications in the treatment of stress and obesity, with the physiologically stable dicarba bond. Solid phase peptide synthesis was used to prepare an A-chain analogue in which the two cysteine residues that form the intramolecular bond were replaced with allylglycine. On-resin microwave-mediated ring closing metathesis was then employed to generate the dicarba bridge. Subsequent cleavage of the peptide from the solid support, purification of two isomers and their combination with the B-chain via two intermolecular disulfide bonds, then furnished two isomers of dicarba-H3 relaxin. These were characterized by CD spectroscopy, which suggested a structural similarity to the native peptide. Additional analysis by solution NMR spectroscopy also identified the likely cis/trans form of the analogs. Both peptides demonstrated binding affinities that were equivalent to native H3 relaxin on RXFP1 and RXFP3 expressing cells. However, although the cAMP activity of the analogs on RXFP3 expressing cells was similar to the native peptide, the potency on RXFP1 expressing cells was slightly lower. The data confirmed the use of a dicarba bond as a useful isosteric replacement of the disulfide bond.
C-terminus of the B-chain of relaxin-3 is important for receptor activity
Publisher: Public Library of Science (PLoS)
Date: 11-12-2013
Solid Phase Synthesis of an Analogue of Insulin, A0:R glargine, That Exhibits Decreased Mitogenic Activity
Publisher: Springer Science and Business Media LLC
Date: 09-07-2010
Chemical synthesis and orexigenic activity of rat/mouse relaxin-3
Publisher: Springer Science and Business Media LLC
Date: 03-2013
DOI: 10.1007/S00726-013-1478-0
Abstract: The insulin-like peptide, relaxin-3 was first identified just a decade ago via a genomic database search and is now recognized to be a key neuropeptide with several roles including the regulation of arousal, stress responses and neuroendocrine homeostasis. It also has significant potential as a drug to treat stress and obesity. Its actions are mediated via its cognate G protein-coupled receptor, RXFP3, which is found in abundant numbers in the brain. However, much remains to be determined with respect to the mechanism of neurological action of this peptide. Consequently, the chemical synthesis of the rat and mouse (which share identical primary structures) two-chain, three disulfide peptide was undertaken and the resulting peptide subjected to detailed in vitro and in vivo assay. Use of efficient solid-phase synthesis methods provided the two regioselectively S-protected A- and B-chains which were readily combined via sequential disulfide bond formation. The synthetic rat/mouse relaxin-3 was obtained in high purity and good overall yield. It demonstrated potent orexigenic activity in rats in that central intracerebroventricular infusion led to significantly increased food intake and water drinking.
Structure of the R3/I5 chimeric relaxin peptide, a selective GPCR135 and GPCR142 agonist
Publisher: Elsevier BV
Date: 08-2008
Antiviral Peptides as Promising Therapeutics against SARS-CoV-2
Publisher: American Chemical Society (ACS)
Date: 23-10-2020
Effects of C-Terminal B-Chain Modifications in a Relaxin 3 Agonist Analogue.
Publisher: American Chemical Society (ACS)
Date: 22-10-2020
Signal transduction pathways activated by insulin-like peptide 5 at the relaxin family peptide RXFP4 receptor.
Publisher: Wiley
Date: 13-07-2016
DOI: 10.1111/BPH.13522
The structural and functional role of the B-chain C-terminal arginine in the relaxin-3 peptide antagonist, R3(BΔ23-27)R/I5
Publisher: Wiley
Date: 18-12-2009
DOI: 10.1111/J.1747-0285.2008.00756.X
Abstract: Relaxin-3, a member of the insulin superfamily, is involved in regulating stress and feeding behavior. It is highly expressed in the brain and is the endogenous ligand for the receptor RXFP3. As relaxin-3 also interacts with the relaxin receptor RXFP1, selective agonists and antagonists are crucial for studying the physiological function(s) of the relaxin-3/RXFP3 pair. The analog R3(BDelta23-27)R/I5, in which a C-terminally truncated human relaxin-3 (H3) B-chain is combined with the INSL5 A-chain, is a potent selective RXFP3 antagonist and has an Arg residue remaining on the B-chain C-terminus as a consequence of the recombinant protein production process. To investigate the role of this residue in the RXFP3 receptor binding and activation, the analogs R3(BDelta23-27)R/I5 and R3(BDelta23-27)R containing the B-chain C-terminal Arg as well as R3(BDelta23-27)/I5 and R3(BDelta23-27), both lacking the Arg, were chemically assembled and their secondary structure and receptor activity assessed. The peptides generally had a similar conformation but those with the extra Arg residue displayed a significantly increased affinity for the RXFP3. Interestingly, in contrast to R3(BDelta23-27)R and R3(BDelta23-27)R/I5, the peptide R3(BDelta23-27) is a weak agonist. This suggests that the C-terminal Arg, although increasing the affinity, alters the manner in which the peptide binds to the receptor and thereby prevents activation, giving R3(BDelta23-27)R/I5 its potent antagonistic activity.
B7-33 replicates the vasoprotective functions of human relaxin-2 (serelaxin).
Publisher: Elsevier BV
Date: 07-2017
DOI: 10.1016/J.EJPHAR.2017.05.005
Abstract: Recombinant H2 relaxin (serelaxin) has gained considerable attention as a new vasoprotective drug, largely due to its potential therapeutic effects in heart failure and fibrosis. However, serelaxin is laborious and costly to produce. A single-chain peptidomimetic, B7-33, has been developed to overcome these problems but little is known about its biological actions in the vascular system. This study first compared the rapid vascular effects of an acute bolus injection of B7-33 compared with serelaxin. Male Wistar rats received a tail vein injection of placebo (20mM sodium acetate), B7-33 (13.3μg/kg) or serelaxin (26.6μg/kg). Three hours later vascular function in the mesenteric artery, small renal artery and abdominal aorta was assessed by wire myography. B7-33 and serelaxin selectively enhanced bradykinin-mediated endothelium-dependent relaxation in the rat mesenteric artery by increasing endothelium-derived hyperpolarization, but had no overall effects on relaxation in the small renal artery or aorta. We then compared the actions of B7-33 and serelaxin in an ex vivo model of vascular disease using virgin female mouse mesenteric arteries pre-incubated in placental trophoblast conditioned media to induce endothelial dysfunction characteristic of preecl sia. Co-incubation of these arteries in trophoblast conditioned media with B7-33 or serelaxin (15, 30nM) prevented the development of endothelial dysfunction. In conclusion, equimolar doses of B7-33 replicated the acute beneficial vascular effects of serelaxin in rat mesenteric arteries and also prevented endothelial dysfunction induced by placental trophoblast conditioned media in mouse mesenteric arteries. Therefore, B7-33 should be considered as a cost-effective vasoactive therapeutic in cardiovascular diseases, including preecl sia.
Inorganic-organic based novel nano-conjugate material for effective cobalt(II) ions capturing from wastewater
Publisher: Elsevier BV
Date: 09-2017
ML290 is a biased allosteric agonist at the relaxin receptor RXFP1.
Publisher: Springer Science and Business Media LLC
Date: 07-06-2017
DOI: 10.1038/S41598-017-02916-5
Abstract: Activation of the relaxin receptor RXFP1 has been associated with improved survival in acute heart failure. ML290 is a small molecule RXFP1 agonist with simple structure, long half-life and high stability. Here we demonstrate that ML290 is a biased agonist in human cells expressing RXFP1 with long-term beneficial actions on markers of fibrosis in human cardiac fibroblasts (HCFs). ML290 did not directly compete with orthosteric relaxin binding and did not affect binding kinetics, but did increase binding to RXFP1. In HEK-RXFP1 cells, ML290 stimulated cAMP accumulation and p38MAPK phosphorylation but not cGMP accumulation or ERK1/2 phosphorylation although prior addition of ML290 increased p-ERK1/2 responses to relaxin. In human primary vascular endothelial and smooth muscle cells that endogenously express RXFP1, ML290 increased both cAMP and cGMP accumulation but not p-ERK1/2. In HCFs, ML290 increased cGMP accumulation but did not affect p-ERK1/2 and given chronically activated MMP-2 expression and inhibited TGF-β1-induced Smad2 and Smad3 phosphorylation. In vascular cells, ML290 was 10x more potent for cGMP accumulation and p-p38MAPK than for cAMP accumulation. ML290 caused strong coupling of RXFP1 to Gα s and Gα oB but weak coupling to Gα i3 . ML290 exhibited signalling bias at RXFP1 possessing a signalling profile indicative of vasodilator and anti-fibrotic properties.
Design and development of analogues of dimers of insulin-like peptide 3 B-chain as high-affinity antagonists of the RXFP2 receptor.
Publisher: Wiley
Date: 2011
DOI: 10.1002/BIP.21484
Abstract: Insulin-like peptide 3 (INSL3) is one of 10 members of the human relaxin-insulin superfamily of peptides. It is a peptide hormone that is expressed by fetal and postnatal testicular Leydig cells and postnatal ovarian thecal cells. It mediates testicular descent during fetal life and suppresses sperm apoptosis in adult males, whereas, in females, it causes oocyte maturation. INSL3 has also been shown to promote thyroid tumor growth and angiogenesis in human. These actions of INSL3 are mediated through its G protein-coupled receptor, RXFP2. INSL3, a two-chained peptide, binds to its receptor primarily via its B-chain, whereas elements of the A-chain are essential for receptor activation. In an attempt to design a high-affinity antagonist with potential clinical application as an anticancer agent as well as a contraceptive, we have previously prepared a synthetic parallel dimer of INSL3 B-chain and demonstrated that it binds to RXFP2 with high affinity. In this work, we undertook full pharmacological characterization of this peptide and show that it can antaogonize INSL3-mediated cAMP signaling through RXFP2. Further refinement by truncation of 18 residues yielded a minimized analogue that retained full binding affinity and INSL3 antagonism. It is an attractive lead peptide for in vivo evaluation as an inhibitor of male and female fertility and of INSL3-mediated carcinogenesis.
Modulation of hippocampal theta oscillations and spatial memory by relaxin-3 neurons of the nucleus incertus
Publisher: Cold Spring Harbor Laboratory
Date: 30-10-2009
DOI: 10.1101/LM.1438109
Abstract: Hippoc al theta rhythm is thought to underlie learning and memory, and it is well established that “pacemaker” neurons in medial septum (MS) modulate theta activity. Recent studies in the rat demonstrated that brainstem-generated theta rhythm occurs through a multisynaptic pathway via the nucleus incertus (NI), which is the primary source of the neuropeptide relaxin-3 (RLN3). Therefore, this study examined the possible contribution of RLN3 to MS activity, and associated hippoc al theta activity and spatial memory. In anesthetized and conscious rats, we identified the ability of intraseptal RLN3 signaling to modulate neuronal activity in the MS and hippoc us and promote hippoc al theta rhythm. Behavioral studies in a spontaneous alternation task indicated that endogenous RLN3 signaling within MS promoted spatial memory and exploratory activity significantly increased c-Fos immunoreactivity in RLN3-producing NI neurons. Anatomical studies demonstrated axons/terminals from NI/RLN3 neurons make close contact with septal GABAergic (and cholinergic) neurons, including those that project to the hippoc us. In summary, RLN3 neurons of the NI can modulate spatial memory and underlying hippoc al theta activity through axonal projections to pacemaker neurons of the MS. NI/RLN3 neurons are highly responsive to stress and express corticotropin-releasing factor type-1 receptors, suggesting that the effects observed could be an important component of memory processing associated with stress responses.
De novo design and synthesis of cyclic and linear peptides to mimic the binding cassette of human relaxin
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2009.03840.X
Abstract: A synthetic cyclic regioselectively addressable functionalized template was used to prepare six analogs that presented the side chains of the key receptor-binding residues of each of H2 and H3 relaxins and insulin-like peptide 3. None showed any binding affinity for RXFP1, RXFP2, or RXFP3, indicating that the key residues were either incorrectly oriented or that additional residues are required for receptor binding.
Colokinetic effect of an insulin‐like peptide 5‐related agonist of the RXFP4 receptor
Publisher: Wiley
Date: 27-01-2020
DOI: 10.1111/NMO.13796
Abstract: Insulin-like peptide 5 (INSL5) is a hormone stored in colonic enteroendocrine cells that also contain the unrelated hormones, GLP-1 and PYY. It acts at the relaxin family peptide 4, RXFP4, receptor. RXFP4 is expressed by enteric neurons in the colon, and it has been speculated that INSL5, through its action on enteric neurons, might be involved in the control of colonic contractions. Similar to insulin and relaxin, INSL5 consists of A and B peptide chains linked by three disulfide bonds, two between the chains and one intrinsic to the A chain. Because of its complex structure, it is difficult to synthesize and to prepare peptide analogues to investigate its roles. We have recently developed a potent simplified peptide analogue, INSL5-A13 (INSL5 analogue 13). In the present work, we have investigated the actions of INSL5-A13 in mice. We investigated the ability of INSL5-A13 to increase the speed of emptying of a bead from the colon, after expulsion had been slowed by the peripherally restricted opioid agonist, loperamide (1 mg/kg). INSL5-A13 was a full agonist at the mouse RXFP4 expressed in HEK cells, with an EC50 of ~9 nmol/L. INSL5-A13 caused an acceleration of colorectal bead propulsion in mice constipated by loperamide in the dose range 0.2 to 60 µg/kg, with an EC50 of ~6 µg/kg in vivo. It also accelerated bead propulsion in untreated mice. Bead expulsion was not accelerated in RXFP4-/- mice. Our data suggest that RXFP4 agonists could be useful in the treatment of constipation.
Effect of dimerized melittin on gastric cancer cells and antibacterial activity
Publisher: Springer Science and Business Media LLC
Date: 16-05-2018
DOI: 10.1007/S00726-018-2587-6
Abstract: Melittin is the peptide toxin found in bee venom and is effective against cancer cells. To enhance its activity, a branched dimeric form of melittin was designed. The monomeric form of the peptide was more cytotoxic against gastric cancer cells at low concentrations (1-5 μM) than the dimer form, while the cytotoxic effect was comparable at higher concentrations (10 μM). Confocal microscopy showed that both the monomer and dimer forms of melittin with fluorescent label at the C terminus penetrated the cytoplasm and localized at the cell nucleus and disrupted the cell membrane. The results indicated that both peptides localized in the nucleus and no significant difference in penetration was observed between monomer and dimer of melittin. Although the C and N termini are important for melittin activity, using C terminus for dimerization of the peptide resulted in similar activity for the monomer and dimer against bacteria and gastric cancer cells.
Total Solid-Phase Synthesis of Biologically Active Drosophila Insulin-Like Peptide 2 (DILP2)
Publisher: CSIRO Publishing
Date: 2017
DOI: 10.1071/CH16626
Abstract: In the fruit fly Drosophila melanogaster, there are eight insulin-like peptides (DILPs) with DILPs 1–7 interacting with a sole insulin-like receptor tyrosine kinase (DInR) while DILP8 interacts with a single G protein-coupled receptor (GPCR), Lgr3. Loss-of-function dilp mutation studies show that the neuropeptide DILP2 has a key role in carbohydrate and lipid metabolism as well as longevity and reproduction. A better understanding of the processes whereby DILP2 mediates its specific actions is required. Consequently we undertook to prepare DILP2 as part of a larger, detailed structure–function relationship study. Use of our well established insulin-like peptide synthesis protocol that entails separate solid-phase assembly of each of the A- and B-chains with selective cysteine S-protection followed by sequential S-deprotection and simultaneous disulfide bond formation produced DILP2 in good overall yield and high purity. The synthetic DILP2 was shown to induce significant DInR phosphorylation and downstream signalling, with it being more potent than human insulin. This peptide will be a valuable tool to provide further insights into its binding to the insulin receptor, the subsequent cell signalling, and role in insect metabolism.
Engineering of chimeric peptides as antagonists for the G protein-coupled receptor, RXFP4
Publisher: Springer Science and Business Media LLC
Date: 28-11-2019
DOI: 10.1038/S41598-019-53707-Z
Abstract: Insulin-like peptide 5 (INSL5) is a very important pharma target for treating human conditions such as anorexia and diabetes. However, INSL5 with two chains and three disulfide bridges is an extremely difficult peptide to assemble by chemical or recombinant means. In a recent study, we were able to engineer a simplified INSL5 analogue 13 which is a relaxin family peptide receptor 4 (RXFP4)-specific agonist. To date, however, no RXFP4-specific antagonist (peptide or small molecule) has been reported in the literature. The focus of this study was to utilize the non-specific RXFP3/RXFP4 antagonist ΔR3/I5 as a template to rationally design an RXFP4 specific antagonist. Unexpectedly, we demonstrated that ΔR3/I5 exhibited partial agonism at RXFP4 when expressed in CHO cells which is associated with only partial antagonism of INSL5 analogue activation. In an attempt to improve RXFP4 specificity and antagonist activity we designed and chemically synthesized a series of analogues of ΔR3/I5. While all the chimeric analogues still demonstrated partial agonism at RXFP4, one peptide (Analogue 17) exhibited significantly improved RXFP4 specificity. Importantly, analogue 17 has a simplified structure which is more amenable to chemical synthesis. Therefore, analogue 17 is an ideal template for further development into a specific high affinity RXFP4 antagonist which will be an important tool to probe the physiological role of RXFP4/INSL5 axis.
Engineering of a Biologically Active Insulin Dimer.
Publisher: American Chemical Society (ACS)
Date: 19-11-2021
DOI: 10.1021/ACS.JMEDCHEM.1C01594
Abstract: The growing epidemic of diabetes means that there is a need for therapies that are more efficacious, safe, and convenient. Here, we report the efficient synthesis of a novel disulfide dimer of human insulin tethered at the N-terminus of its B-chain through placement of a cysteine residue. The resulting peptide was shown to bind to both the insulin receptor isoform B and insulin-like growth factor-1 receptor with comparable affinity to native insulin. In
Amyloid aggregation and membrane activity of the antimicrobial peptide uperin 3.5
Publisher: Wiley
Date: 23-02-2018
DOI: 10.1002/PEP2.24052
Wnt signaling mediates TLR pathway and promote unrestrained adipogenesis and metaflammation: Therapeutic targets for obesity and type 2 diabetes
Publisher: Elsevier BV
Date: 02-2020
DOI: 10.1016/J.PHRS.2019.104602
Abstract: Diabesity is the combination of type 2 diabetes and obesity characterized by chronic low-grade inflammation. The Wnt signaling act as an evolutionary pathway playing crucial role in regulating cellular homeostasis and energy balance from hypothalamus to metabolic organs. Aberrant activity of certain appendages in the canonical and non-canonical Wnt system deregulates metabolism and leads to adipose tissue expansion, this key event initiates metabolic stress causing metaflammation and obesity. Metaflammation induced obesity initiates abnormal development of adipocytes mediating through the non-canonical Wnt signaling inhibition of canonical Wnt pathway to fan the flames of adipogenesis. Moreover, activation of toll like receptor (TLR)-4 signaling in metabolic stress invites immune cells to release pro-inflammatory cytokines for recruitment of macrophages in adipose tissues, further causes polarization of macrophages into M1(classically activated) and M2 (alternatively activated) subtypes. These events end with chronic low-grade inflammation which interferes with insulin signaling in metabolic tissues to develop type 2 diabetes. However, there is a dearth in understanding the exact mechanism of Wnt-TLR axis during diabesity. This review dissects the molecular facets of Wnt and TLRs that modulates cellular components during diabesity and provides current progress, challenges and alternative therapeutic strategies at preclinical and clinical level.
The complex binding mode of the peptide hormone H2 relaxin to its receptor RXFP1.
Publisher: Springer Science and Business Media LLC
Date: 18-04-2016
DOI: 10.1038/NCOMMS11344
Abstract: H2 relaxin activates the relaxin family peptide receptor-1 (RXFP1), a class A G-protein coupled receptor, by a poorly understood mechanism. The ectodomain of RXFP1 comprises an N-terminal LDLa module, essential for activation, tethered to a leucine-rich repeat (LRR) domain by a 32-residue linker. H2 relaxin is hypothesized to bind with high affinity to the LRR domain enabling the LDLa module to bind and activate the transmembrane domain of RXFP1. Here we define a relaxin-binding site on the LDLa-LRR linker, essential for the high affinity of H2 relaxin for the ectodomain of RXFP1, and show that residues within the LDLa-LRR linker are critical for receptor activation. We propose H2 relaxin binds and stabilizes a helical conformation of the LDLa-LRR linker that positions residues of both the linker and the LDLa module to bind the transmembrane domain and activate RXFP1.
Binding conformation and determinants of a single-chain peptide antagonist at the relaxin-3 receptor RXFP3
Publisher: Elsevier BV
Date: 10-2018
Amyloid Beta (Aβ) Peptide and Factors that Play Important Roles in Alzheimer’s Disease
Publisher: Bentham Science Publishers Ltd.
Date: 28-03-2016
DOI: 10.2174/0929867323666160229113911
Abstract: Amyloid beta peptide (Aβ) is recognised as a main feature of Alzheimer's disease (AD). Increasing evidence suggests that small soluble oligomers of Aβ are the toxic form of the peptide and may instigate AD. Different factors including some key residues within Aβ molecule, the cell membrane, prion protein and metals play important roles in developing AD. Significant progress has been made to understand these factors and elucidate the mechanism of cytotoxicity of Aβ. This review summarizes recent findings in the area of Aβ and AD research, and this current knowledge could enable medicinal chemists to design and develop therapeutics to treat AD.
Synthesis of fluorescent analogs of relaxin family peptides and their preliminary in vitro and in vivo characterization.
Publisher: Frontiers Media SA
Date: 2013
Total Chemical Synthesis of an Intra‐A‐Chain Cystathionine Human Insulin Analogue with Enhanced Thermal Stability
Publisher: Wiley
Date: 20-10-2016
Abstract: Despite recent advances in the treatment of diabetes mellitus, storage of insulin formulations at 4 °C is still necessary to minimize chemical degradation. This is problematic in tropical regions where reliable refrigeration is not ubiquitous. Some degradation byproducts are caused by disulfide shuffling of cystine that leads to covalently bonded oligomers. Consequently we examined the utility of the non‐reducible cystine isostere, cystathionine, within the A‐chain. Reported herein is an efficient method for forming this mimic using simple monomeric building blocks. The intra‐A‐chain cystathionine insulin analogue was obtained in good overall yield, chemically characterized and demonstrated to possess native binding affinity for the insulin receptor isoform B. It was also shown to possess significantly enhanced thermal stability indicating potential application to next‐generation insulin analogues.
Glycosylation Improves the Proteolytic Stability of Exenatide
Publisher: American Chemical Society (ACS)
Date: 16-05-2023
Chemical Glycosylation and Its Application to Glucose Homeostasis-Regulating Peptides
Publisher: Frontiers Media SA
Date: 12-04-2021
DOI: 10.3389/FCHEM.2021.650025
Abstract: Peptides and proteins are attractive targets for therapeutic drug development due to their exquisite target specificity and low toxicity profiles. However, their complex structures give rise to several challenges including solubility, stability, aggregation, low bioavailability, and poor pharmacokinetics. Numerous chemical strategies to address these have been developed including the introduction of several natural and non-natural modifications such as glycosylation, lipidation, cyclization and PEGylation. Glycosylation is considered to be one of the most useful modifications as it is known to contribute to increasing the stability, to improve solubility, and increase the circulating half-lifves of these biomolecules. However, cellular glycosylation is a highly complex process that generally results in heterogenous glycan structures which confounds quality control and chemical and biological assays. For this reason, much effort has been expended on the development of chemical methods, including by solid phase peptide synthesis or chemoenzymatic processes, to enable the acquisition of homogenous glycopeptides to greatly expand possibilities in drug development. In this mini-review, we highlight the importance of such chemical glycosylation methods for improving the biophysical properties of naturally non-glycosylated peptides as applied to the therapeutically essential insulin and related peptides that are used in the treatment of diabetes.
Synthesis, conformational analysis and biological properties of a dicarba derivative of the antimicrobial peptide, brevinin-1BYa
Publisher: Springer Science and Business Media LLC
Date: 11-02-2011
DOI: 10.1007/S00249-011-0679-2
Abstract: Brevinin-1BYa (FLPILASLAAKFGPKLFCLVTKKC), first isolated from skin secretions of the frog Rana boylii, displays broad-spectrum antimicrobial activity and potent haemolytic activity. This study investigates the effects on conformation and biological activity of replacement of the intramolecular disulphide bridge in the peptide by a non-reducible dicarba bond. Dicarba-brevinin-1BYa was prepared by microwave irradiation of [Agl(18),Agl(24)]-brevinin-1BYa (Agl = allylglycine) in the presence of a second generation Grubbs' catalyst. Circular dichroism spectroscopy in 50% trifluoroethanol-water indicated that the degree of α-helicity of the dicarba derivative (22%) was less than that of brevinin-1BYa (27%) but comparable to that of the acyclic derivative [Ser(18),Ser(24)]-brevinin-1BYa (23%). Dicarba-brevinin-1BYa showed a two-fold increase in potency against reference strains of Escherichia coli, Staphylococcus aureus, and Candida albicans compared with the native peptide and displayed potent bactericidal activity against clinical isolates of methicillin-resistant S. aureus (MRSA) and multidrug-resistant Acinetobacter baumannii (MIC in the range 1-8 μM). Compared with brevinin-1BYa and [Ser(18),Ser(24)]-brevinin-1BYa, the dicarba derivative was associated with increased cytotoxicity against human erythrocytes (2.5-fold), MDA-MB-231 breast carcinoma cells (1.3-fold) and HepG2 hepatoma-derived cells (1.5-fold).
2‐Nitroveratryl as a Photocleavable Thiol‐Protecting Group for Directed Disulfide Bond Formation in the Chemical Synthesis of Insulin
Publisher: Wiley
Date: 24-06-2014
Abstract: Chemical synthesis of peptides can allow the option of sequential formation of multiple cysteines through exploitation of judiciously chosen regioselective thiol-protecting groups. We report the use of 2-nitroveratryl (oNv) as a new orthogonal group that can be cleaved by photolysis under ambient conditions. In combination with complementary S-pyridinesulfenyl activation, disulfide bonds are formed rapidly in situ. The preparation of Fmoc-Cys(oNv)-OH is described together with its use for the solid-phase synthesis of complex cystine-rich peptides, such as insulin.
Covalent conjugation of cationic antimicrobial peptides with a beta-lactam antibiotic core
Publisher: Wiley
Date: 07-03-2018
DOI: 10.1002/PEP2.24059
ChemInform Abstract: Sequential Formation of Regioselective Disulfide Bonds in Synthetic Peptides with Multiple Disulfide Bonds
Publisher: Wiley
Date: 27-04-2015
Identification, Synthesis, Conformation and Activity of an Insulin-like Peptide from a Sea Anemone
Publisher: MDPI AG
Date: 29-11-2021
DOI: 10.3390/BIOM11121785
Abstract: The role of insulin and insulin-like peptides (ILPs) in vertebrate animals is well studied. Numerous ILPs are also found in invertebrates, although there is uncertainty as to the function and role of many of these peptides. We have identified transcripts with similarity to the insulin family in the tentacle transcriptomes of the sea anemone Oulactis sp. (Actiniaria: Actiniidae). The translated transcripts showed that these insulin-like peptides have highly conserved A- and B-chains among in iduals of this species, as well as other Anthozoa. An Oulactis sp. ILP sequence (IlO1_i1) was synthesized using Fmoc solid-phase peptide synthesis of the in idual chains, followed by regioselective disulfide bond formation of the intra-A and two interchain disulfide bonds. Bioactivity studies of IlO1_i1 were conducted on human insulin and insulin-like growth factor receptors, and on voltage-gated potassium, sodium, and calcium channels. IlO1_i1 did not bind to the insulin or insulin-like growth factor receptors, but showed weak activity against KV1.2, 1.3, 3.1, and 11.1 (hERG) channels, as well as NaV1.4 channels. Further functional studies are required to determine the role of this peptide in the sea anemone.
Structural Dynamics of a Lytic Peptide Interacting with a Supported Lipid Bilayer
Publisher: Elsevier BV
Date: 03-2011
Association of alpha-helix peptides that have gamma-cyclodextrin and pyrene units in their side chain, and induction of dissociation of the association dimer by external stimulant molecules
Publisher: Royal Society of Chemistry (RSC)
Date: 2000
DOI: 10.1039/B001090L
Development of a Single-Chain Peptide Agonist of the Relaxin-3 Receptor Using Hydrocarbon Stapling.
Publisher: American Chemical Society (ACS)
Date: 08-08-2016
DOI: 10.1021/ACS.JMEDCHEM.6B00265
Abstract: Structure-activity studies of the insulin superfamily member, relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist which will facilitate study of the function of relaxin-3.
C-Terminal Modifications Broaden Activity of the Proline-Rich Antimicrobial Peptide, Chex1-Arg20
Publisher: CSIRO Publishing
Date: 2015
DOI: 10.1071/CH15169
Abstract: A series of N- and C-terminal modifications of the monomeric proline-rich antimicrobial peptide, Chex1-Arg20, was obtained via different chemical strategies using Fmoc/tBu solid-phase peptide synthesis in order to study their effects on a panel of Gram-negative bacteria. In particular, C-terminal modifications with hydrazide or alcohol functions extended their antibacterial activity from E. coli and K. pneumoniae to other Gram-negative species, A. baumannii and P. aeruginosa. Furthermore, these analogues did not show cytotoxicity towards mammalian cells. Hence, such modifications may aid in the development of more potent proline-rich antimicrobial peptides with a greater spectrum of activity against Gram-negative bacteria than the parent peptide.
Chemically synthesized dicarba H2 relaxin analogues retain strong RXFP1 receptor activity but show an unexpected loss of in vitro serum stability.
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C5OB01539A
Abstract: Replacement of a disulfide bond with a non-reducible dicarba bond in an insulin-like peptide, relaxin, did not significantly alter functional activity but resulted in unexpected dramatic decrease in vitro serum stability.
Analgesic effect of central relaxin receptor activation on persistent inflammatory pain in mice: behavioral and neurochemical data
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 16-06-2021
Investigation of interactions at the extracellular loops of the relaxin family peptide receptor 1 (RXFP1)
Publisher: Elsevier BV
Date: 12-2014
Native Chemical Ligation to Minimize Aspartimide Formation during Chemical Synthesis of Small LDLa Protein.
Publisher: Wiley
Date: 27-11-2015
Abstract: The inhibition of the G protein-coupled receptor, relaxin family peptide receptor 1 (RXFP1), by a small LDLa protein may be a potential approach for prostate cancer treatment. However, it is a significant challenge to chemically produce the 41-residue and three-disulfide cross-bridged LDLa module which is highly prone to aspartimide formation due to the presence of several aspartic acid residues. Known palliative measures, including addition of HOBt to piperidine for N(α) -deprotection, failed to completely overcome this side reaction. For this reason, an elegant native chemical ligation approach was employed in which two segments were assembled for generating the linear LDLa protein. Acquisition of correct folding was achieved by using either a regioselective disulfide bond formation or global oxidation strategies. The final synthetic LDLa protein obtained was characterized by NMR spectroscopic structural analysis after chelation with a Ca(2+) ion and confirmed to be equivalent to the same protein obtained by recombinant DNA production.
Relaxin-3/RXFP3 system regulates alcohol-seeking
Publisher: Proceedings of the National Academy of Sciences
Date: 02-12-2013
Abstract: Relapse and hazardous drinking represent the most difficult clinical problems in treating patients with alcohol use disorders. Increasing our understanding of the brain circuits and chemicals that regulate alcohol intake and relapse offers the potential for more targeted therapeutic approaches to assist in relapse prevention. Using a rat model of alcohol use and alcohol-seeking, we provide the first evidence that a neuropeptide, namely relaxin-3, acts upon specific receptors (relaxin family peptide 3) within the brain to regulate alcohol self-administration and relapse-like behavior. In the case of relapse-like alcohol-seeking, this system appears particularly involved in stress-mediated relapse via actions within a brain region called the bed nucleus of the stria terminalis.
Understanding VPAC receptor family peptide binding and selectivity
Publisher: Springer Science and Business Media LLC
Date: 16-11-2022
DOI: 10.1038/S41467-022-34629-3
Abstract: The vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) receptors are key regulators of neurological processes. Despite recent structural data, a comprehensive understanding of peptide binding and selectivity among different subfamily receptors is lacking. Here, we determine structures of active, Gs-coupled, VIP-VPAC1R, PACAP27-VPAC1R, and PACAP27-PAC1R complexes. Cryo-EM structural analyses and molecular dynamics simulations (MDSs) reveal fewer stable interactions between VPAC1R and VIP than for PACAP27, more extensive dynamics of VIP interaction with extracellular loop 3, and receptor-dependent differences in interactions of conserved N-terminal peptide residues with the receptor core. MD of VIP modelled into PAC1R predicts more transient VIP-PAC1R interactions in the receptor core, compared to VIP-VPAC1R, which may underlie the selectivity of VIP for VPAC1R over PAC1R. Collectively, our work improves molecular understanding of peptide engagement with the PAC1R and VPAC1R that may benefit the development of novel selective agonists.
The importance of tryptophan B28 in H2 relaxin for RXFP2 binding and activation
Publisher: Springer Science and Business Media LLC
Date: 26-10-2013
Fluorescent Ion Efflux Screening Assay for Determining Membrane-Active Peptides
Publisher: CSIRO Publishing
Date: 2017
DOI: 10.1071/CH16659
Abstract: A major global health threat is the emergence of antibiotic-resistant microbes. Coupled with a lack of development of modified antibiotics, there is a need to develop new antimicrobial molecules and screening assays for them. In this study, we provide proof of concept that a large unilamellar vesicle (LUV) method used to study chloride ion efflux facilitated by ionophores and surfactant-like molecules that disrupt membrane integrity can be adapted to identify membrane-interactive antimicrobial peptides (AMPs) and to screen relative activity of AMPs. Lucigenin was encapsulated in LUVs in the presence of Cl– ion (NaCl), which quenches fluorescence, and then incubated with AMPs in 100 mM NaNO3 buffer. Upon AMP membrane interaction or disruption, the Cl– ion is exchanged with the NO3– ion, and the resultant lucigenin fluorescence is indicative of relative AMP activity. Seven AMPs were synthesized by solid-phase peptide chemistry and incubated with LUVs of different phospholipid compositions. Each AMP resulted in lucigenin fluorescence, which was dose dependent, and the relative fluorescence correlated with the minimum inhibitory concentration and minimum bactericidal concentration values for the corresponding peptide. Furthermore, using mammalian model phospholipid LUVs, lucigenin-induced fluorescence also correlated with the AMP cytotoxicity half-maximal inhibitory concentration values. The proline-rich AMP, Chex1-Arg20, which is non-lytic but interacts with the bacterial membrane resulted in lucigenin fluorescence of bacterial membrane model LUVs but not of mammalian membrane model LUVs. The fluorescent ion efflux assay developed here should have applicability for most AMPs and could be tailored to target particular bacterial species membrane composition, potentially leading to the identification of novel membrane-interactive AMPs. The rapid high-throughput method also allows for screening of relative AMP activity and toxicity before biological testing.
Regioselective disulfide solid phase synthesis, chemical characterization and in vitro receptor binding activity of equine relaxin
Publisher: Springer Science and Business Media LLC
Date: 19-05-2006
Development of Novel High-Affinity Antagonists for the Relaxin Family Peptide Receptor 1
Publisher: American Chemical Society (ACS)
Date: 03-05-2023
Inside Cover: A One‐Pot Chemically Cleavable Bis‐Linker Tether Strategy for the Synthesis of Heterodimeric Peptides (Angew. Chem. Int. Ed. 47/2016)
Publisher: Wiley
Date: 20-09-2016
Cellular Disulfide Bond Formation in Bioactive Peptides and Proteins
Publisher: MDPI AG
Date: 14-01-2015
DOI: 10.3390/IJMS16011791
Membrane interactions of proline-rich antimicrobial peptide, Chex1-Arg20, multimers
Publisher: Elsevier BV
Date: 06-2016
DOI: 10.1016/J.BBAMEM.2016.02.035
Abstract: The increasing prevalence of antibiotic-resistant pathogens requires the development of new antibiotics. Proline-rich antimicrobial peptides (PrAMPs), including native apidaecins, Bac7, and oncocins or designed A3APO, show multi-modal actions against pathogens together with immunostimulatory activities. The interactions of the designed PrAMP, Chex1-Arg20, and its dimeric and tetrameric oligomers with different model membranes were investigated by circular dichroism spectroscopy, dynamic light scattering, zeta potential, differential scanning calorimetry, and dye leakage. Chex1-Arg20 oligomers showed stronger affinity and preferential binding to negatively charged phospholipid bilayers and led to lipid aggregation and neutralization. Fluorescence microscopy of negatively charged giant unilamellar vesicles with AlexFluor-647-labeled Chex1-Arg20 dimers and tetramers displayed aggregation at a peptide/lipid low ratio of 1:200 and at higher peptide concentrations (1:100/1:50) for Chex1-Arg20 monomer. Such interactions, aggregation, and neutralization of PrAMP oligomers additionally showed the importance of interactions of PrAMPs with negatively charged membranes.
Assessment of renal fibrosis and anti-fibrotic agents using a novel diagnostic and stain-free second-harmonic generation platform
Publisher: Wiley
Date: 28-04-2021
Chemical synthesis and biological evaluation of an antimicrobial peptide gonococcal growth inhibitor
Publisher: Springer Science and Business Media LLC
Date: 04-05-2012
DOI: 10.1007/S00726-012-1305-Z
Abstract: Gonococcal growth inhibitor 1 (GGI-1) is a 44-residue peptide with potent anti-Legionella activity. It has been isolated from Staphylococcus haemolyticus but, to date, its chemical synthesis has not been reported. Acquisition of this peptide via this means would enable a more detailed examination of its antimicrobial properties. However, its synthesis represents a significant challenge because of two predicted "difficult sequences" within the peptide. Its successful solid-phase assembly is reported in this paper, and was accomplished by use of simple palliative measures including the introduction of a single pseudo-proline isostere in order to counteract on-resin aggregation. The peptide had moderate antimicrobial activity against Escherichia coli but was inactive against another Gram-negative bacterium and two Gram-positive bacteria (Bacillus species). It had significant haemolytic activity, with a H(50) (concentration of peptide that causes 50 % haemolysis) of 20 and 125 μM for two blood s les from different donors. An alternative therapeutic index to that proposed for GGI-1 in a recent publication is proposed.
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.
Multi-Component Mechanism of H2 Relaxin Binding to RXFP1 through NanoBRET Kinetic Analysis.
Publisher: Elsevier BV
Date: 2018
Proline-rich antimicrobial peptides: potential therapeutics against antibiotic-resistant bacteria.
Publisher: Springer Science and Business Media LLC
Date: 21-08-2014
DOI: 10.1007/S00726-014-1820-1
Abstract: The increasing resistance of pathogens to antibiotics causes a huge clinical burden that places great demands on academic researchers and the pharmaceutical industry for resolution. Antimicrobial peptides, part of native host defense, have emerged as novel potential antibiotic alternatives. Among the different classes of antimicrobial peptides, proline-rich antimicrobial peptides, predominantly sourced from insects, have been extensively investigated to study their specific modes of action. In this review, we focus on recent developments in these peptides. They show a variety of modes of actions, including mechanism shift at high concentration, non-lytic mechanisms, as well as possessing different intracellular targets and lipopolysaccharide binding activity. Furthermore, proline-rich antimicrobial peptides display the ability to not only modulate the immune system via cytokine activity or angiogenesis but also possess properties of penetrating cell membranes and crossing the blood brain barrier suggesting a role as potential novel carriers. Ongoing studies of these peptides will likely lead to the development of more potent antimicrobial peptides that may serve as important additions to the armoury of agents against bacterial infection and drug delivery.
A Lipidated Single-B-Chain Derivative of Relaxin Exhibits Improved In Vitro Serum Stability without Altering Activity
Publisher: MDPI AG
Date: 04-2023
DOI: 10.3390/IJMS24076616
Abstract: Human relaxin-2 (H2 relaxin) is therapeutically very important due to its strong anti-fibrotic, vasodilatory, and cardioprotective effects. Therefore, relaxin’s receptor, relaxin family peptide receptor 1 (RXFP1), is a potential target for the treatment of fibrosis and related disorders, including heart failure. H2 relaxin has a complex two-chain structure (A and B) and three disulfide bridges. Our laboratory has recently developed B7-33 peptide, a single-chain agonist based on the B-chain of H2 relaxin. However, the peptide B7-33 has a short circulation time in vitro in serum (t1/2 = ~6 min). In this study, we report structure-activity relationship studies on B7-33 utilizing different fatty-acid conjugations at different positions. We have shown that by fatty-acid conjugation with an appropriate spacer length, the in vitro half-life of B7-33 can be increased from 6 min to 60 min. In the future, the lead lipidated molecule will be studied in animal models to measure its PK/PD properties, which will lead to their pre-clinical applications.
The effect of tailing lipidation on the bioactivity of antimicrobial peptides and their aggregation tendency
Publisher: Wiley
Date: 24-02-2023
DOI: 10.1002/AGT2.329
Abstract: Antimicrobial peptides (AMPs) are potentially powerful alternatives to conventional antibiotics in combating multidrug resistance, given their broad spectrum of activity. They mainly interact with cell membranes through surface electrostatic potentials and the formation of secondary structures, resulting in permeability and destruction of target microorganism membranes. Our earlier work showed that two leading AMPs, MSI‐78 (4–20) and pardaxin (1–22), had potent antimicrobial activity against a range of bacteria. It is known that the attachment of moderate‐length lipid carbon chains to cationic peptides can further improve the functionality of these peptides through enhanced interactions with the membrane lipid bilayer, inducing membrane curvature, destabilization, and potential leakage. Thus, in this work, we aimed to investigate the antimicrobial activity, oligomerization propensity, and lipid‐membrane binding interactions of a range of N‐terminal lipidated analogs of MSI‐78 (4–20) and pardaxin (1–22). Molecular modeling results suggest that aggregation of the N‐lipidated AMPs may impart greater structural stability to the peptides in solution and a greater depth of lipid bilayer insertion for the N‐lipidated AMPs over the parental peptide. Our experimental and computational findings provide insights into how N‐terminal lipidation of AMPs may alter their conformations, with subsequent effects on their functional properties in regard to their self‐aggregation behavior, membrane interactions, and antimicrobial activity.
ChemInform Abstract: The Relaxin Peptide Family - Structure, Function and Clinical Applications
Publisher: Wiley
Date: 25-08-2011
Design, synthesis and pharmacological evaluation of cyclic mimetics of the insulin-like peptide 3 (INSL3) B-chain
Publisher: Wiley
Date: 2007
DOI: 10.1002/PSC.807
Abstract: Insulin-like peptide 3 (INSL3) is a peptide hormone belonging to the relaxin-insulin superfamily of peptides that plays important roles in testes descent, oocyte maturation and the control of male germ cell apoptosis. These actions are mediated via a specific G-protein coupled receptor, LGR8. Previous structure-activity studies have shown that the key binding site of INSL3 is situated within its B-chain. Recent studies in our laboratory have led to the identification of a cyclic peptide mimetic 2 of the INSL3 B-chain, which we have shown to compete with the binding of [33P]-relaxin to LGR8 expressed in HEK293T cells, and to inhibit cAMP-mediated signaling in these cells, i.e. it is an antagonist of INSL3. In order to further define the structure-activity relationships of cyclic analogues of the INSL3 B-chain, we used a structure-based approach to design a series of cyclic, disulfide-constrained INSL3 B-chain mimetics. To do this, we first created a model of the 3D structure of INSL3 using the crystal structure of human relaxin as a template. This model of INSL3 was then used as a template to design a series of disulfide-constrained mimetics of the INSL3 B-chain. The peptides were synthesized by solid-phase peptide synthesis using pseudoproline dipeptides to improve the synthesis outcome. Of the seven prepared INSL3 B-chain mimetics, three compounds were found to have partial displacement activity, while four were able to completely displace [33P]-relaxin from LGR8, including compounds that were markedly shorter than compound 2. The best of these, mimetic 6, showed significantly greater affinity for LGR8 than compound 2, but still displayed around 1000-fold less affinity for LGR8 than native INSL3. Analysis of selected mimetics for their alpha-helical content using circular dichroism (CD) spectroscopy revealed that, generally, the mimetics showed less than expected helicity. The inability of the compounds to display true native INSL3 structure is likely contributing to their reduced receptor binding affinity. We are currently examining alternative INSL3 B-chain mimetics that might better present key receptor binding residues in the native INSL3-like conformation.
Innentitelbild: A One‐Pot Chemically Cleavable Bis‐Linker Tether Strategy for the Synthesis of Heterodimeric Peptides (Angew. Chem. 47/2016)
Publisher: Wiley
Date: 20-09-2016
Improved chemical synthesis and demonstration of the relaxin receptor binding affinity and biological activity of mouse relaxin
Publisher: American Chemical Society (ACS)
Date: 11-04-2007
DOI: 10.1021/BI700238H
Abstract: The primary stored and circulating form of relaxin in humans, human gene-2 (H2) relaxin, has potent antifibrotic properties with rapidly occurring efficacy. However, when administered to experimental models of fibrosis, H2 relaxin can only be applied over short-term (2-4 week) periods, due to rodents mounting an antibody response to the exogenous human relaxin, resulting in delayed clearance and, hence, increased and variable circulating levels. To overcome this problem, the current study investigated the therapeutic potential of mouse relaxin over long-term exposure in vivo. Mouse relaxin is unique among the known relaxins in that it possesses an extra residue within the C-terminal region of its A-chain. To enable a detailed assessment of its receptor interaction and biological properties, it was chemically synthesized in good overall yield by the separate preparation of each of its A- and B-chains followed by regioselective formation of each of the intramolecular and two intermolecular disulfide bonds. Murine relaxin was shown to bind with high affinity to the human, mouse, and rat RXFP1 (primary relaxin) receptor but with a slightly lower affinity to that of H2 relaxin. When administered to relaxin-deficient mice (which undergo an age-dependent progression of organ fibrosis) over a 4 month treatment period, mouse relaxin was able to significantly inhibit the progression of collagen accumulation in several organs including the lung, kidney, testis, and skin (all p < 0.05 vs untreated group), consistent with the actions of H2 relaxin. These combined data demonstrate that mouse relaxin can effectively inhibit collagen deposition and accumulation (fibrosis) over long-term treatment periods.
Insulin-like peptide 5 is an orexigenic gastrointestinal hormone
Publisher: Proceedings of the National Academy of Sciences
Date: 15-07-2014
Abstract: Hormonal factors from specialized enteroendocrine cells in the gut epithelium link the availability of dietary nutrients to energy utilization and storage. Many gut hormones also affect behaviors such as appetite and foraging, conveying for ex le the satiating effects of food consumption. Here we identify insulin-like peptide 5 (Insl5) as a product of colonic endocrine L-cells, and show that levels were elevated in calorie-restricted mice and reduced after feeding. Consistent with this profile Insl5 administration stimulated food intake in mice, indicating it should join ghrelin as only the second identified gut hormone that enhances appetite. Modulating the Insl5 axis presents a new strategy for the treatment of metabolic disease and obesity.
Producing Fuels and Fine Chemicals from Biomass Using Nanomaterials
Publisher: CRC Press
Date: 28-10-2013
DOI: 10.1201/B15585
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.
The C-terminus of the B-chain of human insulin-like peptide 5 is critical for cognate RXFP4 receptor activity.
Publisher: Springer Science and Business Media LLC
Date: 11-12-2015
DOI: 10.1007/S00726-015-2144-5
Abstract: Insulin-like peptide 5 (INSL5) is an orexigenic peptide hormone belonging to the relaxin family of peptides. It is expressed primarily in the L-cells of the colon and has a postulated key role in regulating food intake. Its G protein-coupled receptor, RXFP4, is a potential drug target for treating obesity and anorexia. We studied the effect of modification of the C-terminus of the A and B-chains of human INSL5 on RXFP4 binding and activation. Three variants of human INSL5 were prepared using solid phase peptide synthesis and subsequent sequential regioselective disulfide bond formation. The peptides were synthesized as C-terminal acids (both A- and B-chains with free C-termini, i.e., the native form), amides (both chains as the C-terminal amide) and one analog with the C-terminus of its A-chain as the amide and the C-terminus of the B-chain as the acid. The results showed that C-terminus of the B-chain is more important than that of the A-chain for RXFP4 binding and activity. Amidation of the A-chain C-terminus does not have any effect on the INSL5 activity. The difference in RXFP4 binding and activation between the three peptides is believed to be due to electrostatic interaction of the free carboxylate of INSL5 with a positively charged residue (s), either situated within the INSL5 molecule itself or in the receptor extracellular loops.
Systematic comparison of activity and mechanism of antimicrobial peptides against nosocomial pathogens
Publisher: Elsevier BV
Date: 03-2022
DOI: 10.1016/J.EJMECH.2022.114135
Abstract: The World Health Organisation has deemed several multi-drug resistant (MDR) nosocomial bacterial pathogens to be of significant threat to human health. A stark increase in morbidity, mortality and the burden to healthcare systems around the world can be attributed to the development of resistance in these bacteria. Accordingly, alternative antimicrobial agents have been sought as an attractive means to combat MDR pathogens, with one such ex le being antimicrobial peptides (AMPs). Given the reported activity of AMPs, including Pardaxin, MSI-78, dermaseptin-PC (DMPC) and Cecropin B, it is important to understand their activities and modes of action against bacteria for further AMP design. In this study, we compared these AMPs against a panel of nosocomial bacterial pathogens, followed by detailed mechanistic studies. It was found that Pardaxin (1-22) and MSI-78 (4-20) displayed the most pronounced antimicrobial activity against the tested bacteria. The mechanistic studies by membrane permeability and molecular dynamics simulation further confirmed the strong membrane interaction and structure of Pardaxin (1-22) and MSI-78 (4-20), which contributed to their potent activity. This study demonstrated a structure and activity guidance for further design of Pardaxin (1-22) and MSI-78 (4-20) as therapeutics against MDR pathogens. The different effects of DMPC (1-19) and Cecropin B (1-21) on membrane integrity and phospholipid membrane interactions provided critical information for the rational design of next-generation analogues with specificity against either Gram-negative or Gram-positive bacteria.
The anti-fibrotic actions of relaxin are mediated through AT2 R-associated protein phosphatases via RXFP1-AT2 R functional crosstalk in human cardiac myofibroblasts.
Publisher: Wiley
Date: 16-04-2020
Noncovalent Peptide Stapling Using Alpha-Methyl-l -Phenylalanine for α-Helical Peptidomimetics
Publisher: American Chemical Society (ACS)
Date: 13-07-2023
DOI: 10.1021/JACS.3C02743
Traceable Peptidic Ligands that Target Ghrelin Receptors
Publisher: Bentham Science Publishers Ltd.
Date: 31-12-2020
DOI: 10.2174/1389203721666200702131457
Abstract: Ghrelin is a 28-amino acid octanoylated peptide hormone that is implicated in many physiological and pathophysiological processes. Specific visualization of ghrelin and its cognate receptor using traceable ligands is crucial in elucidating the localization, functions, and expression pattern of the peptide’s signaling pathway. Here 12 representative radio- and fluorescently-labeled peptide-based ligands are reviewed for in vitro and in vivo imaging studies. In particular, the focus is on their structural features, pharmacological properties, and applications in further biochemical research.
The Chemical Synthesis of Insulin: From the Past to the Present
Publisher: Bentham Science Publishers Ltd.
Date: 03-2011
Fluorescence imaging of the interaction of amyloid beta 40 peptides with live cells and model membrane
Publisher: Elsevier BV
Date: 09-2018
DOI: 10.1016/J.BBAMEM.2018.01.024
Abstract: Amyloid beta peptides (Aβ) found in plaques in the brain have been widely recognised as a hallmark of Alzheimer's disease although the underlying mechanism is still unknown. Aβ40 and Aβ40(A2T) peptides were synthesized and their effects on neuronal cells are reported together with the effect of tetramer forms of the peptides. ThT assay revealed that mutation affected the lag time and aggregation and the presence of lipid vesicles changed the fibril formation profile for both peptides. The A2T mutation appeared to reduce cytotoxicity and lessen binding of Aβ40 peptides to neuronal cells. Fluorescence microscopy of the interaction between Aβ40 peptides and giant unilamellar vesicles revealed that both peptides led to formation of smaller vesicles although the tetramer of Aβ(A2T) appeared to promote vesicle aggregation. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.
Chemically prepared novel dicarba analogs of human relaxin-3, a recently discovered neuropeptide, exhibit full biological activity
Publisher: Elsevier BV
Date: 2010
Intramolecular acyl transfer in peptide and protein ligation and synthesis
Publisher: Wiley
Date: 30-01-2015
DOI: 10.1002/PSC.2749
Abstract: Intramolecular acyl transfer equilibrium in peptides and proteins has stimulated the development of new methodologies for ligation, aggregation suppression or difficult peptide synthesis. Native chemical ligation or aggregation suppression methodologies are based on an X-to-N acyl transfer of a peptide chain (X = S, O). The reverse reaction from N-to-X has led to exciting developments in solving key synthetic problems such as peptide thioester preparation using Fmoc/tBu strategy. Depending on the target peptide or protein, variations of these methods, which are also based on acyl transfer equilibriums, are now available. In this review, we provide a detailed overview of development and utility of methodologies in peptide chemistry that are based on the control of intramolecular equilibrium. To this end, we outline the scaffolds that are favorable for acyl transfer, the conditions for controlling both sides of the acyl transfer equilibrium and their applications to peptide and protein chemistry. Additional new methodologies have been developed for the synthesis of difficult peptides such as peptide alcohols or head-to-tail cyclic peptides. Promising new applications of intramolecular acyl transfer reactions are also highlighted.
Cyclodecapeptides to mimic the radical site of tyrosyl-containing proteins
Publisher: Wiley
Date: 2006
DOI: 10.1002/PSC.769
Abstract: Tyrosyl radicals are involved in many biologically important processes. The development of model compounds to mimic radical enzyme active sites, such as galactose oxidase (GO), has widely contributed to an enhanced understanding of their spectral properties, structural attributes and even reactivity. An emerging approach towards the synthesis of such active site mimetics is the use of peptidic ligands. The potential of cyclodecapeptides to bear phenoxyl radicals has been evaluated through three compounds. LH(4) (2+) is a cyclodecapetide containing two histidine residues (mimicking His(496) and His(581) of GO) and two tyrosine residues (mimicking Tyr(495) and the Tyr(272)* radical of GO). L(tBu)H(4) (2+) and L(OMe)H(4) (2+) incorporate 2,4,6-protected phenols in place of each tyrosine in LH(4) (2+). The deprotonation constants of each peptide determined by potentiometric titrations showed that there are some interactions between the acido-basic residues. Cyclic voltammetric studies revealed that only the peptides incorporating 2,4,6-protected phenolates exhibit reversible redox couples and are thus precursors of radicals stable enough to persist in solution. These studies also showed L(OMe2-) to possess the lower oxidation potential, indicating that this peptide, in its radical form, is the most stabilized. The electrochemically generated radical species have been characterized by EPR spectroscopy.
Adsorption of Amyloidogenic Peptides to Functionalized Surfaces Is Biased by Charge and Hydrophilicity
Publisher: American Chemical Society (ACS)
Date: 19-09-2019
DOI: 10.1021/ACS.LANGMUIR.9B02063
Abstract: Surfaces are abundant in living systems, such as in the form of cellular membranes, and govern many biological processes. In this study, the adsorption of the amyloidogenic model peptides GNNQQNY, NNFGAIL, and VQIVYK as well as the amyloid-forming antimicrobial peptide uperin 3.5 (U3.5) were studied at low concentrations (100 μM) to different surfaces. The technique of a quartz crystal microbalance with dissipation monitoring (QCM-D) was applied as it enables the monitoring of mass binding to sensors at nanogram sensitivity. Gold-coated quartz sensors were used as unmodified gold surfaces or functionalized with self-assembled monolayers (SAMs) of alkanethiols (terminated as methyl, amino, carboxyl, and hydroxyl) resulting in different adsorption affinities of the peptides. Our objective was to evaluate the underlying role of the nature and feature of interfaces in biological systems which could concentrate peptides and impact or trigger peptide aggregation processes. In overall, the largely hydrophobic peptides adsorbed with preference to hydrophobic or countercharged surfaces. Further, the glycoprotein lubricin (LUB) was tested as an antiadhesive coating. Despite its hydrophilicity, the adsorption of peptides to LUB coated sensors was similar to the adsorption to unmodified gold surfaces, which indicates that some peptides diffused through the LUB layer to reach the underlying gold sensor surface. The LUB protein-antiadhesive is thus more effective as a biomaterial coating against larger biomolecules than small peptides under the conditions used here. This study provides directions toward a better understanding of amyloid peptide adsorption to biologically relevant interfaces, such as cellular membranes.
The 9-Fluorenylmethoxycarbonyl (Fmoc) Group in Chemical Peptide Synthesis – Its Past, Present, and Future
Publisher: CSIRO Publishing
Date: 2019
DOI: 10.1071/CH19427
Abstract: The chemical formation of the peptide bond has long fascinated and challenged organic chemists. It requires not only the activation of the carboxyl group of an amino acid but also the protection of the Nα-amino group. The more than a century of continuous development of ever-improved protecting group chemistry has been married to dramatic advances in the chemical synthesis of peptides that, itself, was substantially enhanced by the development of solid-phase peptide synthesis by R. B. Merrifield in the 1960s. While the latter technology has continued to undergo further refinement and improvement in both its chemistry and automation, the development of the base-labile 9-fluorenylmethoxycarbonyl (Fmoc) group and its integration into current synthesis methods is considered a major landmark in the history of the chemical synthesis of peptides. The many beneficial attributes of the Fmoc group, which have yet to be surpassed by any other Nα-protecting group, allow very rapid and highly efficient synthesis of peptides, including ones of significant size and complexity, making it an even more valuable resource for research in the post-genomic world. This review charts the development and use of this Nα-protecting group and its adaptation to address the need for more green chemical peptide synthesis processes.
Relaxin and its role in fibrotic diseases
Publisher: The Royal Society of Chemistry
Date: 21-11-2013
DOI: 10.1039/9781849737081-00060
Abstract: Relaxin is a member of the insulin family of peptides. It is produced in the corpus luteum and/or placenta during pregnancy in mammals and is secreted into the blood where it has numerous essential endocrine functions. In addition to long-recognized roles in remodeling of reproductive tissues, it has an important role as a cardiovascular hormone during pregnancy with potent vasodilatory and renal actions. Additionally, relaxin has potent effects on collagen turnover in connective tissue. The positive effects of relaxin in induced fibrosis models show a significant role in ameliorating kidney, heart and lung fibrosis. Much work has thus been undertaken on the structure and function relationship of this peptide with the goal of understanding the molecular basis for relaxin's action and developing mimetics with potential therapeutic applications in fibrosis.
Signalling profiles of H 3 relaxin, H 2 relaxin and R 3(BΔ 23–27)R /I 5 acting at the relaxin family peptide receptor 3 (RXFP 3)
Publisher: Wiley
Date: 15-05-2014
DOI: 10.1111/BPH.12623
Central relaxin-3 receptor (RXFP3) activation decreases anxiety- and depressive-like behaviours in the rat
Publisher: Elsevier BV
Date: 05-2013
DOI: 10.1016/J.BBR.2013.01.034
Abstract: Relaxin-3 is a recently discovered neuropeptide and the results of earlier anatomical and pharmacological studies suggest it plays a physiological role in modulating functions such as arousal, learning and memory, food intake and neuroendocrine homeostasis. Relaxin-3 is also postulated to modulate affective behaviour, based on high densities of the relaxin-3 G-protein coupled receptor (RXFP3) in brain areas involved in stress and mood/anxiety, including the central amygdala, bed nucleus of the stria terminalis and hypothalamic paraventricular nucleus (PVN) and strong activation of relaxin-3 neurons by stressors, via activation of corticotropin-releasing factor receptor-1 (CRF1). This study assessed the effect of central administration of a newly developed RXFP3-selective agonist, on anxiety- and depressive-like behaviour in rats. Adult, male Sprague-Dawley rats administered 5 μg [R3A(11-24,C15→A)B] (referred to as RXFP3-A2), intracerebroventricularly, demonstrated decreased anxiety-like behaviour in the light-dark box and elevated plus maze, but not in the open field. Notably, in the repeat forced swim test, central RXFP3-A2 administration decreased immobility in rats that had been subjected to the 'stress' of former exposure to the anxiety tests, but not in experimentally naïve rats. These data implicate relaxin-3/RXFP3 signalling in the modulation of effects of acute (anxiety) and cumulative (depression) neurogenic stressors on behaviour and suggest a potential for RXFP3 agonists as anxiolytic and anti-depressant agents. In addition, our results demonstrate that exposure of adult Sprague-Dawley rats to tests of anxiety-like behaviour (∼10-14 days prior) can significantly increase immobility time in the repeat forced swim test.
The chemically synthesized human relaxin-2 analog, B-R13/17K H2, is an RXFP1 antagonist
Publisher: Springer Science and Business Media LLC
Date: 31-12-2010
DOI: 10.1007/S00726-009-0454-1
Abstract: Relaxin is a pleiotropic hormone which exerts its biological functions through its G-protein coupled receptor, RXFP1. While relaxin is well known for its reproductive and antifibrotic roles, recent studies suggest that it is produced by cancer cells and acts on RXFP1 to induce growth and metastasis. Furthermore, more recently Silvertown et al. demonstrated that lentiviral production of a human gene-2 (H2) relaxin analog reduced the growth of prostate xenograft tumors. The authors proposed that the lentivirally produced peptide was an RXFP1 antagonist however, the processed form of the peptide produced was not demonstrated. In this study, we have chemically synthesized the H2 relaxin analog, B-R13/17K H2 relaxin, and subjected it to detailed chemical characterization by HPLC, MALDI-TOF mass spectrometry, and amino acid analysis. The biological activity of the synthetic peptide was then tested in three different cell lines. It was found to bind with 500-fold lower affinity than H2 relaxin to RXFP1 receptors over-expressed in HEK-293T cells where it acted as a partial agonist. However, in cells which natively express the RXFP1 receptor, rat renal myofibroblasts and MCF-7 cancer cells, it acted as a full antagonist. Importantly, it was able to significantly inhibit cell invasion induced by H2 relaxin in MCF-7 cells consistent with the results of the lentiviral-driven expression in prostate cancer cells. The relaxin analog, B-R13/17K H2, can now be used as a tool to further understand RXFP1 function, and serve as a template for drug design for a therapeutic to treat prostate and other cancers.
Relaxin‐3 innervation of the intergeniculate leaflet of the rat thalamus – neuronal tract‐tracing and in vitro electrophysiological studies
Publisher: Wiley
Date: 25-02-2013
DOI: 10.1111/EJN.12155
Abstract: Behavioural state is controlled by a range of neural systems that are sensitive to internal and external stimuli. The relaxin-3 and relaxin family peptide receptor 3 (RXFP3) system has emerged as a putative ascending arousal network with putative involvement in regulation of stress responses, neuroendocrine control, feeding and metabolism, circadian activity and cognition. Relaxin-3/γ-aminobutyric acid neuron populations have been identified in the nucleus incertus, pontine raphe nucleus, periaqueductal grey (PAG) and an area dorsal to the substantia nigra. Relaxin-3-positive fibres/terminals densely innervate arousal-related structures in the brainstem, hypothalamus and limbic forebrain, but the functional significance of the heterogeneous relaxin-3 neuron distribution and its inputs to specific brain areas are unclear. Therefore, in this study, we used neuronal tract-tracing and immunofluorescence staining to explore the source of the dense relaxin-3 innervation of the intergeniculate leaflet (IGL) of the thalamus, a component of the neural circadian timing system. Confocal microscopy analysis revealed that relaxin-3-positive neurons retrogradely labelled from the IGL were predominantly present in the PAG and these neurons expressed corticotropin-releasing factor receptor-like immunoreactivity. Subsequently, whole-cell patch-cl recordings revealed heterogeneous effects of RXFP3 activation in the IGL by the RXFP3 agonist, relaxin-3 B-chain/insulin-like peptide-5 A-chain (R3/I5). Identified, neuropeptide Y-positive IGL neurons, known to influence suprachiasmatic nucleus activity, were excited by R3/I5, whereas neurons of unidentified neurotransmitter content were either depolarized or displayed a decrease in action potential firing and/or membrane potential hyperpolarization. Our data identify a PAG to IGL relaxin-3/RXFP3 pathway that might convey stress-related information to key elements of the circadian system and influence behavioural state rhythmicity.
Central relaxin-3 receptor (RXFP3) activation reduces elevated, but not basal, anxiety-like behaviour in C57BL/6J mice
Publisher: Elsevier BV
Date: 10-2015
DOI: 10.1016/J.BBR.2015.06.010
Abstract: Anxiety disorders are among the most prevalent neuropsychiatric conditions, but their precise aetiology and underlying pathophysiological processes remain poorly understood. In light of putative anatomical and functional interactions of the relaxin-3/RXFP3 system with anxiety-related neural circuits, we assessed the ability of central administration of the RXFP3 agonist, RXFP3-A2, to alter anxiety-like behaviours in adult C57BL/6J mice. We assessed how RXFP3-A2 altered performance in tests measuring rodent anxiety-like behaviour (large open field (LOF), elevated plus maze (EPM), light/dark (L/D) box, social interaction). We examined effects of RXFP3-A2 on low 'basal' anxiety, and on elevated anxiety induced by the anxiogenic benzodiazepine, FG-7142 and explored endogenous relaxin-3/RXFP3 signalling modulation by testing effects of an RXFP3 antagonist, R3(B1-22)R, on these behaviours. Intracerebroventricular (icv) injection of RXFP3-A2 (1 nmol, 15 min pre-test) did not alter anxiety-like behaviour under 'basal' conditions in the LOF, EPM or L/D box, but reduced elevated indices of FG-7142-induced (30 mg/kg, ip) anxiety-like behaviour in the L/D box and a single-chamber social interaction test. Furthermore, R3(B1-22)R (4 nmol, icv, 15 min pre-test) increased anxiety-like behaviour in the EPM (reflected by reduced entries into the open arms), but not consistently in the LOF, L/D box or social interaction tests, suggesting endogenous signaling only weakly participates in regulating 'basal' anxiety-like behaviour, in line with previous studies of relaxin-3 and RXFP3 gene knockout mice. Overall, these data suggest exogenous RXFP3 agonists can reduce elevated (FG-7142-induced) levels of anxiety in mice data important for gauging how conserved such effects are, with a view to modelling human pathophysiology and the likely therapeutic potential of RXFP3-targeted drugs.
Total Chemical Synthesis of Palmitoyl-Conjugated Insulin
Publisher: American Chemical Society (ACS)
Date: 06-04-2023
Total Chemical Synthesis of an Intra‐A‐Chain Cystathionine Human Insulin Analogue with Enhanced Thermal Stability
Publisher: Wiley
Date: 20-10-2016
Use of a Temporary “Solubilizing” Peptide Tag for the Fmoc Solid-Phase Synthesis of Human Insulin Glargine via Use of Regioselective Disulfide Bond Formation
Publisher: American Chemical Society (ACS)
Date: 24-06-2009
DOI: 10.1021/BC900181A
Abstract: Solid-phase peptide synthesis has been refined to a stage where efficient preparation of long and complex peptides is now achievable. However, the postsynthesis handling of poorly soluble peptides often remains a significant hindrance to their purification and further use. Several synthetic schemes have been developed for the preparation of such peptides containing modifications to aid their solubility. However, these require the use of complex chemistry or yield non-native sequences. We describe a simple approach based on the use of penta-lysine "tags" that are linked to the C-terminus of the peptide of interest via a base-labile linker. After ready purification of the now freely solubilized peptide, the "tag" is removed by simple, brief base treatment giving the native sequence in much higher overall yield. The applicability of the method was demonstrated by the novel preparation of insulin glargine via solid-phase synthesis of each of the two chains--including the notoriously poorly soluble A-chain--followed by their combination in solution via regioselective disulfide bond formation. At the conclusion of the chain combination, the solubilizing peptide tag was removed from the A-chain to provide synthetic human glargine in nearly 10% overall yield. This approach should facilitate the development of new insulin analogues as well as be widely applicable to the improved purification and acquisition of otherwise poorly soluble synthetic peptides.
The actions of relaxin family peptides on signal transduction pathways activated by the relaxin family peptide receptor RXFP4.
Publisher: Springer Science and Business Media LLC
Date: 26-11-2016
DOI: 10.1007/S00210-016-1321-8
Abstract: The relaxin family peptide receptor 4 (RXFP4) is a G protein-coupled receptor (GPCR) expressed in the colorectum with emerging roles in metabolism and appetite regulation. It is activated by its cognate ligand insulin-like peptide 5 (INSL5) that is expressed in enteroendocrine L cells in the gut. Whether other evolutionarily related peptides such as relaxin-2, relaxin-3, or INSL3 activate RXFP4 signal transduction mechanisms with a pattern similar to or distinct from INSL5 is still unclear. In this study, we compare the signaling pathways activated by various relaxin family peptides to INSL5. We found that, like INSL5, relaxin-3 activated ERK1/2, p38MAPK, Akt, and S6RP phosphorylations leading to increased cell proliferation and also caused GRK and β-arrestin-mediated receptor internalization. Interestingly, relaxin-3 was slightly more potent than INSL5 in ERK1/2 and Akt phosphorylations, but both peptides were almost equipotent in adenylyl cyclase inhibition, S6RP phosphorylation, and cell proliferation. In addition, relaxin-3 showed greater efficacy only in Akt phosphorylation but not in the other pathways investigated. In contrast, no signaling activity or receptor internalization mechanisms were observed following relaxin-2 and INSL3. In conclusion, relaxin-3 is a high-efficacy agonist at RXFP4 with a comparable signal transduction profile to INSL5.
Amphipathic α-helical peptide, HP (2–20), and its analogues derived from Helicobacter pylori: Pore formation mechanism in various lipid compositions
Publisher: Elsevier BV
Date: 2008
DOI: 10.1016/J.BBAMEM.2007.09.020
Abstract: In a previous study, we determined that HP(2-20) (residues 2-20 of parental HP derived from the N-terminus of Helicobacter pylori Ribosomal Protein L1) and its analogue, HPA3, exhibit broad-spectrum antimicrobial activity. The primary objective of the present study was to gain insight into the relevant mechanisms of action using analogues of HP(2-20) together with model liposomes of various lipid compositions and electron microscopy. We determined that these analogues, HPA3 and HPA3NT3, exert potent antibacterial effects in low-salt buffer and antifungal activity against chitin-containing fungi, while having little or no hemolytic activity or cytotoxicity against mammalian cell lines. Our examination of the interaction of HP(2-20) and its analogues with liposomes showed that the peptides disturb both neutral and negatively-charged membranes, as demonstrated by the release of encapsulated fluorescent markers. The release of fluorescent markers induced by HP(2-20) and its analogues was inversely related to marker size. The pore created by HP(2-20) shows that the radius is approximately 1.8 nm, whereas HPA3, HPA3NT3, and melittin have apparent radii between 3.3 and 4.8 nm. Finally, as shown by electron microscopy, the liposomes and various microbial cells treated with HPA3 and HPA3NT3 showed oligomerization and blebbing similar to that seen with melittin, while HP(2-20) exhibited flabbiness. These results suggest that HP(2-20) may exert its antibiotic effects through a small pore (about 1.8 nm), whereas HPA3 and HPA3NT3 formed pores of a size consistent with those formed by melittin.
Molecule-responsive fluorescent sensors of alpha-helix peptides bearing alpha-cyclodextrin, pyrene and nitrobenzene units in their side chains
Publisher: Springer Science and Business Media LLC
Date: 2002
Chemical Synthesis and Characterization of a Nonfibrillating Glycoglucagon.
Publisher: American Chemical Society (ACS)
Date: 08-09-2021
DOI: 10.1021/ACS.BIOCONJCHEM.1C00419
Abstract: The current commercially available glucagon formulations for the treatment of severe hypoglycemia must be reconstituted immediately prior to use, owing to the susceptibility of glucagon to fibrillation and aggregation in an aqueous solution. This results in the inconvenience of handling, misuse, and wastage of this drug. To address these issues, we synthesized a glycosylated glucagon analogue in which the 25th residue (Trp) was replaced with a cysteine (Cys) and a Br-disialyloligosaccharide was conjugated at the Cys thiol moiety. The resulting analogue, glycoglucagon, is a highly potent full agonist at the glucagon receptor. Importantly, glycoglucagon exhibits markedly reduced propensity for fibrillation and enhanced thermal and metabolic stability. This novel analogue is thus a valuable lead for producing stable liquid glucagon formulations that will improve patient compliance and minimize wastage.
Melittin peptides exhibit different activity on different cells and model membranes
Publisher: Springer Science and Business Media LLC
Date: 09-09-2014
DOI: 10.1007/S00726-014-1833-9
Abstract: Melittin (MLT) is a lytic peptide with a broad spectrum of activity against both eukaryotic and prokaryotic cells. To understand the role of proline and the thiol group of cysteine in the cytolytic activity of MLT, native MLT and cysteine-containing analogs were prepared using solid phase peptide synthesis. The antimicrobial and cytolytic activities of the monomeric and dimeric MLT peptides against different cells and model membranes were investigated. The results indicated that the proline residue was necessary for antimicrobial activity and cytotoxicity and its absence significantly reduced lysis of model membranes and hemolysis. Although lytic activity against model membranes decreased for the MLT dimer, hemolytic activity was increased. The native peptide and the MLT-P14C monomer were mainly unstructured in buffer while the dimer adopted a helical conformation. In the presence of neutral and negatively charged vesicles, the helical content of the three peptides was significantly increased. The lytic activity, therefore, is not correlated to the secondary structure of the peptides and, more particularly, on the propensity to adopt helical conformation.
Relaxin family peptides: structure-activity relationship studies.
Publisher: Wiley
Date: 19-01-2017
DOI: 10.1111/BPH.13684
Structure of human insulin-like peptide 5 and characterization of conserved hydrogen bonds and electrostatic interactions within the relaxin framework
Publisher: Portland Press Ltd.
Date: 14-04-2009
DOI: 10.1042/BJ20082353
Abstract: INSL5 (insulin-like peptide 5) is a two-chain peptide hormone related to insulin and relaxin. It was recently discovered through searches of expressed sequence tag databases and, although the full biological significance of INSL5 is still being elucidated, high expression in peripheral tissues such as the colon, as well as in the brain and hypothalamus, suggests roles in gut contractility and neuroendocrine signalling. INSL5 activates the relaxin family peptide receptor 4 with high potency and appears to be the endogenous ligand for this receptor, on the basis of overlapping expression profiles and their apparent co-evolution. In the present study, we have used solution-state NMR to characterize the three-dimensional structure of synthetic human INSL5. The structure reveals an insulin/relaxin-like fold with three helical segments that are braced by three disulfide bonds and enclose a hydrophobic core. Furthermore, we characterized in detail the hydrogen-bond network and electrostatic interactions between charged groups in INSL5 by NMR-monitored temperature and pH titrations and undertook a comprehensive structural comparison with other members of the relaxin family, thus identifying the conserved structural features of the relaxin fold. The B-chain helix, which is the primary receptor-binding site of the relaxins, is longer in INSL5 than in its close relative relaxin-3. As this feature results in a different positioning of the receptor-activation domain ArgB23 and TrpB24, it may be an important contributor to the difference in biological activity observed for these two peptides. Overall, the structural studies provide mechanistic insights into the receptor selectivity of this important family of hormones.
Multimerization of a Proline-Rich Antimicrobial Peptide, Chex-Arg20, Alters Its Mechanism of Interaction with the Escherichia coli Membrane
Publisher: Elsevier BV
Date: 09-2015
DOI: 10.1016/J.CHEMBIOL.2015.08.011
Abstract: A3-APO, a de novo designed branched dimeric proline-rich antimicrobial peptide (PrAMP), is highly effective against a variety of in vivo bacterial infections. We undertook a selective examination of the mechanism for the Gram-negative Escherichia coli bacterial membrane interaction of the monomer (Chex-Arg20), dimer (A3-APO), and tetramer (A3-APO disulfide-linked dimer). All three synthetic peptides were effective at killing E. coli. However, the tetramer was 30-fold more membrane disruptive than the dimer while the monomer showed no membrane activity. Using flow cytometry and high-resolution fluorescent microscopy, it was observed that dimerization and tetramerization of the Chex-Arg20 monomer led to an alteration in the mechanism of action from non-lytic/membrane hyperpolarization to membrane disruption/depolarization. Our findings show that the membrane interaction and permeability of Chex-Arg20 was altered by multimerization.
Elucidation of relaxin-3 binding interactions in the extracellular loops of RXFP3
Publisher: Frontiers Media SA
Date: 2013
The relaxin receptor as a therapeutic target - perspectives from evolution and drug targeting.
Publisher: Elsevier BV
Date: 07-2018
DOI: 10.1016/J.PHARMTHERA.2018.02.008
Abstract: The peptide relaxin was first identified as an important circulating hormone during pregnancy over 90 years ago. Research over many years defined the numerous biological roles that relaxin plays throughout pregnancy in many mammalian species. These important biological actions have led to the testing of relaxin as a therapeutic agent for a number of indications. The discovery of the relaxin receptor, RXFP1, in 2002 facilitated the better understanding of the cellular targets of relaxin, its mechanism of action and enabled the development of relaxin mimetics and screening for small molecule agonists. Additionally, the rapid expansion of the genome databases and bioinformatics tools has significantly advanced our understanding of the evolution of the relaxin/RXFP1 signaling system. It is now clear that the relaxin-RXFP1 signaling axis is far more ancient than previously appreciated with important roles for invertebrate relaxin-like peptides in reproductive and non-reproductive functions. This review summarizes these advances as well as developments in drug targeting of RXFP1. Hence the complex mode of activation of RXFP1 is discussed as is the discovery and development of a peptide mimetic and small molecule agonist. Detailed signaling studies are summarized which highlight the cell specific signaling of a peptide mimetic and biased signaling of a small molecule agonist. These studies highlight the complexities of targeting peptide GPCRs such as RXFP1.
Guest-induced diminishment in fluorescence quenching and molecule sensing ability of a novel cyclodextrin-peptide conjugate [7]
Publisher: American Chemical Society (ACS)
Date: 07-07-2001
DOI: 10.1021/JA0105921
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.
Evaluation of Potential DnaK Modulating Proline-Rich Antimicrobial Peptides Identified by Computational Screening
Publisher: Frontiers Media SA
Date: 13-04-2022
DOI: 10.3389/FCHEM.2022.875233
Abstract: The day is rapidly approaching where current antibiotic therapies will no longer be effective due to the development of multi-drug resistant bacteria. Antimicrobial peptides (AMPs) are a promising class of therapeutic agents which have the potential to help address this burgeoning problem. Proline-rich AMPs (PrAMPs) are a sub-class of AMPs, that have multiple modes of action including modulation of the bacterial protein folding chaperone, DnaK. They are highly effective against Gram-negative bacteria and have low toxicity to mammalian cells. Previously we used an in silico approach to identify new potential PrAMPs from the DRAMP database. Four of these peptides, antibacterial napin, attacin-C, P9, and PP30, were each chemically assembled and characterized. Together with synthetic oncocin as a reference, each peptide was then assessed for antibacterial activity against Gram-negative/Gram-positive bacteria and for in vitro DnaK modulation activity. We observed that these peptides directly modulate DnaK activity independently of eliciting or otherwise an antibiotic effect. Based on our findings, we propose a change to our previously established PrAMP definition to remove the requirement for antimicrobial activity in isolation, leaving the following classifiers: & % proline, modulation of DnaK AND/OR the 70S ribosome, net charge of +1 or more, produced in response to bacterial infection AND/OR with pronounced antimicrobial activity.
The Relaxin Peptide Family – Structure, Function and Clinical Applications
Publisher: Bentham Science Publishers Ltd.
Date: 03-2011
DOI: 10.2174/092986611794578396
Abstract: The relaxin peptide family in humans consists of seven members, relaxin-1, -2 and -3 and insulin-like (INSL) peptides 3, 4, 5 and 6. It is an offshoot of the large insulin superfamily. Each member consists of two chains, commonly referred to as A and B, which are held together by two inter-chain disulfide bonds and another intra-chain disulfide bond present within the A chain. The cysteine residues present in each chain, together with the distinctive disulfide bonding pattern, are conserved across all members of the superfamily. The chemical synthesis of these complex peptides poses a significant challenge. In the past, random combination of the two synthetic S-reduced chains under oxidizing conditions was utilized to form the three disulfide bonds. Nowadays, with the aid of highly efficient solid phase peptide synthesis methodologies, in conjunction with selective S-thiol-protecting groups, combination of in idual A- and B- chains by sequential chemical formation of each of the three disulfide bonds is now possible resulting in good yields of these peptides. The relaxin peptide family members bind to G-protein coupled receptors (GPCRs) which have been classified as relaxin family peptide (RXFP) receptors. The various unique receptor-ligand interactions are outlined in this review, together with the physiological roles of the relaxin peptide family members and lastly their past and present clinical applications.
Single chain peptide agonists of relaxin receptors
Publisher: Elsevier BV
Date: 05-2019
DOI: 10.1016/J.MCE.2019.01.008
Abstract: There are seven human relaxin family peptides that have two chains (A and B) and three disulfide bonds. The target receptors for four of these peptides are known as relaxin family peptide receptors, RXFP1-RXFP4. Detailed structure-activity relationship (SAR) studies of relaxin family peptides have been reported over the years and have led to the design of new analogs with agonistic and antagonistic properties. This review briefly summarizes the SAR of human relaxin 2 (H2 relaxin) and human relaxin 3 (H3 relaxin) leading to the design and development of single-B-chain only agonists, B7-33 and peptide 5. The physiological functions of these new peptides agonists in cellular and animal models are also described.
The Roles of the A- and B-Chains of Human Relaxin-2 and -3 on Their Biological Activity
Publisher: Bentham Science Publishers Ltd.
Date: 12-2010
DOI: 10.2174/138920310794557736
Abstract: Two members of the human insulin/relaxin superfamily, relaxins-2 and 3 (H2 and H3 respectively), are separated by nearly 75 years in terms of chronological identification but are both the subject of intense recent biological study. The physiological effects of H2 relaxin include vasodilatory, anti-inflammatory, extracellular matrix remodeling, and angiogenic and anti-ischemic. Because of its potent systemic and renal vasodilatory effects, it is currently undergoing phase III clinical trial for the treatment of acute heart failure. In contrast, H3 relaxin is a highly conserved neuropeptide that has rapidly emerged as an important regulator of homeostatic physiology and complex behaviors. Because of their immense clinical potential, an understanding of the structural features that control their functions is critical for rational drug design and development. The native receptor for H2 relaxin is RXFP1. It also strongly binds to the related receptor, RXFP2. The native receptor for H3 relaxin is the unrelated receptor, RXFP3 however, it also has high affinity for another related receptor, RXFP4. Interestingly, H3 relaxin also has a high affinity for RXFP1 and can interact with RXFP2 with a significantly lower affinity. H3 relaxin thus interacts with all four of the relaxin family receptors. Previous studies have shown that H2 and H3 relaxins interact with their receptors primarily using their B-chain specific residues. However, more recent studies suggest that the role of the respective A and B chains for their activity is both peptide- and receptor-dependent. This mini-review summarizes these recent findings on the structure-activity relationships of H2 and H3 relaxins.
Synthesis, conformation, and activity of human insulin-like peptide 5 (INSL5)
Publisher: Wiley
Date: 11-07-2008
Sequential Formation of Regioselective Disulfide Bonds in Synthetic Peptides with Multiple Disulfide Bonds
Publisher: Humana Press
Date: 2013
DOI: 10.1007/978-1-62703-544-6_5
Abstract: Numerous methods have been developed for the formation of disulfide bonds in recombinant DNA-derived and chemically synthesized peptides and proteins, but only a few have found widespread acceptance. The choice of method(s) for formation of disulfide in synthetic peptides and proteins needs to be tailored for each in idual polypeptide in such a way so that the reaction conditions are selective, efficient, and safe and give the maximum yield. Here we describe the sequential formation of three disulfide bonds regioselectively which has been optimized for the synthesis of two-chained, heterodimeric polypeptide members of the insulin-relaxin superfamily.
Minimum active structure of insulin-like peptide 5
Publisher: American Chemical Society (ACS)
Date: 21-11-2013
DOI: 10.1021/JM400924P
Abstract: Insulin-like peptide 5 (INSL5) is a complex two-chain peptide hormone constrained by three disulfide bonds in a pattern identical to insulin. High expression of INSL5 in the colon suggests roles in activation of colon motility and appetite control. A more recent study indicates it may have significant roles in the regulation of insulin secretion and β-cell homeostasis. This peptide thus has considerable potential for the treatment of eating disorders, obesity, and/or diabetes. However, the synthesis of INSL5 is extremely challenging either by chemical or recombinant means. The A-chain is very poorly soluble and the B-chain is highly aggregating in nature which, together, makes their postsynthesis handling and purification very difficult. Given these difficulties, we have developed a highly active INSL5 analogue that has a much simpler structure with two disulfide bonds and is thus easier to assemble compared to native INSL5. This minimized peptide represents an attractive new mimetic for investigating the functional role of INSL5.
818: A single-chain derivative of the relaxin hormone (b7-33) protects cytotrophoblasts from hyperglycemia-induced preeclampsia phenotype and induces the survival pathway
Publisher: Elsevier BV
Date: 2017
Chimeric relaxin peptides highlight the role of the A-chain in the function of H2 relaxin
Publisher: Elsevier BV
Date: 05-2012
DOI: 10.1016/J.PEPTIDES.2012.02.021
Abstract: Human gene-2 (H2) relaxin is a member of the insulin-relaxin peptide superfamily. Because of the potential clinical applications of H2 relaxin, there is a need for novel analogs that have improved biological activity and receptor specificity. In this respect, we have chemically assembled chimeric peptides consisting of the B-chain of H2 relaxin in combination with A-chains from other insulin/relaxin family members. The peptides were prepared using solid phase peptide synthesis together with regioselective disulfide bond formation and characterized by RP-HPLC, MALDI-TOF MS and amino acid analysis. Their in vitro activity was assessed in RXFP1 or RXFP2 expressing cells. Replacement of the H2 relaxin A-chain resulted in parallel losses of binding affinity and activity on RXFP1. Not surprisingly H1A-H2B demonstrated the highest activity as the H1 A-chain shares high homology with H2 relaxin whereas INSLA-H2B, which shows low homology, had very poor activity. Importantly A-chain replacements had a dramatic effect on RXFP2 activity similar to previous results demonstrating different modes of activation of A-chain variants on RXFP1 and RXFP2. H3A-H2B is particularly interesting as it displays moderate activity at RXFP1 but poor activity at RXFP2 indicating that it may be a template for specific RXFP1 agonist development. Our study confirms that the activity of H2 relaxin at both RXFP1 and RXFP2 relies on interactions with both the B- and A-chains, and also provide new biochemical insights into the mechanism of relaxin action that the A-chain needs to be in native or near-native form for strong RXFP1 or RXFP2 agonist activity.
Computational screening of 645 antiviral peptides against the receptor-binding domain of the spike protein in SARS-CoV-2
Publisher: Elsevier BV
Date: 09-2021
Challenges in the design of insulin and relaxin/insulin-like peptide mimetics.
Publisher: Elsevier BV
Date: 06-2017
DOI: 10.1016/J.BMC.2017.09.030
Abstract: Peptidomimetics are designed to overcome the poor pharmacokinetics and pharmacodynamics associated with the native peptide or protein on which they are based. The design of peptidomimetics starts from developing structure-activity relationships of the native ligand-target pair that identify the key residues that are responsible for the biological effect of the native peptide or protein. Then minimization of the structure and introduction of constraints are applied to create the core active site that can interact with the target with high affinity and selectivity. Developing peptidomimetics is not trivial and often challenging, particularly when peptides' interaction mechanism with their target is complex. This review will discuss the challenges of developing peptidomimetics of therapeutically important insulin superfamily peptides, particularly those which have two chains (A and B) and three disulfide bonds and whose receptors are known, namely insulin, H2 relaxin, H3 relaxin, INSL3 and INSL5.
Expression of the relaxin family peptide 4 receptor by enterochromaffin cells of the mouse large intestine
Publisher: Springer Science and Business Media LLC
Date: 21-05-2022
DOI: 10.1007/S00441-022-03635-8
Abstract: The gastrointestinal hormone, insulin-like peptide 5 (INSL5), is found in large intestinal enteroendocrine cells (EEC). One of its functions is to stimulate nerve circuits that increase propulsive activity of the colon through its receptor, the relaxin family peptide 4 receptor (RXFP4). To investigate the mechanisms that link INSL5 to stimulation of propulsion, we have determined the localisation of cells expressing Rxfp4 in the mouse colon, using a reporter mouse to locate cells expressing the gene. The fluorescent signal indicating the location of Rxfp4 expression was in EEC, the greatest overlap of Rxfp4 -dependent labelling being with cells containing 5-HT. In fact, 90% of 5-HT cells were positive for Rxfp4 labelling. A small proportion of cells with Rxfp4 -dependent labelling was 5-HT-negative, 11–15% in the distal colon and rectum, and 35% in the proximal colon. Of these, some were identified as L-cells by immunoreactivity for oxyntomodulin. Rxfp4 -dependent fluorescence was also found in a sparse population of nerve endings, where it was colocalised with CGRP. We used the RXFP4 agonist, INSL5-A13, to activate the receptor and probe the role of the 5-HT cells in which it is expressed. INSL5-A13 administered by i.p. injection to conscious mice caused an increase in colorectal propulsion that was antagonised by the 5-HT 3 receptor blocker, alosetron, also given i.p. We conclude that stimuli that excite INSL5-containing colonic L-cells release INSL5 that, through RXFP4, excites 5-HT release from neighbouring endocrine cells, which in turn acts on 5-HT 3 receptors of enteric sensory neurons to elicit propulsive reflexes.
C‐Terminal Modification and Multimerization Increase the Efficacy of a Proline‐Rich Antimicrobial Peptide
Publisher: Wiley
Date: 16-11-2016
Abstract: Two series of branched tetramers of the proline-rich antimicrobial peptide (PrAMP), Chex1-Arg20, were prepared to improve antibacterial selectivity and potency against a panel of Gram-negative nosocomial pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. First, tetramerization was achieved by dithiomaleimide (DTM) conjugation of two C-terminal-cysteine bearing dimers that also incorporated C-terminal peptide chemical modification. DTM-linked tetrameric peptides containing a C-terminal hydrazide moiety on each dimer exhibited highly potent activities in the minimum inhibitory concentration (MIC) range of 0.49-2.33 μm. A second series of tetrameric analogues with C-terminal hydrazide modification was prepared by using alternative conjugation linkers including trans-1,4-dibromo-2-butene, α,α'-dibromo-p-xylene, or 6-bismaleimidohexane to determine the effect of length on activity. Each displayed potent and broadened activity against Gram-negative nosocomial pathogens, particularly the butene-linked tetrameric hydrazide. Remarkably, the greatest MIC activity is against P. aeruginosa (0.77 μm/8 μg mL
AT1R-AT2R-RXFP1 Functional Crosstalk in Myofibroblasts
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 11-09-2019
Abstract: Studies have shown that the hormone serelaxin, which has organ-protective actions mediated via relaxin family peptide receptor 1 (RXFP1), its cognate G protein–coupled receptor, requires the angiotensin II type 2 receptor (AT 2 R) to ameliorate renal fibrogenesis in vitro and in vivo . In this study, the authors describe a functional interaction between RXFP1, AT 2 R, and the angiotensin II type 1 receptor (AT 1 R), all of which are expressed on extracellular matrix–producing myofibroblasts, the cellular basis of progressive fibrosis. The crosstalk between these G protein–coupled receptors allows antagonists acting at each receptor to directly or allosterically block the antifibrotic actions of agonists acting at AT 2 R or RXFP1. These findings have significant therapeutic implications for a mechanistic understanding of the concomitant use of drugs acting at each receptor. Recombinant human relaxin-2 (serelaxin), which has organ-protective actions mediated via its cognate G protein–coupled receptor relaxin family peptide receptor 1 (RXFP1), has emerged as a potential agent to treat fibrosis. Studies have shown that serelaxin requires the angiotensin II (AngII) type 2 receptor (AT 2 R) to ameliorate renal fibrogenesis in vitro and in vivo . Whether its antifibrotic actions are affected by modulation of the AngII type 1 receptor (AT 1 R), which is expressed on myofibroblasts along with RXFP1 and AT 2 R, is unknown. We examined the signal transduction mechanisms of serelaxin when applied to primary rat renal and human cardiac myofibroblasts in vitro , and in three models of renal- or cardiomyopathy-induced fibrosis in vivo . The AT 1 R blockers irbesartan and candesartan abrogated antifibrotic signal transduction of serelaxin via RXFP1 in vitro and in vivo . Candesartan also ameliorated serelaxin’s antifibrotic actions in the left ventricle of mice with cardiomyopathy, indicating that candesartan’s inhibitory effects were not confined to the kidney. We also demonstrated in a transfected cell system that serelaxin did not directly bind to AT 1 Rs but that constitutive AT 1 R–RXFP1 interactions could form. To potentially explain these findings, we also demonstrated that renal and cardiac myofibroblasts expressed all three receptors and that antagonists acting at each receptor directly or allosterically blocked the antifibrotic effects of either serelaxin or an AT 2 R agonist (compound 21). These findings have significant implications for the concomitant use of RXFP1 or AT 2 R agonists with AT 1 R blockers, and suggest that functional interactions between the three receptors on myofibroblasts may represent new targets for controlling fibrosis progression.
Site of fluorescent label modifies interaction of melittin with live cells and model membranes
Publisher: Elsevier BV
Date: 10-2015
DOI: 10.1016/J.BBAMEM.2015.06.004
Abstract: The mechanism of membrane disruption by melittin (MLT) of giant unilamellar vesicles (GUVs) and live cells was studied using fluorescence microscopy and two fluorescent synthetic analogues of MLT. The N-terminus of one of these was acylated with thiopropionic acid to enable labeling with maleimido-AlexaFluor 430 to study the interaction of MLT with live cells. It was compared with a second analogue labeled at P14C. The results indicated that the fluorescent peptides adhered to the membrane bilayer of phosphatidylcholine GUVs and inserted into the plasma membrane of HeLa cells. Fluorescence and light microscopy revealed changes in cell morphology after exposure to MLT peptides and showed bleb formation in the plasma membrane of HeLa cells. However, the membrane disruptive effect was dependent upon the location of the fluorescent label on the peptide and was greater when MLT was labeled at the N-terminus. Proline at position 14 appeared to be important for antimicrobial activity, hemolysis and cytotoxicity, but not essential for cell membrane disruption.
Probing the functional domains of relaxin-3 and the creation of a selective antagonist for RXFP3/GPCR135 over relaxin receptor RXFP1/LGR7
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2008.03790.X
Abstract: Both relaxin-3 and its receptor (RXFP3, also known as GPCR135) are predominantly expressed in brain regions known to play important roles in processing sensory signals. Recent studies have shown that relaxin-3 is involved in the regulation of stress and feeding behaviors. The mechanisms underlying the involvement of relaxin-3/RXFP3 in the regulation of stress, feeding, and other potential functions remain to be studied. Since relaxin-3 also activates the relaxin receptor (RXFP1, also known as LGR7), which is also expressed in the brain, selective RXFP3 agonists and antagonists are crucial for study of the physiological functions of relaxin-3 and RXFP3 in vivo. The finding that the B chain of relaxin-3 is an agonist for RXFP3 (albeit at low potency) but not RXFP1 suggests that the B chain of relaxin-3 plays a dominant role for RXFP3 binding and activation. Chimeric peptide studies using the B chain from relaxin-3 and the A chains from different members of the insulin and relaxin family have confirmed this hypothesis and led to the generation of R3/I5 (a chimeric peptide with relaxin-3 B chain and INSL5 A chain) as a selective agonist for RXFP3 over RXFP1. Truncation of the C-terminus of the B chain of R3/I5 results in a high-affinity antagonist, R3(BDelta23-27)R/I5, for RXFP3 over RXFP1. R3(BDelta23-27)R/I5 has pA2 values of 9.15 and 9.6 for human and rat RXFP3, respectively, but has no affinity or agonistic activity for the human and rat RXFP1. Ongoing and future in vivo studies using the selective agonist and antagonist for RXFP3 will shed light on the physiological role of the relaxin-3 system.
Identification of key residues essential for the structural fold and receptor selectivity within the A-chain of human gene-2 (H2) relaxin
Publisher: Elsevier BV
Date: 11-2012
Imaging the action of antimicrobial peptides on living bacterial cells
Publisher: Springer Science and Business Media LLC
Date: 27-03-2013
DOI: 10.1038/SREP01557
Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone
Publisher: Elsevier BV
Date: 04-2012
DOI: 10.1016/J.BBRC.2012.02.141
Abstract: Diethylenetriamine pentaacetic acid (DTPA) is a popular chelator agent for enabling the labeling of peptides for their use in structure-activity relationship study and biodistribution analysis. Solid phase peptide synthesis was employed to couple this commercially available chelator at the N-terminus of either the A-chain or B-chain of H2 relaxin. The coupling of the DTPA chelator at the N-terminus of the B-chain and subsequent loading of a lanthanide (europium) ion into the chelator led to a labeled peptide (Eu-DTPA-(B)-H2) in low yield and having very poor water solubility. On the other hand, coupling of the DTPA and loading of Eu at the N-terminus of the A-chain led to a water-soluble peptide (Eu-DTPA-(A)-H2) with a significantly improved final yield. The conjugation of the DTPA chelator at the N-terminus of the A-chain did not have any impact on the secondary structure of the peptide determined by circular dichroism spectroscopy (CD). On the other hand, it was not possible to determine the secondary structure of Eu-DTPA-(B)-H2 because of its insolubility in phosphate buffer. The B-chain labeled peptide Eu-DTPA-(B)-H2 required solubilization in DMSO prior to carrying out binding assays, and showed lower affinity for binding to H2 relaxin receptor, RXFP1, compared to the water-soluble A-chain labeled peptide Eu-DTPA-(A)-H2. The mono-Eu-DTPA labeled A-chain peptide, Eu-DTPA-(A)-H2, thus can be used as a valuable probe to study ligand-receptor interactions of therapeutically important H2 relaxin analogs. Our results show that it is critical to choose an approriate site for incorporating chelators such as DTPA. Otherwise, the bulky size of the chelator, depending on the site of incorporation, can affect yield, solubility, structure and pharmacological profile of the peptide.
Preliminary structure-function relationship studies on insulin-like peptide 5 (INSL5)
Publisher: Springer Science and Business Media LLC
Date: 17-01-2013
B7‐33, a Functionally Selective Relaxin Receptor 1 Agonist, Attenuates Myocardial Infarction–Related Adverse Cardiac Remodeling in Mice
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 21-04-2020
Abstract: Human relaxin‐2 is a peptide hormone capable of pleiotropic effects in several organ systems. Its recombinant formulation (serelaxin) has been demonstrated to reduce infarct size and prevent excessive scar formation in animal models of cardiac ischemia‐reperfusion injury. B7‐33, a synthetically designed peptide analogous to B‐chain of relaxin‐2, invokes signaling at relaxin family peptide receptor 1 (cognate receptor for relaxin‐2) by preferentially phosphorylating the mitogen‐activated protein kinase extracellular signal‐regulated kinase 1/2. We sought to investigate the effects of B7‐33 treatment post ischemia‐reperfusion injury in mice. Adult male CD 1 mice were subjected to ischemia‐reperfusion via ligation of left anterior descending artery for 30 minutes, followed by 24 hours or 7 days of reperfusion. Echocardiography was performed to assess cardiac function, and cardiac tissue was stained to determine infarct size at 24 hours. B7‐33 significantly reduced infarct size (21.99% versus 45.32% P =0.02) and preserved fractional shortening (29% versus 23% P =0.02) compared with vehicle. The difference in fractional shortening further increased at 7 days post myocardial infarction (29% versus 20% for B7‐33 and vehicle groups, respectively). In vitro , primary cardiomyocytes were isolated from adult hearts and subjected to simulated ischemia‐reperfusion injury (simulated ischemia reoxygenation). B7‐33 (50 and 100 nmol/L) improved cell survival and reduced the expression of GRP 78 (glucose regulated protein), an endoplasmic reticulum stress marker. Subsequently, B7‐33 (100 nmol/L) reduced tunicamycin (2.5 μg/mL) induced upregulation of GRP 78 in an extracellular signal‐regulated kinase 1/2–dependent manner. B7‐33 confers acute cardioprotection and limits myocardial infarction–related adverse remodeling in mice by attenuating cardiomyocyte death and endoplasmic reticulum stress as well as preserving cardiac function.
ChemInform Abstract: Intramolecular Acyl Transfer in Peptide and Protein Ligation and Synthesis
Publisher: Wiley
Date: 23-04-2015
Human Insulin-like Peptide 5 (INSL5). Identification of a Simplified Version of Two-Chain Analog A13
Publisher: American Chemical Society (ACS)
Date: 19-10-2020
Design, synthesis and characterization of a fluorescently labeled functional analog of full-length human ghrelin
Publisher: Elsevier BV
Date: 12-2020
Further Developments towards a Minimal Potent Derivative of Human Relaxin-2
Publisher: MDPI AG
Date: 11-08-2023
Abstract: Human relaxin-2 (H2 relaxin) is a peptide hormone with potent vasodilatory and anti-fibrotic effects, which is of interest for the treatment of heart failure and fibrosis. H2 relaxin binds to the Relaxin Family Peptide Receptor 1 (RXFP1). Native H2 relaxin is a two-chain, three-disulfide-bond-containing peptide, which is unstable in human serum and difficult to synthesize efficiently. In 2016, our group developed B7-33, a single-chain peptide derived from the B-chain of H2 relaxin. B7-33 demonstrated poor affinity and potency in HEK cells overexpressing RXFP1 however, it displayed equivalent potency to H2 relaxin in fibroblasts natively expressing RXFP1, where it also demonstrated the anti-fibrotic effects of the native hormone. B7-33 reversed organ fibrosis in numerous pre-clinical animal studies. Here, we detail our efforts towards a minimal H2 relaxin scaffold and attempts to improve scaffold activity through Aib substitution and hydrocarbon stapling to re-create the peptide helicity present in the native H2 relaxin.
Distinct but overlapping binding sites of agonist and antagonist at the relaxin family peptide 3 (RXFP3) receptor
Publisher: Elsevier BV
Date: 10-2018
Spawning induction of the high-value white teatfish sea cucumber, Holothuria fuscogilva, using recombinant relaxin-like gonad stimulating peptide (RGP)
Publisher: Elsevier BV
Date: 2022
Correction to “Atomic Force Microscopy Reveals the Mechanobiology of Lytic Peptide Action on Bacteria”
Publisher: American Chemical Society (ACS)
Date: 06-06-2016
Structure and activity in the relaxin family of peptides.
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2009.03955.X
Abstract: The availability of improved peptide synthesis procedures, convenient and sensitive assays for receptor binding and activation, together with advances in methods for structural characterization, has enabled the key structural features of the relaxin family of peptides responsible for biological activity to be defined. Not surprisingly, despite the similarities in primary amino acid sequences, different structural domains and residues are involved in the binding and activation at the four known relaxin family peptide receptors (RXFP1 to -4). Most of our knowledge on structure and function relates to the relaxin-RXFP1, insulin-like peptide 3 (INSL3)-RXFP2, and relaxin-3-RXFP3 systems, with information accumulating not only on the critical ligand structures but also the domains and residues on the receptor itself that are required for specificity and activation. These studies provide the framework for the design of small-molecule mimetics. While the B-chain cassette R-X-X-X-R-X-X-I, defined by Büllesbach and Schwabe, is essential for binding and activation of RXFP1, it is now recognized that the A chain, particularly the N-terminal domain, is also critical for receptor specificity. Studies of the various endogenous ligand-receptor pairs have led to the design of potent and specific agonists and antagonists. The relaxin-3 A chain-INSL5 B chain chimeric peptide and analogs with C-terminal truncations of the B chain, developed by Liu and colleagues at Johnson & Johnson, have provided selective agonist and antagonist peptides that are proving invaluable for in vivo studies of the relaxin-3-RXFP3 system.
Relaxin Inhibits the Cardiac Myofibroblast NLRP3 Inflammasome as Part of Its Anti-Fibrotic Actions via the Angiotensin Type 2 and ATP (P2X7) Receptors
Publisher: MDPI AG
Date: 25-06-2022
DOI: 10.3390/IJMS23137074
Abstract: Chronic NLRP3 inflammasome activation can promote fibrosis through its production of interleukin (IL)-1β and IL-18. Conversely, recombinant human relaxin (RLX) can inhibit the pro-fibrotic interactions between IL-1β, IL-18 and transforming growth factor (TGF)-β1. Here, the broader extent by which RLX targeted the myofibroblast NLRP3 inflammasome to mediate its anti-fibrotic effects was elucidated. Primary human cardiac fibroblasts (HCFs), stimulated with TGF-β1 (to promote myofibroblast (HCMF) differentiation), LPS (to prime the NLRP3 inflammasome) and ATP (to activate the NLRP3 inflammasome) (T+L+A) or benzoylbenzoyl-ATP (to activate the ATP receptor P2X7R) (T+L+Bz), co-expressed relaxin family peptide receptor-1 (RXFP1), the angiotensin II type 2 receptor (AT2R) and P2X7R, and underwent increased protein expression of toll-like receptor (TLR)-4, NLRP3, caspase-1, IL-1β and IL-18. Whilst RLX co-administration to HCMFs significantly prevented the T+L+A- or T+L+Bz-stimulated increase in these end points, the inhibitory effects of RLX were annulled by the pharmacological antagonism of either RXFP1, AT2R, P2X7R, TLR-4, reactive oxygen species (ROS) or caspase-1. The RLX-induced amelioration of left ventricular inflammation, cardiomyocyte hypertrophy and fibrosis in isoproterenol (ISO)-injured mice, was also attenuated by P2X7R antagonism. Thus, the ability of RLX to ameliorate the myofibroblast NLRP3 inflammasome as part of its anti-fibrotic effects, appeared to involve RXFP1, AT2R, P2X7R and the inhibition of TLR-4, ROS and caspase-1.
Total Chemical Synthesis of a Nonfibrillating Human Glycoinsulin.
Publisher: American Chemical Society (ACS)
Date: 18-12-2020
DOI: 10.1021/JACS.9B11424
Abstract: Glycosylation is an accepted strategy to improve the therapeutic value of peptide and protein drugs. Insulin and its analogues are life-saving drugs for all type I and 30% of type II diabetic patients. However, they can readily form fibrils which is a significant problem especially for their use in insulin pumps. Because of the solubilizing and hydration effects of sugars, it was thought that glycosylation of insulin could inhibit fibril formation and lead to a more stable formulation. Since enzymatic glycosylation results in heterogeneous products, we developed a novel chemical strategy to produce a homogeneous glycoinsulin (disialo-glycoinsulin) in excellent yield (∼60%). It showed a near-native binding affinity for insulin receptors A and B
The Chemical Synthesis of Relaxin and Related Peptides
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2009.03951.X
Abstract: Successful methods for the chemical assembly of insulin-like peptides allow the detailed study of their structure and function relationships. However, the two-chain, three-disulfide bond structure of this family of peptides, which includes relaxin, has long represented a significant challenge with respect to their chemical synthesis. Early efforts involved the random combination of the two synthetic S-reduced chains under oxidizing conditions to spontaneously form the three disulfide bonds. Such an approach, while generally effective for native sequences, is critically dependent upon the presence of intact secondary structures within the in idual chains which guide the subsequent folding and oxidation pathway. This limitation prevents the use of this approach for the preparation of analogs in which these secondary elements are either absent or modified. Nowadays, the use of highly efficient solid-phase peptide synthesis methodologies together with selective S-thiol-protecting groups allows the acquisition of in idual chains that can be combined by effective sequential chemically directed formation of each of the three disulfide bonds. These approaches have allowed the high-yield assembly of an array of insulin-like peptides which, in turn, has provided considerable and valuable structural and biological information.
Role of the intra-A-chain disulfide bond of insulin-like peptide 3 in binding and activation of its receptor, RXFP2
Publisher: Elsevier BV
Date: 09-2010
DOI: 10.1016/J.PEPTIDES.2010.05.021
Abstract: INSL3 is a member of the insulin-IGF-relaxin superfamily and plays a key role in male fetal development and in adult germ cell maturation. It is the cognate ligand for RXFP2, a leucine-rich repeat containing G-protein coupled receptor. To date, and in contrast to our current knowledge of the key structural features that are required for the binding of INSL3 to RXFP2, comparatively little is known about the key residues that are required to elicit receptor activation and downstream cell signaling. Early evidence suggests that these are contained principally within the A-chain. To further explore this hypothesis, we have undertaken an examination of the functional role of the intra-A-chain disulfide bond. Using solid-phase peptide synthesis together with regioselective disulfide bond formation, two analogs of human INSL3 were prepared in which the intra-chain disulfide bond was replaced, one in which the corresponding Cys residues were substituted with the isosteric Ser and the other in which the Cys were removed altogether. Both of these peptides retained nearly full RXFP2 receptor binding but were devoid of cAMP activity (receptor activation), indicating that the intra-A-chain disulfide bond makes a significant contribution to the ability of INSL3 to act as an RXFP2 agonist. Replacement of the disulfide bond with a metabolically stable dicarba bond yielded two isomers of INSL3 that each exhibited bioactivity similar to native INSL3. This study highlights the critical structural role played by the intra-A-chain disulfide bond of INSL3 in mediating agonist actions through the RXFP2 receptor.
(Re)Defining the Proline-Rich Antimicrobial Peptide Family and the Identification of Putative New Members.
Publisher: Frontiers Media SA
Date: 12-2020
DOI: 10.3389/FCHEM.2020.607769
Abstract: As we rapidly approach a post-antibiotic era in which multi-drug resistant bacteria are ever-pervasive, antimicrobial peptides (AMPs) represent a promising class of compounds to help address this global issue. AMPs are best-known for their membrane-disruptive mode of action leading to bacteria cell lysis and death. However, many AMPs are also known to be non-lytic and have intracellular modes of action. Proline-rich AMPs (PrAMPs) are one such class, that are generally membrane permeable and inhibit protein synthesis leading to a bactericidal outcome. PrAMPs are highly effective against Gram-negative bacteria and yet show very low toxicity against eukaryotic cells. Here, we review both the PrAMP family and the past and current definitions for this class of peptides. Computational analysis of known AMPs within the DRAMP database ( dr .cpu-bioinfor.org/ ) and assessment of their PrAMP-like properties have led us to develop a revised definition of the PrAMP class. As a result, we subsequently identified a number of unknown and unclassified peptides containing motifs of striking similarity to known PrAMP-based DnaK inhibitors and propose a series of new sequences for experimental evaluation and subsequent addition to the PrAMP family.
Atomic Force Microscopy Reveals the Mechanobiology of Lytic Peptide Action on Bacteria
Publisher: American Chemical Society (ACS)
Date: 26-05-2015
DOI: 10.1021/ACS.LANGMUIR.5B01011
Abstract: Increasing rates of antimicrobial-resistant medically important bacteria require the development of new, effective therapeutics, of which antimicrobial peptides (AMPs) are among the promising candidates. Many AMPs are membrane-active, but their mode of action in killing bacteria or in inhibiting their growth remains elusive. This study used atomic force microscopy (AFM) to probe the mechanobiology of a model AMP (a derivative of melittin) on living Klebsiella pneumoniae bacterial cells. We performed in situ biophysical measurements to understand how the melittin peptide modulates various biophysical behaviors of in idual bacteria, including the turgor pressure, cell wall elasticity, and bacterial capsule thickness and organization. Exposure of K. pneumoniae to the peptide had a significant effect on the turgor pressure and Young's modulus of the cell wall. The turgor pressure increased upon peptide addition followed by a later decrease, suggesting that cell lysis occurred and pressure was lost through destruction of the cell envelope. The Young's modulus also increased, indicating that interaction with the peptide increased the rigidity of the cell wall. The bacterial capsule did not prevent cell lysis by the peptide, and surprisingly, the capsule appeared unaffected by exposure to the peptide, as capsule thickness and inferred organization were within the control limits, determined by mechanical measurements. These data show that AFM measurements may provide valuable insights into the physical events that precede bacterial lysis by AMPs.
A One‐Pot Chemically Cleavable Bis‐Linker Tether Strategy for the Synthesis of Heterodimeric Peptides
Publisher: Wiley
Date: 16-08-2016
Real‐time examination of cAMP activity at relaxin family peptide receptors using a BRET‐based biosensor
Publisher: Wiley
Date: 24-09-2018
DOI: 10.1002/PRP2.432
A Novel Antagonist Peptide Reveals a Physiological Role of Insulin-Like Peptide 5 in Control of Colorectal Function
Publisher: American Chemical Society (ACS)
Date: 30-08-2021
Central injection of relaxin-3 receptor (RXFP3) antagonist peptides reduces motivated food seeking and consumption in C57BL/6J mice
Publisher: Elsevier BV
Date: 07-2014
DOI: 10.1016/J.BBR.2014.03.037
Abstract: Behavioural arousal in mammals is regulated by various interacting central monoamine- and peptide-neurotransmitter/receptor systems, which function to maintain awake, alert and active states required for performance of goal-directed activities essential for survival, including food seeking. Existing anatomical and functional evidence suggests the highly-conserved neuropeptide, relaxin-3, which signals via its cognate Gi/o-protein coupled receptor, RXFP3, contributes to behavioural arousal and feeding behaviour in rodents. In studies to investigate this possibility further, adult male C57BL/6J mice were treated with the selective RXFP3 antagonist peptides, R3(B1-22)R/I5(A) and R3(B1-22)R, and motivated food seeking and consumption was assessed as a reflective output of behavioural arousal. Compared to vehicle treatment, intracerebroventricular (icv) injection of RXFP3 antagonists reduced: (i) food anticipatory activity before meal time during food restriction (ii) consumption of highly palatable food (iii) consumption of regular chow during the initial dark phase, and (iv) consumption of regular chow after mild (∼4-h) food deprivation. Effects were not due to sedation and appeared to be specifically mediated via antagonism of relaxin-3/RXFP3 signalling, as RXFP3 antagonist treatment did not alter locomotor activity in wild-type mice or reduce palatable food intake in relaxin-3 deficient (knock-out) mice. Notably, in contrast to similar studies in the rat, icv injection of RXFP3 agonists and infusion into the paraventricular hypothalamic nucleus did not increase food consumption in mice, suggesting species differences in relaxin-3/RXFP3-related signalling networks. Together, our data provide evidence that endogenous relaxin-3/RXFP3 signalling promotes motivated food seeking and consumption, and in light of the established biological and translational importance of other arousal systems, relaxin-3/RXFP3 networks warrant further experimental investigation.
Novel Methods for the Chemical Synthesis of Insulin Superfamily Peptides and of Analogues Containing Disulfide Isosteres
Publisher: American Chemical Society (ACS)
Date: 22-08-2017
DOI: 10.1021/ACS.ACCOUNTS.7B00288
Abstract: The insulin superfamily of peptides is ubiquitous within vertebrates and invertebrates and is characterized by the presence of a set of three disulfide bonds in a unique disposition. With the exception of insulin-like growth factors I and II, which are single chain peptides, the remaining 8 members of the human insulin superfamily are two-chain peptides containing one intramolecular and two intermolecular disulfide bridges. These structural features have long made the chemical synthesis of the peptides a considerable challenge, in particular, including their correct disulfide bond pairing and formation. However, they have also afforded the opportunity to develop modern solid phase synthesis methods for the preparation of such peptides that incorporate novel or improved chemical methods for the controlled introduction of both disulfide bonds and their surrogates, both during and after peptide chain assembly. In turn, this has enabled a detailed probing of the structure and function relationship of this small but complex superfamily of peptides. After initially using and subsequently identifying significant limitations of the approach of simultaneous random chain combination and oxidative folding, our laboratory undertook to develop robust chemical synthesis strategies in concert with orthogonal cysteine S-protecting groups and corresponding regioselective disulfide bond formation. These have included the separate synthesis of each of the two chains or of the two chains linked by an artificial C-peptide that is removed following postoxidative folding. These, in turn, have enabled an increased ease of acquisition in a good yield of not only members of human insulin superfamily but other insulin-like peptides. Importantly, these successful methods have enabled, for the first time, a detailed analysis of the role that the disulfide bonds play in the structure and function of such peptides. This was achieved by selective removal of the disulfide bonds or by the judicious insertion of disulfide isosteres that possess structurally subtle variations in bond length, hydrophobicity, and angle. These include lactam, dicarba, and cystathionine, each of which has required modifications to the peptide synthesis protocols for their successful placement within the peptides. Together, these synthesis improvements and the novel chemical developments of cysteine/cystine analogues have greatly aided in the development of novel insulin-like peptide (INSL) analogues, principally with intra-A-chain disulfide isosteres, possessing not only improved functional properties such as increased receptor selectivity but also, with one important and unexpected exception, greater in vivo half-lives due to stability against disulfide reductases. Such analogues greatly will aid further biochemical and pharmacological analyses to delineate the structure-function relationships of INSLs and also future potential drug development.
Total Chemical Synthesis of a Heterodimeric Interchain Bis-Lactam-Linked Peptide: Application to an Analogue of Human Insulin-Like Peptide 3
Publisher: Hindawi Limited
Date: 28-10-2013
DOI: 10.1155/2013/504260
Abstract: Nonreducible cystine isosteres represent important peptide design elements in that they can maintain a near-native tertiary conformation of the peptide while simultaneously extending the in vitro and in vivo half-life of the biomolecule. Ex les of these cystine mimics include dicarba, diselenide, thioether, triazole, and lactam bridges. Each has unique physicochemical properties that impact upon the resulting peptide conformation. Each also requires specific conditions for its formation via chemical peptide synthesis protocols. While the preparation of peptides containing two lactam bonds within a peptide is technically possible and reported by others, to date there has been no report of the chemical synthesis of a heterodimeric peptide linked by two lactam bonds. To examine the feasibility of such an assembly, judicious use of a complementary combination of amine and acid protecting groups together with nonfragment-based, total stepwise solid phase peptide synthesis led to the successful preparation of an analogue of the model peptide, insulin-like peptide 3 (INSL3), in which both of the interchain disulfide bonds were replaced with a lactam bond. An analogue containing a single disulfide-substituted interchain lactam bond was also prepared. Both INSL3 analogues retained significant cognate RXFP2 receptor binding affinity.
Effect of helix-promoting strategies on the biological activity of novel analogues of the B-chain of INSL3
Publisher: Springer Science and Business Media LLC
Date: 07-12-2010
DOI: 10.1007/S00726-008-0219-2
Abstract: Insulin-like 3 (INSL3) is a novel circulating peptide hormone that is produced by testicular Leydig cells and ovarian thecal and luteal cells. In males, INSL3 is responsible for testicular descent during foetal life and suppresses germ cell apoptosis in adult males, whereas in females, it causes oocyte maturation. Antagonists of INSL3 thus have significant potential clinical application as contraceptives in both males and females. Previous work has shown that the INSL3 receptor binding region is largely confined to the B-chain central alpha-helix of the hormone and a conformationally constrained analogue of this has modest receptor binding and INSL3 antagonist activity. In the present study, we have employed and evaluated several approaches for increasing the alpha-helicity of this peptide in order to better present the key receptor binding residues and increase its affinity for the receptor. Analogues of INSL3 with higher alpha-helicity generally had higher receptor binding affinity although other structural considerations limit their effectiveness.
Synthetic covalently linked dimeric form of h2 relaxin retains native RXFP1 activity and has improved in vitro serum stability
Publisher: Hindawi Limited
Date: 2015
DOI: 10.1155/2015/731852
Abstract: Human (H2) relaxin is a two-chain peptide member of the insulin superfamily and possesses potent pleiotropic roles including regulation of connective tissue remodeling and systemic and renal vasodilation. These effects are mediated through interaction with its cognate G-protein-coupled receptor, RXFP1. H2 relaxin recently passed Phase III clinical trials for the treatment of congestive heart failure. However, its in vivo half-life is short due to its susceptibility to proteolytic degradation and renal clearance. To increase its residence time, a covalent dimer of H2 relaxin was designed and assembled through solid phase synthesis of the two chains, including a judiciously monoalkyne sited B-chain, followed by their combination through regioselective disulfide bond formation. Use of a bisazido PEG 7 linker and “click” chemistry afforded a dimeric H2 relaxin with its active site structurally unhindered. The resulting peptide possessed a similar secondary structure to the native monomeric H2 relaxin and bound to and activated RXFP1 equally well. It had fewer propensities to activate RXFP2, the receptor for the related insulin-like peptide 3. In human serum, the dimer had a modestly increased half-life compared to the monomeric H2 relaxin suggesting that additional oligomerization may be a viable strategy for producing longer acting variants of H2 relaxin.
The cell cycle, cancer development and therapy
Publisher: Springer Science and Business Media LLC
Date: 05-08-2022
DOI: 10.1007/S11033-022-07788-1
Abstract: The process of cell ision plays a vital role in cancer progression. Cell proliferation and error-free chromosomes segregation during mitosis are central events in life cycle. Mistakes during cell ision generate changes in chromosome content and alter the balances of chromosomes number. Any defects in expression of TIF1 family proteins, SAC proteins network, mitotic checkpoint proteins involved in chromosome mis-segregation and cancer development. Here we discuss the function of organelles deal with the chromosome segregation machinery, proteins and correction mechanisms involved in the accurate chromosome segregation during mitosis.
Long-Time-Scale Interaction Dynamics between a Model Antimicrobial Peptide and Giant Unilamellar Vesicles
Publisher: American Chemical Society (ACS)
Date: 14-11-2013
DOI: 10.1021/LA403083M
Abstract: The interaction dynamics between a lytic peptide and a biomembrane was studied using time-lapse fluorescence lifetime imaging microscopy. The model membrane was 1,2-dipalmitoyl-sn-glycero-3-phosphochloine giant unilamellar vesicles (GUVs), and the peptide was the K14 derivative of melittin, to which the polarity-sensitive fluorescent probe AlexaFluor 430 was grafted. The interaction of the peptide with the GUVs resulted in a progressive quenching of the fluorescence lifetime over a period of minutes. From previous photophysics characterization of the peptide, we were able to deconvolve the contribution of three distinct peptide states to the lifetime trajectory and use this data to develop a kinetics model for the interaction process. It was found that the peptide-membrane interaction was well described by a two-step mechanism: peptide monomer adsorption followed by membrane surface migration, assembly, and insertion to form membrane pores. There was an equilibrium exchange between pore and surface monomers at all lipid eptide (L/P) concentration ratios, suggesting that the fully inserted phase was reached, even at low peptide concentrations. In contrast to previous studies, there was no evidence of critical behavior irrespective of L/P ratio, lytic pores were the dominant peptide state at equilibrium and were formed even at very low peptide concentrations. We suggest that this behavior is seen in GUVs because their low curvature means low Laplace pressure. Membrane elasticity is therefore relatively ineffective at d ing the thermal fluctuations of lipid molecules that lead to random molecular-level lipid protrusions and membrane undulations. The transient local membrane deformations that result from these thermal fluctuations create the conditions necessary for facile peptide insertion.
Engineering of a Novel Simplified Human Insulin-Like Peptide 5 Agonist.
Publisher: American Chemical Society (ACS)
Date: 12-02-2016
DOI: 10.1021/ACS.JMEDCHEM.5B01786
Abstract: Insulin-like peptide 5 (INSL5) has recently been discovered as only the second orexigenic gut hormone after ghrelin. As we have previously reported, INSL5 is extremely difficult to assemble and oxidize into its two-chain three-disulfide structure. The focus of this study was to generate structure-activity relationships (SARs) of INSL5 and use it to develop a potent and simpler INSL5 mimetic with RXFP4 agonist activity. A series of human and mouse INSL5 (hINSL5/mINSL5) analogues were designed and chemically synthesized, resulting in a chimeric INSL5 analogue exhibiting more than 10-fold higher potency (0.35 nM) at human RXFP4 compared with native hINSL5 (4.57 nM). The SAR study also identified a key residue (K(A15)) in the A-chain of mINSL5 that contributes to improved RXFP4 affinity and potency of mINSL5 compared with hINSL5. This knowledge ultimately led us to engineer a minimized hINSL5 mimetic agonist that retains native hINSL5-like RXFP4 affinity and potency at human RXFP4. This minimized analogue was synthesized in 17.5-fold higher yield and in less time compared with hINSL5.
The Kinetics of Amyloid Fibrillar Aggregation of Uperin 3.5 Is Directed by the Peptide’s Secondary Structure
Publisher: American Chemical Society (ACS)
Date: 06-08-2019
DOI: 10.1021/ACS.BIOCHEM.9B00536
Abstract: Many peptides aggregate into insoluble β-sheet rich amyloid fibrils. Some of these aggregation processes are linked to age-related diseases, such as Alzheimer's disease and type 2 diabetes. Here, we show that the secondary structure of the peptide uperin 3.5 directs the kinetics and mechanism of amyloid fibrillar aggregation. Uperin 3.5 variants were investigated using thioflavin T fluorescence assays, circular dichroism spectroscopy, and structure prediction methods. Our results suggest that those peptide variants with a strong propensity to form an α-helical secondary structure under physiological conditions are more likely to aggregate into amyloid fibrils than peptides in an unstructured or "random coil" conformation. This conclusion is in good agreement with the hypothesis that an α-helical transition state is required for peptide aggregation into amyloid fibrils. Specifically, uperin 3.5 variants in which charged amino acids were replaced by alanine were richer in α-helical content, leading to enhanced aggregation compared to that of wild type uperin 3.5. However, the addition of 2,2,2-trifluoroethanol as a major co-solute or membrane-mimicking phospholipid environments locked uperin 3.5 to the α-helical conformation preventing amyloid aggregation. Strategies for stabilizing peptides into their α-helical conformation could provide therapeutic approaches for overcoming peptide aggregation-related diseases. The impact of the physiological environment on peptide secondary structure could explain aggregation processes in a cellular environment.
Distinct activation modes of the Relaxin Family Peptide Receptor 2 in response to insulin-like peptide 3 and relaxin.
Publisher: Springer Science and Business Media LLC
Date: 12-06-2017
DOI: 10.1038/S41598-017-03638-4
Abstract: Relaxin family peptide receptor 2 (RXFP2) is a GPCR known for its role in reproductive function. It is structurally related to the human relaxin receptor RXFP1 and can be activated by human gene-2 (H2) relaxin as well as its cognate ligand insulin-like peptide 3 (INSL3). Both receptors possess an N-terminal low-density lipoprotein type a (LDLa) module that is necessary for activation and is joined to a leucine-rich repeat domain by a linker. This linker has been shown to be important for H2 relaxin binding and activation of RXFP1 and herein we investigate the role of the equivalent region of RXFP2. We demonstrate that the linker’s highly-conserved N-terminal region is essential for activation of RXFP2 in response to both ligands. In contrast, the linker is necessary for H2 relaxin, but not INSL3, binding. Our results highlight the distinct mechanism by which INSL3 activates RXFP2 whereby ligand binding mediates reorientation of the LDLa module by the linker region to activate the RXFP2 transmembrane domains in conjunction with the INSL3 A-chain. In contrast, relaxin activation of RXFP2 involves a more RXFP1-like mechanism involving binding to the LDLa-linker, reorientation of the LDLa module and activation of the transmembrane domains by the LDLa alone.
A single-chain derivative of the relaxin hormone is a functionally selective agonist of the G protein-coupled receptor, RXFP1.
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C5SC04754D
Abstract: A single-chain derivative of the relaxin hormone ameliorates fibrosis without side-effects.
Human relaxin-2: historical perspectives and role in cancer biology
Publisher: Springer Science and Business Media LLC
Date: 02-08-2012
DOI: 10.1007/S00726-012-1375-Y
Abstract: One of the most recognised and studied family of peptide hormones is the insulin superfamily. Within this family is the relaxin subfamily which comprises seven members: relaxin-1, -2 and -3 and insulin-like peptides 3, 4, 5 and 6. Besides exhibiting sequence similarities, each member exists as an active A-B heterodimer linked by three disulfide bonds. This mini-review is ided into three broad themes: an overview of all insulin superfamily members (including structural similarities) roles of each superfamily member and finally, a focus on the pleiotropic peptide hormone, human relaxin-2. In addition to promoting vasodilatory effects leading to evaluation in Phase III clinical trials for the treatment of acute heart failure, relaxin has recently been shown to be highly expressed by cancer cells, aiding in their proliferation, invasiveness and metastasis. These contrary effects of relaxin are discussed together with current efforts in the development of relaxin antagonists that may possess future therapeutic potential for the treatment of certain cancers.
Coatings Releasing the Relaxin Peptide Analogue B7-33 Reduce Fibrotic Encapsulation
Publisher: American Chemical Society (ACS)
Date: 12-11-2019
Abstract: The development of antifibrotic materials and coatings that can resist the foreign body response (FBR) continues to present a major hurdle in the advancement of current and next-generation implantable medical devices, biosensors, and cell therapies. From an implant perspective, the most important issue associated with the FBR is the prolonged inflammatory response leading to a collagenous capsule that ultimately blocks mass transport and communication between the implant and the surrounding tissue. Up to now, most attempts to reduce the capsule thickness have focused on providing surface coatings that reduce protein fouling and cell attachment. Here, we present an approach that is based on the sustained release of a peptide drug interfering with the FBR. In this study, the biodegradable polymer poly(lactic-
The A-chain of human relaxin family peptides has distinct roles in the binding and activation of the different relaxin family peptide receptors
Publisher: Elsevier BV
Date: 06-2008
The minimal active structure of human relaxin-2
Publisher: Elsevier BV
Date: 10-2011
Solid-phase synthesis of europium-labeled human INSL3 as a novel probe for the study of ligand-receptor interactions
Publisher: American Chemical Society (ACS)
Date: 05-06-2008
DOI: 10.1021/BC800127P
Abstract: An efficient solid-phase synthesis protocol has been developed which, together with regioselective sequential formation of the three disulfide bonds, enabled the preparation of specifically monolanthanide (europium)-labeled human insulin-like peptide 3 (INSL3) for the study of its interaction with its G-protein-coupled receptor, RXFP2, via time-resolved fluorometry. A commercially available chelator, diethylene triamine pentaacetic acid (DTPA), was coupled to the N-terminus of the INSL3 A-chain on the solid phase, and then a coordination complex between europium ion and DTPA was formed using EuCl 3 to protect the chelator from production of an unidentified adduct during subsequent combination of the A- and B-chains. The labeled peptide was purified in high yield using high-performance liquid chromatography with nearly neutral pH buffers to prevent the liberation of Eu (3+) from the chelator. Using time-resolved fluorometry, saturation binding assays were undertaken to determine the binding affinity (p K d) of labeled INSL3 for RXFP2 in HEK-293T cells stably expressing RXFP2. The dissociation constant of DTPA-labeled INSL3 (9.05 +/- 0.03, n = 3) that was obtained from saturation binding experiments was comparable to that of (125)I-labeled INSL3 (9.59 +/- 0.09, n = 3). The receptor binding affinity (p K i) of human INSL3 was determined to be 9.27 +/- 0.06, n = 3, using Eu-DTPA-INSL3 as a labeled ligand, which again is similar to that obtained when (125)I-INSL3 was used as labeled ligand (9.34 +/- 0.02, n = 4). This novel lanthanide-coordinated, DTPA-labeled INSL3 has excellent sensitivity, stability, and high specific activity, properties that will be particularly beneficial in high-throughput screening of INSL3 analogues in structure-activity studies.
R3(BΔ23-27)R/I5 chimeric peptide, a selective antagonist for GPCR135 and GPCR142 over relaxin receptor LGR7: In vitro and in vivo characterization
Publisher: Elsevier BV
Date: 08-2007
A One‐Pot Chemically Cleavable Bis‐Linker Tether Strategy for the Synthesis of Heterodimeric Peptides
Publisher: Wiley
Date: 16-08-2016
Abstract: Heterodimeric peptides linked by disulfide bonds are attractive drug targets. However, their chemical assembly can be tedious, time‐consuming, and low yielding. Inspired by the cellular synthesis of pro‐insulin in which the two constituent peptide chains are expressed as a single‐chain precursor separated by a connecting C‐peptide, we have developed a novel chemically cleavable bis‐linker tether which allows the convenient assembly of two peptide chains as a single “pro”‐peptide on the same solid support. Following the peptide cleavage and post‐synthetic modifications, this bis‐linker tether can be removed in one‐step by chemical means. This method was used to synthesize a drug delivery‐cargo conjugate, TAT‐PKCi peptide, and a two‐disulfide bridged heterodimeric peptide, thionin (7‐19)‐(24‐32R), a thionin analogue. To our knowledge, this is the first report of a one‐pot chemically cleavable bis‐linker strategy for the facile synthesis of cross‐bridged two‐chain peptides.
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