ORCID Profile
0000-0003-3470-923X
Current Organisations
University of Kentucky
,
University of Queensland
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Publisher: Wiley
Date: 07-03-2014
Publisher: Springer Netherlands
Date: 2016
Publisher: Elsevier BV
Date: 10-1993
DOI: 10.1016/0041-0101(93)90407-A
Abstract: A mouse bioassay, validated for the quantification of ciguatoxin in up to 20 mg of ether extract from fish flesh, revealed that 63 +/- 14% of spiked ciguatoxin was recovered using a standard extraction procedure. Except for extracts from the least toxic of ciguateric fish (0.1-0.5 nmol ciguatoxin-1/kg fish), signs in mice of intoxication by ciguatoxin (hypothermia to below 33 degrees C as well as at least severe diarrhoea or lachrymation or hypersalivation) could be distinguished from the toxic reaction that follows administration of ciguatoxin-free ether extracts. Ciguatoxin recovery was similar for four variants of the ether-water partition, with the 2 M NaC1/ether partition extracting half the contaminants. The method described is selective for ciguatoxin and could be used to quantify natural levels ciguatoxin in the flesh of fish in the absence of a validated in vitro test.
Publisher: Public Library of Science (PLoS)
Date: 15-06-2016
Publisher: Wiley
Date: 21-07-2018
DOI: 10.1111/BPH.13910
Publisher: Wiley
Date: 05-04-2021
DOI: 10.1002/AJP.23256
Publisher: Elsevier BV
Date: 11-2000
Publisher: Springer Science and Business Media LLC
Date: 24-03-2014
DOI: 10.1038/NCOMMS4521
Publisher: Elsevier BV
Date: 1997
Publisher: Royal Society of Chemistry
Date: 2013
Publisher: Elsevier BV
Date: 09-2004
Publisher: Research Square Platform LLC
Date: 18-10-2022
DOI: 10.21203/RS.3.RS-2153130/V1
Abstract: Marine cone snails have attracted researchers from all disciplines but early life stages have received limited attention due to difficulties accessing or rearing juvenile specimens. Here, we document for the first time the culture of Conus magus from eggs through metamorphosis to reveal dramatic shifts in predatory feeding behaviour between post-metamorphic juveniles and adult specimens. Adult C. magus capture fish using a set of paralytic venom peptides combined with a hooked radular tooth used to tether envenomed fish. In contrast, early juveniles feed exclusively on polychaete worms using a unique “sting-and-stalk” foraging behaviour facilitated by short, unbarbed radular teeth and a distinct venom repertoire that induces hypoactivity in prey. Our results demonstrate how coordinated morphological, behavioural and molecular changes facilitate the shift from worm- to fish-hunting in C. magus , and showcase juvenile cone snails as a rich and unexplored source of novel venom peptides for ecological, evolutionary and biodiscovery studies.
Publisher: Frontiers Media SA
Date: 21-09-2021
DOI: 10.3389/FMOLB.2021.742457
Abstract: Venom peptides are potent and selective modulators of voltage-gated ion channels that regulate neuronal function both in health and in disease. We previously identified the spider venom peptide Tap1a from the Venezuelan tarantula Theraphosa apophysis that targeted multiple voltage-gated sodium and calcium channels in visceral pain pathways and inhibited visceral mechano-sensing neurons contributing to irritable bowel syndrome. In this work, alanine scanning and domain activity analysis revealed Tap1a inhibited sodium channels by binding with nanomolar affinity to the voltage-sensor domain II utilising conserved structure-function features characteristic of spider peptides belonging to family NaSpTx1. In order to speed up the development of optimized Na V -targeting peptides with greater inhibitory potency and enhanced in vivo activity, we tested the hypothesis that incorporating residues identified from other optimized NaSpTx1 peptides into Tap1a could also optimize its potency for Na V s. Applying this approach, we designed the peptides Tap1a-OPT1 and Tap1a-OPT2 exhibiting significant increased potency for Na V 1.1, Na V 1.2, Na V 1.3, Na V 1.6 and Na V 1.7 involved in several neurological disorders including acute and chronic pain, motor neuron disease and epilepsy. Tap1a-OPT1 showed increased potency for the off-target Na V 1.4, while this off-target activity was absent in Tap1a-OPT2. This enhanced potency arose through a slowed off-rate mechanism. Optimized inhibition of Na V channels observed in vitro translated in vivo , with reversal of nocifensive behaviours in a murine model of Na V -mediated pain also enhanced by Tap1a-OPT. Molecular docking studies suggested that improved interactions within loops 3 and 4, and C-terminal of Tap1a-OPT and the Na V channel voltage-sensor domain II were the main drivers of potency optimization. Overall, the rationally designed peptide Tap1a-OPT displayed new and refined structure-function features which are likely the major contributors to its enhanced bioactive properties observed in vivo . This work contributes to the rapid engineering and optimization of potent spider peptides multi-targeting Na V channels, and the research into novel drugs to treat neurological diseases.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 03-2002
DOI: 10.1016/S0304-3959(01)00436-5
Abstract: N-type calcium channels modulate the release of key pro-nociceptive neurotransmitters such as glutamate and substance P (SP) in the central nervous system. Considerable research interest has focused on the therapeutic potential of the peptidic omega-conopeptides, GVIA and MVIIA as novel analgesic agents, due to their potent inhibition of N-type calcium channels. Recently, the novel peptidic N-type calcium channel blocker, AM336, was isolated from the venom of the cone snail, Conus catus. Thus, the aims of this study were to (i) document the antinociceptive effects of AM336 (also known as CVID) relative to MVIIA following intrathecal (i.t.) bolus dosing in rats with adjuvant-induced chronic inflammatory pain of the right hindpaw and to (ii) quantify the inhibitory effects of AM336 relative to MVIIA on K+-evoked SP release from slices of rat spinal cord. Both AM336 and MVIIA inhibited the K+-evoked release of the pro-nociceptive neurotransmitter, SP, from rat spinal cord slices in a concentration-dependent manner (EC50 values=21.1 and 62.9 nM, respectively), consistent with the antinociceptive actions of omega-conopeptides. Following acute i.t. dosing, AM336 evoked dose-dependent antinociception (ED50 approximately 0.110 nmol) but the doses required to produce side-effects were an order of magnitude larger than the doses required to produce antinociception. For i.t. doses of MVIIA 0.07 nmol, produced a dose-dependent decrease in antinociception but the incidence and severity of the side-effects continued to increase for all doses of MVIIA investigated, suggesting that dose-titration with MVIIA in the clinical setting, may be difficult.
Publisher: Bentham Science Publishers Ltd.
Date: 04-06-2020
DOI: 10.2174/0929867325666181001112821
Abstract: Low Voltage-Activated (LVA) T-type calcium channels are characterized by transient current and Low Threshold Spikes (LTS) that trigger neuronal firing and oscillatory behavior. Combined with their preferential localization in dendrites and their specific “window current”, T-type calcium channels are considered to be key players in signal lification and synaptic integration. Assisted by the emerging pharmacological tools, the structural determinants of channel gating and kinetics, as well as novel physiological and pathological functions of T-type calcium channels, are being uncovered. In this review, we provide an overview of structural determinants in T-type calcium channels, their involvement in disorders and diseases, the development of novel channel modulators, as well as Structure-Activity Relationship (SAR) studies that lead to rational drug design.
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C8MO00181B
Abstract: Physiological and pathological pain involves a complex interplay of multiple cell types and signaling pathways.
Publisher: Elsevier BV
Date: 08-2004
Publisher: Wiley
Date: 02-04-2019
DOI: 10.1111/MMI.14244
Abstract: DivIVA proteins and their GpsB homologues are late cell ision proteins found in Gram-positive bacteria. DivIVA/GpsB proteins associate with the inner leaflet of the cytosolic membrane and act as scaffolds for other proteins required for cell growth and ision. DivIVA/GpsB proteins comprise an N-terminal lipid-binding domain for membrane association fused to C-terminal domains supporting oligomerization. Despite sharing the same domain organization, DivIVA and GpsB serve different cellular functions: DivIVA plays erse roles in ision site selection, chromosome segregation and controlling peptidoglycan homeostasis, whereas GpsB contributes to the spatiotemporal control of penicillin-binding protein activity. The crystal structures of the lipid-binding domains of DivIVA from Bacillus subtilis and GpsB from several species share a fold unique to this group of proteins, whereas the C-terminal domains of DivIVA and GpsB are radically different. A number of pivotal features identified from the crystal structures explain the functional differences between the proteins. Herein we discuss these structural and functional relationships and recent advances in our understanding of how DivIVA/GpsB proteins bind and recruit their interaction partners, knowledge that might be useful for future structure-based DivIVA/GpsB inhibitor design.
Publisher: Elsevier BV
Date: 1990
DOI: 10.1016/0041-0101(90)90116-O
Abstract: Gambierdiscus toxicus strains isolated from Australia and French Polynesia were grown in modified f2 and f10 nutrient media and the cells extracted for ciguatoxin and maitotoxin. The high nutrient enrichment of f2 media induced aberrant cell morphology in both strains whereas the majority of cells grown in f10 media had the typical lenticular shape of wild G. toxicus cells. The Australian strain grew faster and produced greater cell densities than the French Polynesian strain. Different chromatographic types of maitotoxin were extracted from the two G. toxicus strains and purified to homogeneity using reverse-phase high-performance liquid chromatography. The toxins elicited similar bioassay signs in mice, but produced different death-time vs dose relationships. The maitotoxin extracted from the Australian strain eluted later on both straight-phase and reverse-phase chromatographic columns than did the maitotoxin extracted from the French Polynesian strain. The maitotoxin extracted from the French Polynesian strain was chromatographically identical to reference maitotoxin. For each strain no differences were found between maitotoxins extracted from cells grown in f2 or f10 media. Only one toxin was produced by each strain of G. toxicus. Ciguatoxin was not produced by either strain.
Publisher: Wiley
Date: 06-2004
Publisher: Wiley
Date: 17-11-2015
Abstract: Most venomous predators have evolved complex venom primarily to immobilize their prey and secondarily to defend against predators. In a new paradigm, carnivorous marine gastropods of the genus Conus were shown to rapidly and reversibly switch between two types of venoms in response to predatory or defensive stimulus, suggesting that the defensive use of venom may have a more important role in venom evolution and specialization than previously thought. To further investigate this phenomenon, the defensive repertoire of a vermivorous species, Conus planorbis, was deciphered using second-generation sequencing coupled to high-throughput proteomics. The venom gland transcriptome of C. planorbis revealed 182 unique conotoxin precursors from 25 gene superfamilies, with superfamily T dominating in terms of read and paralog numbers. Analysis of the defense-evoked venom revealed that this vermivorous species uses a similarly complex arsenal to deter aggressors as more recently evolved fish- and mollusk-hunting species, with MS/MS validating 23 conotoxin sequences from six superfamilies. Pharmacological characterization of the defensive venom on human receptors identified the nicotinic acetylcholine receptors as a primary target. This work provides the first insights into the composition and biological activity of specifically evolved defensive venoms in vermivorous cone snails.
Publisher: Elsevier BV
Date: 10-2010
DOI: 10.1016/J.TOXICON.2009.07.026
Abstract: Caribbean ciguatoxin-1 (C-CTX-1) induced, after about 1h exposure, muscle membrane depolarisation and repetitive post-synaptic action potentials (APs) in frog neuromuscular preparations. This depolarising effect was also observed in a Ca(2+)-free medium with a strong enhancement of spontaneous quantal transmitter release, compared with control conditions. The ciguatoxin-induced increase in release could be accelerated when Ca(2+) was present in the extracellular medium. C-CTX-1 also enhanced nerve-evoked quantal acetylcholine (ACh) release. At normal neuromuscular junctions loaded with the fluorescent dye FM1-43, C-CTX-1 induced swelling of nerve terminals, an effect that was reversed by hyperosmotic d-mannitol. In myelinated axons, C-CTX-1 increased nodal membrane excitability, inducing spontaneous and repetitive APs. Also, the toxin enlarged the repolarising phase of APs in control and tetraethylammonium-treated axons. Overall, our data suggest that C-CTX-1 affects nerve excitability and neurotransmitter release at nerve terminals. We conclude that C-CTX-1-induced up-regulation of Na(+) channels and the inhibition of K(+) channels, at low nanomolar concentrations, produce a variety of functional dysfunctions that are in part responsible for the human muscle skeletal symptoms observed in ciguatera. All these dysfunctions seem to result from the subtle balance between ionic currents, intracellular Na(+) and Ca(2+) concentrations, and engaged second messengers.
Publisher: Elsevier BV
Date: 12-1997
DOI: 10.1016/S0969-2126(97)00307-9
Abstract: kappa-PVIIA is a 27-residue polypeptide isolated from the venom of Conus purpurascens and is the first member of a new class of conotoxins that block potassium channels. By comparison to other ion channels of eukaryotic cell membranes, voltage-sensitive potassium channels are relatively simple and methodology has been developed for mapping their interactions with small-peptide toxins. PVIIA, therefore, is a valuable new probe of potassium channel structure. This study of the solution structure and mode of channel binding of PVIIA forms the basis for mapping the interacting residues at the conotoxin-ion channel interface. The three-dimensional structure of PVIIA resembles the triple-stranded beta sheet/cystine-knot motif formed by a number of toxic and inhibitory peptides. Subtle structural differences, predominantly in loops 2 and 4, are observed between PVIIA and other conotoxins with similar structural frameworks, however. Electrophysiological binding data suggest that PVIIA blocks channel currents by binding in a voltage-sensitive manner to the external vestibule and occluding the pore. Comparison of the electrostatic surface of PVIIA with that of the well-characterised potassium channel blocker charybdotoxin suggests a likely binding orientation for PVIIA. Although the structure of PVIIA is considerably different to that of the alphaK scorpion toxins, it has a similar mechanism of channel blockade. On the basis of a comparison of the structures of PVIIA and charybdotoxin, we suggest that Lys19 of PVIIA is the residue which is responsible for physically occluding the pore of the potassium channel.
Publisher: Elsevier BV
Date: 2006
DOI: 10.1016/J.BCMD.2005.10.007
Abstract: The mechanisms underlying the swelling of frog red blood cells (RBC), induced by Pacific (P-CTX-1) and Caribbean (C-CTX-1) ciguatoxins (CTXs), were investigated by measuring the length, width and surface of their elliptic shape. P-CTX-1 (0.5 to 5 nM) and C-CTX-1 (1 nM) induced RBC swelling within 60 min. The CTXs-induced RBC swelling was blocked by apamin (1 microM) and by Sr(2+) (1 mM). P-CTX-1-induced RBC swelling was prevented and inhibited by H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (27 microM), an inhibitor of soluble guanylate cyclase (sGC), and NOS blockade by NG methyl-l-arginine (l-NMA 10 microM). Cytochalasin D (cytD, 10 microM) increased RBC surface and mimicked CTX effect but did not prevent the P-CTX-1-induced l-NMA-sensitive extra increase. Calculations revealed that P-CTX-1 and cytD increase RBC total surface envelop and volume. These data strongly suggest that the molecular mechanisms underlying CTXs-induced RBC swelling involve the NO pathway by an activation of the inducible NOS, leading to sGC activation which modulates intracellular cGMP and regulates L-type Ca(2+) channels. The resulting increase in intracellular Ca(2+) content, in turn, disrupts the actin cytoskeleton, which causes a water influx and triggers a Ca(2+)-activated K(+) current through SK2 isoform channels.
Publisher: Informa UK Limited
Date: 11-2002
DOI: 10.1080/02652030210155378
Abstract: A grey snapper (Lutjanus griseus), a grouper (Serranidae) and a black jack (Caranx lugubris) were implicated in three different ciguatera poisonings in Guadeloupe, French West Indies. A mouse bioassay indicated toxicity for each specimens: 0.5-1, > or = 1 and > 1 MUg g(-1), respectively. After purification by gel filtration chromatography, the s les were analysed by high-performance liquid chromatography coupled to mass spectrometry (LC-MS). The toxin profiles differ from one fish to another. C-CTX-1 was detected at 0.24, 0.90 and 13.8 ng g(-1) flesh in the snapper, grouper and jack, respectively. It contributed only to part of the whole toxicity determined by the mouse bioassay. Other toxins identified were C-CTX-2 (a C-CTX-1 epimer), three additional isomers of C-CTX-1 or-2, and five ciguatoxin congeners (C-CTX-1127, C-CTX-1143 and its isomer C-CTX-1143a, and C-CTX-1157 and its isomer C-CTX-1157b). Putative hydroxy-polyether-like compounds were also detected in the flesh of the grouper with [M+ + H]+ ions at m/z 851.51, 857.50, 875.51, 875.49 and 895.54 Da. Some of these compounds have the same mass range as some known dinoflagellate toxins. In conclusion, this study confirms the usefulness of LC-MS analysis to determine the ciguatoxins levels and the toxin profile in fish flesh hazardous to humans.
Publisher: Elsevier BV
Date: 10-2003
Publisher: MDPI AG
Date: 02-10-2020
DOI: 10.3390/BIOMEDICINES8100391
Abstract: Voltage-gated sodium (NaV) channel subtypes, including NaV1.7, are promising targets for the treatment of neurological diseases, such as chronic pain. Cone snail-derived µ-conotoxins are small, potent NaV channel inhibitors which represent potential drug leads. Of the 22 µ-conotoxins characterised so far, only a small number, including KIIIA and CnIIIC, have shown inhibition against human NaV1.7. We have recently identified a novel µ-conotoxin, SxIIIC, from Conus striolatus. Here we present the isolation of native peptide, chemical synthesis, characterisation of human NaV channel activity by whole-cell patch-cl electrophysiology and analysis of the NMR solution structure. SxIIIC displays a unique NaV channel selectivity profile (1.4 1.3 1.1 ≈ 1.6 ≈ 1.7 1.2 1.5 ≈ 1.8) when compared to other µ-conotoxins and represents one of the most potent human NaV1.7 putative pore blockers (IC50 152.2 ± 21.8 nM) to date. NMR analysis reveals the structure of SxIIIC includes the characteristic α-helix seen in other µ-conotoxins. Future investigations into structure-activity relationships of SxIIIC are expected to provide insights into residues important for NaV channel pore blocker selectivity and subsequently important for chronic pain drug development.
Publisher: Elsevier BV
Date: 09-2009
DOI: 10.1016/J.BMC.2009.07.063
Abstract: A number of omega-conotoxin GVIA mimetics based on an anthranilamide core were prepared and tested for their affinity for rat brain Ca(v)2.2 channels. Features such as the presence of hydroxyl and fluoro substituents on the tyrosine side chain mimic, the length of the chains on the lysine/arginine side chain mimics and the use of diguanidino and diamino substituents rather than mono-guanidine/mono-amine substitution were examined. The diguanidinylated compounds proved to be the most active and deletion of the hydroxyl substituent had a limited influence on activity. The SAR associated with variation in the lysine/arginine side chain mimics was not strong. The introduction of a fluoro substituent into the tyrosine mimic produced the most active compound prepared in this study (2g), with an EC(50) at rat brain Ca(v)2.2 channels of 6 microM.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 19-06-2018
DOI: 10.1126/SCISIGNAL.AAO2060
Abstract: The membrane-binding protein annexin A6 suppresses mechanically induced pain in a mouse model of osteoarthritis.
Publisher: Springer Science and Business Media LLC
Date: 24-06-2021
DOI: 10.1038/S41598-021-91919-4
Abstract: The venom duct origins of predatory and defensive venoms has not been studied for hook-and-line fish hunting cone snails despite the pharmacological importance of their venoms. To better understand the biochemistry and evolution of injected predatory and defensive venoms, we compared distal, central and proximal venom duct sections across three specimens of C. striatus ( Pionoconus ) using proteomic and transcriptomic approaches. A total of 370 conotoxin precursors were identified from the whole venom duct transcriptome. Milked defensive venom was enriched with a potent cocktail of proximally expressed inhibitory α-, ω- and μ-conotoxins compared to milked predatory venom. In contrast, excitatory κA-conotoxins dominated both the predatory and defensive venoms despite their distal expression, suggesting this class of conotoxin can be selectively expressed from the same duct segment in response to either a predatory or defensive stimuli. Given the high abundance of κA-conotoxins in the Pionoconus clade, we hypothesise that the κA-conotoxins have evolved through adaptive evolution following their repurposing from ancestral inhibitory A superfamily conotoxins to facilitate the dietary shift to fish hunting and species radiation in this clade.
Publisher: Elsevier BV
Date: 1981
Publisher: Elsevier BV
Date: 09-2007
DOI: 10.1016/J.BBRC.2007.06.138
Abstract: Conotoxins are highly constrained peptide toxins that exhibit pharmaceutically relevant biological activities. We herein report the extent of absorption and profile of distribution of a native alpha-conotoxin, MII and a lipophilic analogue of MII (N-LaaMII) after intravenous (iv) and oral administration to male Sprague-Dawley rats. N-LaaMII is formed by coupling 2-amino-D,L-dodecanoic acid (Laa) to the N-terminus of MII and has previously been shown to exhibit significantly improved permeability across Caco-2 cell monolayers compared to the native MII while maintaining the potency in inhibition of nAChRs of the parent peptide. Both peptides crossed the GI tract after oral administration (approximately 6% after 30 m). While Laa conjugation did not significantly improve absorption, it did greatly increase the accumulation of the compound in the liver after iv administration. Neither peptide crossed the blood-brain barrier to any significant extent. This is the first study of the in vivo biodistribution of an alpha-conotoxin after oral administration.
Publisher: MDPI AG
Date: 13-02-2014
Publisher: Oxford University Press (OUP)
Date: 08-04-2014
Publisher: Elsevier BV
Date: 09-2002
DOI: 10.1016/S0041-0101(02)00146-0
Abstract: Optimised gradient reversed-phase high-performance liquid chromatography electrospray ionisation mass spectrometry (LC/MS) methods, in combination with a [3H]-brevetoxin binding assay (RLB), revealed multiple ciguatoxins in a partially purified extract of a highly toxic Lutjanus sebae (red emperor) from the Indian Ocean. Two major ciguatoxins of 1140.6 Da (I-CTX-1 and -2) and two minor ciguatoxins of 1156.6 Da (I-CTX-3 and -4) were identified. Accurate mass analysis revealed that I-CTX-1 and -2 and Caribbean C-CTX-1 had indistinguishable masses (1140.6316 Da, at 0.44 ppm resolution). Toxicity estimated from LC/MS/RLB responses indicated that I-CTX-1 and -2 were both approximately 60% the potency of Pacific ciguatoxin-1 (P-CTX-1). In contrast to ciguatoxins of the Pacific where the more oxidised ciguatoxins are more potent, I-CTX-3 and -4 were approximately 20% of P-CTX-1 potency. Interconversion in dilute acid or on storage, typical of spiroketal and hemiketal functionality found in P-CTXs and C-CTXs, respectively, was not observed to occur between I-CTX-1 and -2. The ratio of CTX-1 and -2 varied depending on the fish extract being analysed. These results suggest that I-CTX-1 and -2 may arise from separate dinoflagellate precursors that may be oxidatively biotransformed to I-CTX-3 and -4 in fish.
Publisher: Wiley
Date: 03-2000
DOI: 10.1002/(SICI)1099-1352(200003/04)13:2<55::AID-JMR488>3.0.CO;2-O
Publisher: Elsevier BV
Date: 2005
DOI: 10.1016/J.NEUROSCIENCE.2004.09.039
Abstract: We evaluated the effects of Ala-7-conantokin-G (Con-G(A7)) and ifenprodil on the modulation by spermine of [(3)H]MK801 binding to human cortical membranes. Human cortical tissue was obtained at autopsy and stored at -80 degrees C until assay. Both Con-G(A7) and ifenprodil inhibited [(3)H]MK801 binding, but spermine affected these inhibitions differently. Con-G(A7) IC(50) changed little with spermine concentration, indicative of a non-competitive interaction, whereas the rightward shift in ifenprodil IC(50) with increasing spermine concentration suggested partial competition. When the two agents were tested against the biphasic activation of [(3)H]MK801 binding by spermine, they again differed in their effects. In the activation phase Con-G(A7) was a non-competitive inhibitor of spermine activation, and may even enhance the spermine EC(50), while the ifenprodil data indicated a partially competitive interaction. Both agents were non-competitive in the inhibitory phase. Overall, the data suggest that Con-G(A7) and ifenprodil interact differently with the polyamine modulation of the glutamate-N-methyl-D-aspartate receptor.
Publisher: Springer Netherlands
Date: 24-12-2011
DOI: 10.1007/978-94-007-0265-3_3
Abstract: Natural product ligands have contributed significantly to the deorphanisation of TRP ion channels. Furthermore, natural product ligands continue to provide valuable leads for the identification of ligands acting at "orphan" TRP channels. Additional naturally occurring modulators at TRP channels can be expected to be discovered in future, aiding in our understanding of not only their pharmacology and physiology, but also the therapeutic potential of this fascinating family of ion channels.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 08-08-2012
Abstract: The human norepinephrine transporter (NET) is implicated in many neurological disorders and is a target of tricyclic antidepressants and nisoxetine (NX). We used molecular docking simulations to guide the identification of residues likely to affect substrate transport and ligand interactions at NET. Mutations to alanine identified a hydrophobic pocket in the extracellular cavity of NET, comprising residues Thr80, Phe317, and Tyr317, which was critical for efficient norepinephrine (NE) transport. This secondary NE substrate site (NESS-2) overlapped the NX binding site, comprising Tyr84, Phe317, and Tyr317, and was positioned ∼11 Å extracellular to the primary site for NE (NESS-1). Thr80 in NESS-2 appeared to be critical in positioning NE for efficient translocation to NESS-1. Three residues identified as being involved in gating the reverse transport of NE (Arg81, Gln314, and Asp473) did not affect NE affinity for NESS-1. Mutating residues adjacent to NESS-2 abolished NET expression (D75A and L76A) or appeared to affect NET folding (S419A), suggesting important roles in stabilizing NET structure, whereas W308A and F388A at the top of NESS-2 abolished both NE transport and NX binding. Our findings are consistent with a multistep model of substrate transport by NET, for which a second, shallow extracellular NE substrate site (NESS-2) is required for efficient NE transport by NET.
Publisher: Oxford University Press (OUP)
Date: 03-2014
DOI: 10.5740/JAOACINT.SGEMANGER
Abstract: Ciguatoxins are potent neurotoxins with a significant public health impact. Cytotoxicity assays have allowed the most sensitive means of detection of ciguatoxin-like activity without reliance on mouse bioassays and have been invaluable in studying outbreaks. An improvement of these cell-based assays is presented here in which rapid flow cytometric detection of ciguatoxins and saxitoxins is demonstrated using fluorescent voltage sensitive dyes. A depolarization response can be detected directly due to ciguatoxin alone however, an approximate 1000-fold increase insensitivity is observed in the presence of veratridine. These results demonstrate that flow cytometric assessment of ciguatoxins is possible at levels approaching the trace detection limits of our earlier cytotoxicity assays, however, with a significant reduction in analysis time. Preliminary results are also presented for detection of brevetoxins and for automation and throughput improvements to a previously described method for detecting saxitoxins in shellfish extracts.
Publisher: Royal Society of Chemistry (RSC)
Date: 2022
DOI: 10.1039/D1MD00331C
Abstract: Experimental and theoretical evidence that the blockade of Ca V 2.2 ion channels by TCAs is partially responsible for their analgesic effects.
Publisher: Springer Science and Business Media LLC
Date: 08-1995
DOI: 10.1007/BF00176780
Publisher: Elsevier BV
Date: 12-2017
DOI: 10.1016/J.NEUROPHARM.2017.04.008
Abstract: κ-Hexatoxins (κ-HXTXs) are a family of excitotoxic insect-selective neurotoxins from Australian funnel-web spiders that are lethal to a wide range of insects, but display no toxicity towards vertebrates. The prototypic κ-HXTX-Hv1c selectively blocks native and expressed cockroach large-conductance calcium-activated potassium (BK
Publisher: MDPI AG
Date: 06-07-2021
DOI: 10.3390/MD19070387
Abstract: Ciguatera fish poisoning (CFP) and neurotoxic shellfish poisoning syndromes are induced by the consumption of seafood contaminated by ciguatoxins and brevetoxins. Both toxins cause sensory symptoms such as paresthesia, cold dysesthesia and painful disorders. An intense pruritus, which may become chronic, occurs also in CFP. No curative treatment is available and the pathophysiology is not fully elucidated. Here we conducted single-cell calcium video-imaging experiments in sensory neurons from newborn rats to study in vitro the ability of Pacific-ciguatoxin-2 (P-CTX-2) and brevetoxin-1 (PbTx-1) to sensitize receptors and ion channels, (i.e., to increase the percentage of responding cells and/or the response litude to their pharmacological agonists). In addition, we studied the neurotrophin release in sensory neurons co-cultured with keratinocytes after exposure to P-CTX-2. Our results show that P-CTX-2 induced the sensitization of TRPA1, TRPV4, PAR2, MrgprC, MrgprA and TTX-r NaV channels in sensory neurons. P-CTX-2 increased the release of nerve growth factor and brain-derived neurotrophic factor in the co-culture supernatant, suggesting that those neurotrophins could contribute to the sensitization of the aforementioned receptors and channels. Our results suggest the potential role of sensitization of sensory receptors/ion channels in the induction or persistence of sensory disturbances in CFP syndrome.
Publisher: Elsevier BV
Date: 11-2000
DOI: 10.1016/S0168-1605(00)00382-2
Abstract: Ciguatera is an important form of human poisoning caused by the consumption of seafood. The disease is characterised by gastrointestinal, neurological and cardiovascular disturbances. In cases of severe toxicity, paralysis, coma and death may occur. There is no immunity, and the toxins are cumulative. Symptoms may persist for months or years, or recur periodically. The epidemiology of ciguatera is complex and of central importance to the management and future use of marine resources. Ciguatera is an important medical entity in tropical and subtropical Pacific and Indian Ocean regions, and in the tropical Caribbean. As reef fish are increasingly exported to other areas, it has become a world health problem. The disease is under-reported and often misdiagnosed. Lipid-soluble, polyether toxins known as ciguatoxins accumulated in the muscles of certain subtropical and tropical marine finfish cause ciguatera. Ciguatoxins arise from biotransformation in the fish of less polar ciguatoxins (gambiertoxins) produced by Gambierdiscus toxicus, a marine dinoflagellate that lives on macroalgae, usually attached to dead coral. The toxins and their metabolites are concentrated in the food chain when carnivorous fish prey on smaller herbivorous fish. Humans are exposed at the end of the food chain. More than 400 species of fish can be vectors of ciguatoxins, but generally only a relatively small number of species are regularly incriminated in ciguatera. Ciguateric fish look, taste and smell normal, and detection of toxins in fish remains a problem. More than 20 precursor gambiertoxins and ciguatoxins have been identified in G. toxicus and in herbivorous and carnivorous fish. The toxins become more polar as they undergo oxidative metabolism and pass up the food chain. The main Pacific ciguatoxin (P-CTX-1) causes ciguatera at levels=0.1 microg/kg in the flesh of carnivorous fish. The main Caribbean ciguatoxin (C-CTX-1) is less polar and 10-fold less toxic than P-CTX-1. Ciguatoxins activate sodium ion (Na ) channels, causing cell membrane excitability and instability. Worldwide coral bleaching is now well documented, and there is a strong association between global warming and the bleaching and death of coral. This, together with natural environmental factors such as earthquakes and hurricanes, and man-made factors such as tourism, dock construction, sewage and eutrophication, may create more favourable environments for G. toxicus. While low levels of G. toxicus are found throughout tropical and subtropical waters, the presence of bloom numbers is unpredictable and patchy. Only certain genetic strains produce ciguatoxins, and environmental triggers for increasing toxin production are unknown.
Publisher: Elsevier BV
Date: 02-2009
Publisher: Springer Science and Business Media LLC
Date: 28-11-2019
DOI: 10.1038/S41598-019-54186-Y
Abstract: Cone snails use separately evolved venoms for prey capture and defence. While most use a harpoon for prey capture, the Gastridium clade that includes the well-studied Conus geographus and Conus tulipa , have developed a net hunting strategy to catch fish. This unique feeding behaviour requires secretion of “nirvana cabal” peptides to d en the escape response of targeted fish allowing for their capture directly by mouth. However, the active components of the nirvana cabal remain poorly defined. In this study, we evaluated the behavioural effects of likely nirvana cabal peptides on the teleost model, Danio rerio (zebrafish). Surprisingly, the conantokins (NMDA receptor antagonists) and/or conopressins (vasopressin receptor agonists and antagonists) found in C . geographus and C . tulipa venom failed to produce a nirvana cabal-like effect in zebrafish. In contrast, low concentrations of the non-competitive adrenoceptor antagonist ρ-TIA found in C . tulipa venom (EC 50 = 190 nM) dramatically reduced the escape response of zebrafish larvae when added directly to aquarium water. ρ-TIA inhibited the zebrafish α 1 -adrenoceptor, confirming ρ-TIA has the potential to reverse the known stimulating effects of norepinephrine on fish behaviour. ρ-TIA may act alone and not as part of a cabal, since it did not synergise with conopressins and/or conantokins. This study highlights the importance of using ecologically relevant animal behaviour models to decipher the complex neurobiology underlying the prey capture and defensive strategies of cone snails.
Publisher: American Chemical Society (ACS)
Date: 10-04-2009
DOI: 10.1021/BI9000326
Abstract: AlphaD-conotoxins are peptide inhibitors of nicotinic acetylcholine receptors (nAChRs) first described from Conus vexillum (alphaD-VxXIIA-C and renamed here to alphaD-VxXXA, alphaD-VxXXB, and alphaD-VxXXC). In this study, we report cDNA sequences encoding D-superfamily conopeptides identified in the Clade XII Conidae Conus vexillum, Conus capitaneus, Conus mustelinus, and Conus miles, together with partial sequences of corresponding peptides from this family. The D-superfamily signal peptide sequences display greater heterogeneity than reported for other conotoxin superfamilies. Phylogenetic analysis of the relationships among alphaD-conotoxin precursors reveals two distinct groups containing either an EMM or AVV signal peptide sequence motif. Homodimer and heterodimer combinations of predicted mature toxin sequences likely account for the partial amino acid sequences and mass values observed for several of the native dimeric peptide components identified in C. capitaneus, C. miles, and C. mustelinus venom. The discovery of the precursors and several novel conotoxins from different species defines this large conotoxin family and expands our understanding of sequence ersification mechanisms in Conus species.
Publisher: Springer Science and Business Media LLC
Date: 24-02-2011
DOI: 10.1007/S00726-010-0516-4
Abstract: The remarkable potency and pharmacological ersity of animal venoms has made them an increasingly valuable source of lead molecules for drug and insecticide discovery. Nevertheless, most of the chemical ersity encoded within these venoms remains uncharacterized, despite decades of research, in part because of the small quantities of venom available. However, recent advances in the miniaturization of bioassays and improvements in the sensitivity of mass spectrometry and NMR spectroscopy have allowed unprecedented access to the molecular ersity of animal venoms. Here, we discuss these technological developments in the context of establishing a high-throughput pipeline for venoms-based drug discovery.
Publisher: Wiley
Date: 2005
DOI: 10.1002/BIP.20302
Abstract: The chi-conopeptides MrIA and MrIB are 13-residue peptides with two disulfide bonds that inhibit human and rat norepinephrine transporter systems and are of significant interest for the design of novel drugs involved in pain treatment. In the current study we have determined the solution structure of MrIA using NMR spectroscopy. The major element of secondary structure is a beta-hairpin with the two strands connected by an inverse gamma-turn. The residues primarily involved in activity have previously been shown to be located in the turn region (Sharpe, I. A. Palant, E. Schroder, C. I. Kaye, D. M. Adams, D. J. Alewood, P. F. Lewis, R. J. J Biol Chem 2003, 278, 40317-40323), which appears to be more flexible than the beta-strands based on disorder in the ensemble of calculated structures. Analogues of MrIA with N-terminal truncations indicate that the N-terminal residues play a role in defining a stable conformation and the native disulfide connectivity. In particular, noncovalent interactions between Val3 and Hyp12 are likely to be involved in maintaining a stable conformation. The N-terminus also affects activity, as a single N-terminal deletion introduced additional pharmacology at rat vas deferens, while deleting the first two amino acids reduced chi-conopeptide potency.
Publisher: Annual Reviews
Date: 09-2009
DOI: 10.1146/ANNUREV.GENOM.9.081307.164356
Abstract: Throughout evolution, numerous proteins have been convergently recruited into the venoms of various animals, including centipedes, cephalopods, cone snails, fish, insects (several independent venom systems), platypus, scorpions, shrews, spiders, toxicoferan reptiles (lizards and snakes), and sea anemones. The protein scaffolds utilized convergently have included AVIT/colipase rokineticin, CAP, chitinase, cystatin, defensins, hyaluronidase, Kunitz, lectin, lipocalin, natriuretic peptide, peptidase S1, phospholipase A 2 , sphingomyelinase D, and SPRY. Many of these same venom protein types have also been convergently recruited for use in the hematophagous gland secretions of invertebrates (e.g., fleas, leeches, kissing bugs, mosquitoes, and ticks) and vertebrates (e.g., v ire bats). Here, we discuss a number of overarching structural, functional, and evolutionary generalities of the protein families from which these toxins have been frequently recruited and propose a revised and expanded working definition for venom. Given the large number of striking similarities between the protein compositions of conventional venoms and hematophagous secretions, we argue that the latter should also fall under the same definition.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 28-10-2014
Abstract: Ligand binding and conformational changes that accompany signaling from G protein-coupled receptors (GPCRs) have mostly focused on the role of transmembrane helices and intracellular loop regions. However, recent studies, including several GPCRs cocrystallized with bound ligands, suggest that the extracellular surface (ECS) of GPCRs plays an important role in ligand recognition, selectivity, and binding, as well as potentially contributing to receptor activation and signaling. This study applied alanine-scanning mutagenesis to investigate the role of the complete ECS of the α1B-adrenoreceptor on norepinephrine (NE) potency, affinity, and efficacy. Half (24 of 48) of the ECS mutations significantly decreased NE potency in an inositol 1-phosphate assay. Most mutations reduced NE affinity (17) determined from [(3)H]prazosin displacement studies, whereas four mutations at the entrance to the NE binding pocket enhanced NE affinity. Removing the influence of NE affinity and receptor expression levels on NE potency gave a measure of NE efficacy, which was significantly decreased for 11 of 48 ECS mutants. These different effects tended to cluster to different regions of the ECS, which is consistent with different regions of the ECS playing discrete functional roles. Exposed ECS residues at the entrance to the NE binding pocket mostly affected NE affinity, whereas buried or structurally significant residues mostly affected NE efficacy. The broad potential for ECS mutations to affect GPCR function has relevance for the increasing number of nonsynonymous single nucleotide polymorphisms now being identified in GPCRs.
Publisher: American Chemical Society (ACS)
Date: 30-07-2009
DOI: 10.1021/NP900174N
Abstract: The plant Momordica cochinchinensis has traditionally been used in Chinese medicine to treat a variety of illnesses. A range of bioactive molecules have been isolated from this plant, including peptides, which are the focus of this study. Here we report the isolation and characterization of two novel peptides, MCoCC-1 and MCoCC-2, containing 33 and 32 amino acids, respectively, which are toxic against three cancer cell lines. The two peptides are highly homologous to one another, but show no sequence similarity to known peptides. Elucidation of the three-dimensional structure of MCoCC-1 suggests the presence of a cystine knot motif, also found in a family of trypsin inhibitor peptides from this plant. However, unlike its structural counterparts, MCoCC-1 does not inhibit trypsin. MCoCC-1 has a well-defined structure, characterized mainly by a triple-stranded antiparallel beta-sheet, but unlike the majority of cystine knot proteins MCoCC-1 contains a disordered loop presumably as a result of flexibility in a localized region of the molecule. Of the cell lines tested, MCoCC-1 is the most toxic against a human melanoma cell line (MM96L) and is nonhemolytic to human erythrocytes. The role of these peptides within the plant remains to be determined.
Publisher: Elsevier BV
Date: 10-2015
DOI: 10.1016/J.BCP.2014.07.025
Abstract: Neuronal nicotinic acetylcholine receptors (nAChRs) are a erse class of ligand-gated ion channels involved in neurological conditions such as neuropathic pain and Alzheimer's disease. α-Conotoxin [A10L]PnIA is a potent and selective antagonist of the mammalian α7 nAChR with a key binding interaction at position 10. We now describe a molecular analysis of the receptor-ligand interactions that determine the role of position 10 in determining potency and selectivity for the α7 and α3β2 nAChR subtypes. Using electrophysiological and radioligand binding methods on a suite of [A10L]PnIA analogs we observed that hydrophobic residues in position 10 maintained potency at both subtypes whereas charged or polar residues abolished α7 binding. Molecular docking revealed dominant hydrophobic interactions with several α7 and α3β2 receptor residues via a hydrophobic funnel. Incorporation of norleucine (Nle) caused the largest (8-fold) increase in affinity for the α7 subtype (Ki=44nM) though selectivity reverted to α3β2 (IC50=0.7nM). It appears that the placement of a single methyl group determines selectivity between α7 and α3β2 nAChRs via different molecular determinants.
Publisher: Springer Netherlands
Date: 2017
Publisher: Wiley
Date: 22-02-2016
DOI: 10.1002/PSC.2857
Abstract: Peptide dendrimers are a novel class of macromolecules of emerging interest with the potential of delayed renal clearance due to their molecular size and enhanced activity due to the multivalency effect. In this work, an active analogue of the disulfide-rich χ-conotoxin χ-MrIA (χ-MrIA), a norepinephrine reuptake (norepinephrine transporter) inhibitor, was grafted onto a polylysine dendron. Dendron decoration was achieved by employing copper-catalyzed alkyne-azide cycloaddition with azido-PEG chain-modified χ-MrIA analogues, leading to homogenous 4-mer and 8-mer χ-MrIA dendrimers with molecular weights ranging from 8 to 22 kDa. These dendrimers were investigated for their impact on peptide secondary structure, in vitro functional activity, and potential anti-allodynia in vivo. NMR studies showed that the χ-MrIA tertiary structure was maintained in the χ-MrIA dendrimers. In a functional norepinephrine transporter reuptake assay, χ-MrIA dendrimers showed slightly increased potency relative to the azido-PEGylated χ-MrIA analogues with similar potency to the parent peptide. In contrast to χ-MrIA, no anti-allodynic action was observed when the χ-MrIA dendrimers were administered intrathecally in a rat model of neuropathic pain, suggesting that the larger dendrimer structures are unable to diffuse through the spinal column tissue and reach the norepinephrine transporter. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Publisher: Elsevier BV
Date: 1983
Publisher: Bentham Science Publishers Ltd.
Date: 09-2006
DOI: 10.2174/157340606778250216
Abstract: Highly selective Ca(v)2.2 voltage-gated calcium channel (VGCC) inhibitors have emerged as a new class of therapeutics for the treatment of chronic and neuropathic pain. Cone snail venoms provided the first drug in class with FDA approval granted in 2005 to Prialt (omega-conotoxin MVIIA, Elan) for the treatment of neuropathic pain. Since this pioneering work, major efforts underway to develop alternative small molecule inhibitors of Ca(v)2.2 calcium channel have met with varied success. This review focuses on the properties of the Ca(v)2.2 calcium channel in different pain states, the action of omega-conotoxins GVIA, MVIIA and CVID, describing their structure-activity relationships and potential as leads for the design of improved Ca(v)2.2 calcium channel therapeutics, and finally the development of small molecules for the treatment of chronic pain.
Publisher: Elsevier BV
Date: 05-2009
DOI: 10.1016/J.BMCL.2009.03.130
Abstract: We report the synthesis and biological activity of a low molecular weight non-peptidic mimic of the analgesic peptide omega-conotoxin GVIA. The molecular weight of this compound presents a reduction by 193g/mol compared to a previously reported lead. This compound exhibits an EC(50) of 5.8microM and is accessible in only six synthetic steps compared to the original lead (13 steps). We also report several improvements to the original synthetic route.
Publisher: American Chemical Society (ACS)
Date: 10-09-2015
DOI: 10.1021/ACS.JPROTEOME.5B00630
Abstract: Venomous marine cone snails produce a unique and remarkably erse range of venom peptides (conotoxins and conopeptides) that have proven to be invaluable as pharmacological probes and leads to new therapies. Conus catus is a hook-and-line fish hunter from clade I, with ∼20 conotoxins identified, including the analgesic ω-conotoxin CVID (AM336). The current study unravels the venom composition of C. catus with tandem mass spectrometry and 454 sequencing data. From the venom gland transcriptome, 104 precursors were recovered from 11 superfamilies, with superfamily A (especially κA-) conotoxins dominating (77%) their venom. Proteomic analysis confirmed that κA-conotoxins dominated the predation-evoked milked venom of each of six C. catus analyzed and revealed remarkable intraspecific variation in both the intensity and type of conotoxins. High-throughput FLIPR assays revealed that the predation-evoked venom contained a range of conotoxins targeting the nAChR, Cav, and Nav ion channels, consistent with α- and ω-conotoxins being used for predation by C. catus. However, the κA-conotoxins did not act at these targets but induced potent and rapid immobilization followed by bursts of activity and finally paralysis when injected intramuscularly in zebrafish. Our venomics approach revealed the complexity of the envenomation strategy used by C. catus, which contains a mix of both excitatory and inhibitory venom peptides.
Publisher: MDPI AG
Date: 22-10-2012
DOI: 10.3390/MD10102349
Publisher: Wiley
Date: 2012
DOI: 10.1002/BIP.22031
Abstract: Lys2 has previously been implicated as a residue important in binding interactions between omega-conotoxins and the N-type calcium channel. To further assess the importance of this residue, Lys2 to Ala2 derivatives of omega-conotoxins MVIIA and CVID were synthesized and their structures and binding potencies determined. A comparison of the 3D structures of the Ala2 mutants with the parent peptides suggest there are significant structural differences brought about by this substitution. In particular, stabilizing interactions between Lys2 and loop 2 of the omega-conotoxins are removed, leading to greater flexibility in loop 2, which contains Tyr13, a crucial residue for omega-conotoxin binding to the N- and P/Q-type voltage-gated calcium channel (VGCC). The significant drop in binding potencies resulting from replacement of Lys2 thus appears to relate more to entropic factors than to any direct interaction of Lys2 with the VGCC. This has significant implications for the development of a pharmacophore binding model for omega-conotoxins, as removal of Lys2 from consideration suggests that the omega-conotoxins residues that interact with the N-type VGCC reside in loop 2 and 4, and thus cover a significantly smaller and more defined area of the surface of omega-conotoxin than previously thought.
Publisher: American Chemical Society (ACS)
Date: 26-11-1998
DOI: 10.1021/AC980598H
Abstract: Ciguatera is a significant food-borne disease caused by potent polyether toxins (ciguatoxins) which accumulate in the flesh of ciguateric reef fish at risk levels > 0.1 ppb for Pacific ciguatoxins. Research on ciguatera has been severely hindered by the lack of analytical methods that detect and characterize low levels of ciguatoxin in crude extracts of fish. Here we report a new procedure for ciguatoxin analysis based on gradient reversed-phase HPLC/tandem mass spectrometry (HPLC/MS/MS). The method gave a linear response to pure Pacific and Caribbean ciguatoxins (P-CTX-1 and C-CTX-1) and the structurally related brevetoxin (PbTx-2) spiked into crude extracts of fish. Levels equivalent to 40 ppt P-CTX-1, 100 ppt C-CTX-1, and 200 ppt PbTx-2 in fish flesh could be detected by HPLC/MS/MS. Using P-CTX-1 as an internal standard, the analysis of extracts of 30 ciguateric fish from the Caribbean Sea (8 toxic, 12 borderline, and 10 nontoxic by mouse bioassay) confirmed the reliability of the method and allowed an estimated risk level of > 0.25 ppb C-CTX-1 to be established. HPLC/MS/MS provides a sensitive analytical approach, not previously available, for the determination of Pacific and Caribbean ciguatoxins at sub-ppb levels in fish flesh.
Publisher: Wiley
Date: 2012
DOI: 10.1002/BIP.22032
Abstract: μ-Conotoxins are peptide blockers of voltage-gated sodium channels (sodium channels), inhibiting tetrodotoxin-sensitive neuronal (Na(v) 1.2) and skeletal (Na(v) 1.4) subtypes with highest affinity. Structure-activity relationship studies of μ-conotoxins SIIIA, TIIIA, and KIIIA have shown that it is mainly the C-terminal part of the three-loop peptide that is involved in binding to the sodium channel. In this study, we characterize the effect of N- and C-terminal extensions of μ-conotoxins SIIIA, SIIIB, and TIIIA on their potency and selectivity for neuronal versus muscle sodium channels. Interestingly, extending the N- or C-terminal of the peptide by introducing neutral, positive, and/or negatively charged residues, the selectivity of the native peptide can be altered from neuronal to skeletal and the other way around. The results from this study provide further insight into the binding profile of μ-conotoxins at voltage-gated sodium channels, revealing that binding interactions outside the cysteine-stablilized loops can contribute to μ-conotoxin affinity and sodium channel selectivity.
Publisher: American Chemical Society (ACS)
Date: 30-12-2014
DOI: 10.1021/BI400882S
Abstract: α-Conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). Their high selectivity and affinity for the various subtypes of nAChRs have led to significant advances in our understanding of the structure and function of these key ion channels. Here we report the discovery of a novel 4/7 α-conotoxin, MrIC from the venom duct of Conus marmoreus, which acts as an agonist at the endogenous human α7 nAChR in SH-SY5Y cells pretreated with PNU120596 (PNU). This unique agonist activity of MrIC at α7 nAChRs may guide the development of novel α7 nAChR modulators.
Publisher: Elsevier BV
Date: 10-1995
DOI: 10.1016/0041-0101(95)00075-W
Abstract: Purified Lophozozymus pictor toxin (LPTX) shares many properties similar to palytoxin (PTX). LPTX and palytoxin isolated from Palythoa caribaeorum (C-PTX) have similar mol. wts of approx. 2680 on ionspray mass spectrometry (MS). In addition, antibodies against PTX could recognize and bind LPTX. Mixed mode high-performance liquid chromatography (HPLC) of LPTX, C-PTX and H-PTX (isolated from Palythoa tuberculosa) showed a major PTX component common to all three with the characteristic PTX-like UV spectrum at a retention time (Rt) of 17 min. However, LPTX exhibits fluorescence but PTX of equivalent toxicity does not. LPTX showed a unique peak at Rt of approx. 22 min on mixed mode HPLC. In addition, LPTX and C-PTX showed different ion fragmentation patterns on MS/MS. These results suggest that LPTX and the palytoxins are structural isomers, containing at least one difference which gives rise to fluorescence in LPTX.
Publisher: Elsevier BV
Date: 09-2012
DOI: 10.1016/J.BCP.2012.06.024
Abstract: Despite the in vivo lethality of venom, neurotoxicity has not previously been considered a significant complication of envenoming by the Australian pygmy copperhead (Austrelaps labialis). However, recent evidence has emerged demonstrating that this venom contains potent presynaptic and postsynaptic neurotoxicity. The present study describes the isolation and pharmacological characterization of the first postsynaptic neurotoxin, α-EPTX-Al2a, from the venom of A. labialis. α-EPTX-Al2a (8072.77 Da) caused a concentration-dependent block of twitch contractions and a complete block of responses to cholinergic agonists in the chick biventer cervicis nerve-muscle preparation. This action is consistent with postjunctional neurotoxicity. Monovalent tiger snake antivenom prevented the onset of neurotoxicity if applied prior to toxin administration, but was only able to partially reverse neurotoxicity once muscle paralysis had developed. α-EPTX-Al2a produced a potent pseudo-irreversible antagonism of chick muscle nicotinic acetylcholine receptors (nAChRs), with an estimated pA(2) value of 7.902 (K(B) = 12.5 nM). Interestingly, the toxin only produced a modest block of neuronal α7 nAChRs, with an IC(50) of 1.2 μM, and failed to inhibit ganglionic α3β2/α3β4 nAChRs in a fluorescence-based FLIPR assay using SH-SY5Y cells. α-EPTX-Al2a contained 75 amino acid residues with five disulfide bonds that had significant homology to classical long-chain α-neurotoxins. While α-EPTX-Al2a retains most pharmacophore residues critical for binding to muscle-type (α1)(2)βγδ nAChRs it lacks the key Ala(28) and Arg(36) residues important for α7 nAChR affinity. Given that A. labialis venom contains both irreversible presynaptic and postsynaptic neurotoxins, clinicians need to be aware of potential neurotoxic complications associated with pygmy copperhead envenomation.
Publisher: Elsevier BV
Date: 04-2009
Publisher: Springer Science and Business Media LLC
Date: 16-10-2013
Abstract: Conopeptides, often generically referred to as conotoxins, are small neurotoxins found in the venom of predatory marine cone snails. These molecules are highly stable and are able to efficiently and selectively interact with a wide variety of heterologous receptors and channels, making them valuable pharmacological probes and potential drug leads. Recent advances in next-generation RNA sequencing and high-throughput proteomics have led to the generation of large data sets that require purpose-built and dedicated bioinformatics tools for efficient data mining. Here we describe ConoSorter, an algorithm that categorizes cDNA or protein sequences into conopeptide superfamilies and classes based on their signal, pro- and mature region sequence composition. ConoSorter also catalogues key sequence characteristics (including relative sequence frequency, length, number of cysteines, N-terminal hydrophobicity, sequence similarity score) and automatically searches the ConoServer database for known precursor sequences, facilitating identification of known and novel conopeptides. When applied to ConoServer and UniProtKB/Swiss-Prot databases, ConoSorter is able to recognize 100% of known conotoxin superfamilies and classes with a minimum species specificity of 99%. As a proof of concept, we performed a reanalysis of Conus marmoreus venom duct transcriptome and (i) correctly classified all sequences previously annotated, (ii) identified 158 novel precursor conopeptide transcripts, 106 of which were confirmed by protein mass spectrometry, and (iii) identified another 13 novel conotoxin gene superfamilies. Taken together, these findings indicate that ConoSorter is not only capable of robust classification of known conopeptides from large RNA data sets, but can also facilitate de novo identification of conopeptides which may have pharmaceutical importance.
Publisher: Springer Netherlands
Date: 2016
Publisher: MDPI AG
Date: 19-03-2022
Abstract: The defensive use of cone snail venom is hypothesised to have first arisen in ancestral worm-hunting snails and later repurposed in a compartmentalised venom duct to facilitate the dietary shift to molluscivory and piscivory. Consistent with its placement in a basal lineage, we demonstrate that the C. distans venom gland lacked distinct compartmentalisation. Transcriptomics revealed C. distans expressed a wide range of structural classes, with inhibitory cysteine knot (ICK)-containing peptides dominating. To better understand the evolution of the venom gland compartmentalisation, we compared C. distans to C. planorbis, the earliest erging species from which a defence-evoked venom has been obtained, and fish-hunting C. geographus from the Gastridium subgenus that injects distinct defensive and predatory venoms. These comparisons support the hypothesis that venom gland compartmentalisation arose in worm-hunting species and enabled repurposing of venom peptides to facilitate the dietary shift from vermivory to molluscivory and piscivory in more recently erged cone snail lineages.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 09-2013
Publisher: Royal Society of Chemistry (RSC)
Date: 2021
DOI: 10.1039/D1MD00182E
Abstract: Cone snail venoms are richly decorated with posttranslational modifications. We show that tyrosine sulfation and C-terminal amidation increase the structural stability and binding of α-conotoxins.
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C7MB00511C
Abstract: Cone snails use distinct venoms for defence and prey capture. The pharmacology of these neurotoxic peptides have been extensively studied for pharmacological probes, venom evolution mechanisms and potential therapeutics.
Publisher: Wiley
Date: 16-06-2006
DOI: 10.1016/J.FEBSLET.2006.06.011
Abstract: Cone snail venom is a rich source of bioactives, in particular small disulfide rich peptides that disrupt synaptic transmission. Here, we report the discovery of conomap-Vt (Conp-Vt), an unusual linear tetradecapeptide isolated from Conus vitulinus venom. The sequence displays no homology to known conopeptides, but displays significant homology to peptides of the MATP (myoactive tetradecapeptide) family, which are important endogenous neuromodulators in molluscs, annelids and insects. Conp-Vt showed potent excitatory activity in several snail isolated tissue preparations. Similar to ACh, repeated doses of Conp-Vt were tachyphylactic. Since nicotinic and muscarinic antagonists failed to block its effect and Conp-Vt desensitised tissue remained responsive to ACh, it appears that Conp-Vt contractions were non-cholinergic in origin. Finally, biochemical studies revealed that Conp-Vt is the first member of the MATP family with a d-amino acid. Interestingly, the isomerization of L-Phe to D-Phe enhanced biological activity, suggesting that this post-translational modified conopeptide may have evolved for prey capture.
Publisher: Elsevier BV
Date: 10-2010
DOI: 10.1016/J.TOXICON.2009.06.007
Abstract: Ciguatera is a food poisoning identified as the principal risk factor in the consumption of tropical fish in Oceania. The syndrome, which follows ingestion of ciguatoxin-contaminated ciguateric fishes, is characterised by an array of gastrointestinal and neurological features. In this report we examine forensic s les associated with a human fatality using a (3)H-brevetoxin binding assay and reversed-phase HPLC/MS and HPLC/MS/MS. Three Pacific ciguatoxins (P-CTX) were detected in the implicated fish flesh s le by LC-MS/MS, implicating multiple P-CTXs in the fatal case. Additionally, ciguatoxin was identified in a liver s le obtained at post-mortem. The level of ciguatoxin detected (0.14 ppb P-CTX-1 equivalents by binding assay) indicated that at least 10% of the ingested P-CTX-1 remained in the human liver 6 days after the toxic fish was consumed. This study confirms the potential of tropical reef fish to accumulate sufficient P-CTX to be lethal to humans, especially if the liver and viscera are consumed as part of the meal.
Publisher: Elsevier BV
Date: 12-2006
DOI: 10.1016/J.BIOCHI.2006.06.014
Abstract: The venom from Australian elapid snakes contains a complex mixture of polypeptide toxins that adversely affect multiple homeostatic systems within their prey in a highly specific and targeted manner. Included in these toxin families are the recently described venom natriuretic peptides, which display similar structure and vasoactive functions to mammalian natriuretic peptides. This paper describes the identification and detailed comparative analysis of the cDNA transcripts coding for the mature natriuretic peptide from a total of nine Australian elapid snake species. Multiple isoforms were identified in a number of species and represent the first description of a natriuretic peptide from the venom gland for most of these snakes. Two distinct natriuretic peptide isoforms were selected from the common brown snake (Pseudonaja textilis), PtNP-a, and the mulga (Pseudechis australis), PaNP-c, for recombinant protein expression and functional analysis. Only one of these peptides, PtNP-a, displayed cGMP stimulation indicative of normal natriuretic peptide activity. Interestingly, both recombinant peptides demonstrated a dose-dependent inhibition of angiotensin converting enzyme (ACE) activity, which is predictive of the vasoactive effects of the toxin. The natriuretic peptides, however, did not possess any coagulopathic activity, nor did they inhibit or potentiate thrombin, adenosine diphosphate or arachidonic acid induced platelet aggregation. The data presented in this study represent a significant resource for understanding the role of various natriuretic peptides isoforms during the envenomation process by Australian elapid snakes.
Publisher: Wiley
Date: 24-06-2004
DOI: 10.1002/JMR.683
Abstract: alpha-Conotoxins, from cone snails, and alpha-neurotoxins, from snakes, are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) that have overlapping binding sites in the ACh binding pocket. These disulphide-rich peptides are used extensively as tools to localize and pharmacologically characterize specific nAChRs subtypes. Recently, a homology model based on the high-resolution structure of an ACh binding protein (AChBP) allowed the three-fingered alpha-neurotoxins to be docked onto the alpha7 nAChR. To investigate if alpha-conotoxins interact with the nAChR in a similar manner, we built homology models of human alpha7 and alpha3beta2 nAChRs, and performed docking simulations of alpha-conotoxins ImI, PnIB, PnIA and MII using the program GOLD. Docking revealed that alpha-conotoxins have a different mode of interaction compared with alpha-neurotoxins, with surprisingly few nAChR residues in common between their overlapping binding sites. These docking experiments show that ImI and PnIB bind to the ACh binding pocket via a small cavity located above the beta9/beta10 hairpin of the (+)alpha7 nAChR subunit. Interestingly, PnIB, PnIA and MII were found to bind in a similar location on alpha7 or alpha3beta2 receptors mostly through hydrophobic interactions, while ImI bound further from the ACh binding pocket, mostly through electrostatic interactions. These findings, which distinguish alpha-conotoxin and alpha-neurotoxin binding modes, have implications for the rational design of selective nAChR antagonists.
Publisher: Elsevier BV
Date: 03-2021
Publisher: Wiley
Date: 03-1999
DOI: 10.1002/(SICI)1098-2299(199903/04)46:3/4<219::AID-DDR7>3.0.CO;2-S
Publisher: Elsevier BV
Date: 2013
DOI: 10.1016/J.BCP.2012.09.001
Abstract: Their ubiquitous nature, wide cellular distribution and versatile molecular recognition and signalling help make G-protein binding receptors (GPCRs) the most important class of membrane proteins in clinical medicine, accounting for ∼40% of all current therapeutics. A large percentage of current drugs target the endogenous ligand binding (orthosteric) site, which are structurally and evolutionarily conserved, particularly among members of the same GPCR subfamily. With the recent advances in GPCR X-ray crystallography, new opportunities for developing novel subtype selective drugs have emerged. Given the increasing recognition that the extracellular surface conformation changes in response to ligand binding, it is likely that all GPCRs possess an allosteric site(s) capable of regulating GPCR signalling. Allosteric sites are less structurally conserved than their corresponding orthosteric site and thus provide new opportunities for the development of more selective drugs. Constitutive oligomerisation (dimerisation) identified in many of the GPCRs investigated, adds another dimension to the structural and functional complexity of GPCRs. In this review, we compare 60 crystal structures of nine GPCR subtypes (rhodopsin, ß₂-AR, ß₁-AR, A(2a)-AR, CXCR4, D₃R, H₁R, M₂R, M₃R) across four subfamilies of Class A GPCRs, and discuss mechanisms involved in receptor activation and potential allosteric binding sites across the highly variable extracellular surface of these GPCRs. This analysis has identified a new extracellular salt bridge (ESB-2) that might be exploited in the design of allosteric modulators.
Publisher: Wiley
Date: 09-1995
Abstract: Coolia monotis is a benthic dinoflagellate previously thought to be non-toxic. We describe a new toxin, named cooliatoxin, purified from cultures of a strain of C. monotis isolated from Australia. Cooliatoxin is likely a mono-sulphated, polyether toxin (M = 1,062 i.p. LD50 = 1 mg/kg in mice) that induces hypothermia and respiratory failure in mice after a pronounced delay period during which there are no obvious signs of intoxication. These signs in mice are similar to those reported for the shellfish toxin named yessotoxin and the molecular weight of cooliatoxin corresponds to the mono-sulphated form of yessotoxin, suggesting that cooliatoxin may be an analogue of yessotoxin. Cooliatoxin has no effect on the mouse phrenic nerve or diaphragm musculature in vitro but causes initial stimulation and subsequent block of unmylenated nerves in vitro. In isolated guinea pig left atria, cooliatoxin (above 20 nm) induced a slow developing concentration dependent sustained inotropic response. Propranolol or tetrodotoxin reversed the positive inotropic effects, indicating that the majority of the cooliatoxin induced response was mediated by stimulation of nerves associated with the atrial musculature, resulting in the release of noradrenaline. Cooliatoxin induced transient contractions in isolated guinea pig vas deferens preparations. Atria and vas deferens preparations were tachyphylactic to a second equivalent dose of cooliatoxin applied after the effects of the first dose had diminished. The observed in vitro effects of cooliatoxin on peripheral nerves are unlikely to account for the lethal effects in mice and a central action of this toxin is suspected.
Publisher: Elsevier BV
Date: 07-2002
DOI: 10.1016/S0041-0101(02)00088-0
Abstract: We studied the variation in toxin profiles of purified extracts of 10 in idual specimens and two pools of ciguateric Caranx latus. High-performance liquid chromatography/mass spectrometry (HPLC/MS) identified in all in idual s les at least seven Caribbean ciguatoxins (C-CTXs) comprising C-CTX-1 and its epimer C-CTX-2 ([M+H](+) m/z 1141.58), and five new C-CTX congeners with pseudo-molecular ions at m/z 1141.58, 1143.60, 1157.57, 1159.58, and 1127.57. In some s les, additional C-CTX isomers were detected with [M+H](+) ions at m/z 1141.58 (two), 1143.60 (one) and 1157.57 (two). The two low-toxic pools contained only four to six ciguatoxins. The comparison in relative proportions of four different mass classes ([M+H](+) at m/z 1141, 1143, 1157 and 1127) showed that the group at m/z 1157 increased (2-20%) with flesh toxicity. More than 80% of group m/z 1141 comprised C-CTX-1, C-CTX-2 and their isomer C-CTX-1a whose level in this group correlated with fish toxicity. Contrary to low-toxic fishes, high-risk specimens had C-CTX-1 levels <50% and were subjected to large losses of activity on purification indicating that unstable ciguatoxins were present. A possible conversion of C-CTX-1 into C-CTX-1a was identified when flesh was cooked, without changes in toxicity. In conclusion, HPLC/MS characterised 12 C-CTXs accumulated by C. latus at variable levels.
Publisher: Elsevier BV
Date: 08-2006
Publisher: Elsevier BV
Date: 2001
DOI: 10.1016/S0041-0101(00)00161-6
Abstract: Ciguatera is a global disease caused by the consumption of certain warm-water fish (ciguateric fish) that have accumulated orally effective levels of sodium channel activator toxins (ciguatoxins) through the marine food chain. Symptoms of ciguatera include a range of gastrointestinal, neurological and cardiovascular disturbances. This review examines progress in our understanding of ciguatera from the work of Banner in the late 1950s to the present. Similarities and differences in ciguatera in the Pacific Ocean, Indian Ocean and Caribbean Sea are highlighted, and future research directions are suggested.
Publisher: American Chemical Society (ACS)
Date: 29-01-2004
DOI: 10.1021/JM031010O
Abstract: An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the alpha-conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated alpha-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, alpha-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat alpha3beta2 nAChR (IC50 0.3 nM) and was 200-fold less active on the rat alpha7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr16]AnIB showed a 2-fold and 10-fold decrease in activities at alpha3beta2 (IC50 0.6 nM) and alpha7 (IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the alpha7 (IC50 367 nM) than at the alpha3beta2 nAChR (IC50 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-alpha-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for alpha3beta2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor alpha7 affinity.
Publisher: Springer Science and Business Media LLC
Date: 30-01-2014
DOI: 10.1038/NCOMMS4165
Abstract: Poor oral availability and susceptibility to reduction and protease degradation is a major hurdle in peptide drug development. However, drugable receptors in the gut present an attractive niche for peptide therapeutics. Here we demonstrate, in a mouse model of chronic abdominal pain, that oxytocin receptors are significantly upregulated in nociceptors innervating the colon. Correspondingly, we develop chemical strategies to engineer non-reducible and therefore more stable oxytocin analogues. Chemoselective selenide macrocyclization yields stabilized analogues equipotent to native oxytocin. Ultra-high-field nuclear magnetic resonance structural analysis of native oxytocin and the seleno-oxytocin derivatives reveals that oxytocin has a pre-organized structure in solution, in marked contrast to earlier X-ray crystallography studies. Finally, we show that these seleno-oxytocin analogues potently inhibit colonic nociceptors both in vitro and in vivo in mice with chronic visceral hypersensitivity. Our findings have potentially important implications for clinical use of oxytocin analogues and disulphide-rich peptides in general.
Publisher: Frontiers Media SA
Date: 25-11-2020
DOI: 10.3389/FNINS.2020.609005
Abstract: Neuronal nicotinic acetylcholine receptors (nAChRs) are prototypical cation-selective, ligand-gated ion channels that mediate fast neurotransmission in the central and peripheral nervous systems. nAChRs are involved in a range of physiological and pathological functions and hence are important therapeutic targets. Their subunit homology and erse pentameric assembly contribute to their challenging pharmacology and limit their drug development potential. Toxins produced by an extensive range of algae, plants and animals target nAChRs, with many proving pivotal in elucidating receptor pharmacology and biochemistry, as well as providing templates for structure-based drug design. The crystal structures of these toxins with erse chemical profiles in complex with acetylcholine binding protein (AChBP), a soluble homolog of the extracellular ligand-binding domain of the nAChRs and more recently the extracellular domain of human α9 nAChRs, have been reported. These studies have shed light on the erse molecular mechanisms of ligand-binding at neuronal nAChR subtypes and uncovered critical insights useful for rational drug design. This review provides a comprehensive overview and perspectives obtained from structure and function studies of erse plant and animal toxins and their associated inhibitory mechanisms at neuronal nAChRs.
Publisher: Royal Society of Chemistry (RSC)
Date: 2012
DOI: 10.1039/C2OB25133G
Abstract: A dual-pharmacophoric peptide was engineered by grafting the integrin binding RGD motif between the C- and N-termini of a disulfide-rich noradrenaline transporter inhibiting χ-conotoxin resulting in a stable backbone cyclized peptide. The construct maintained two independent biological activities and showed increased plasma stability with no adverse effects observed following administration to rats, highlighting the potential value of pharmacophore grafting into constrained peptide scaffolds.
Publisher: Elsevier BV
Date: 06-2019
DOI: 10.1016/J.BCP.2019.04.025
Abstract: Conorfamides are a poorly studied family of cone snail venom peptides with broad biological activities, including inhibition of glutamate receptors, acid-sensing ion channels, and voltage-gated potassium channels. The aim of this study was to characterize the pharmacological activity of two novel linear conorfamides (conorfamide_As1a and conorfamide_As2a) and their non-amidated counterparts (conopeptide_As1b and conopeptide_As2b) that were isolated from the venom of the Mexican cone snail Conus austini. Although As1a, As2a, As1b and As2b were identified by activity-guided fractionation using a high-throughput fluorescence imaging plate reader (FLIPR) assay assessing α7 nAChR activity, sequence determination revealed activity associated with four linear peptides of the conorfamide rather than the anticipated α-conotoxin family. Pharmacological testing revealed that the amidated peptide variants altered desensitization of acid-sensing ion channels (ASICs) 1a and 3, and the native lysine to arginine mutation differentiating As1a and As1b from As2a and As2b introduced ASIC1a peak current potentiation. Surprisingly, these conorfamides also inhibited α7 and muscle-type nicotinic acetylcholine receptors (nAChR) at nanomolar concentrations. This is the first report of conorfamides with dual activity, with the nAChR activity being the most potent molecular target of any conorfamide discovered to date.
Publisher: American Chemical Society (ACS)
Date: 02-1999
DOI: 10.1021/JM981052Q
Publisher: Proceedings of the National Academy of Sciences
Date: 07-11-2006
Abstract: The tetrodotoxin-resistant voltage-gated sodium channel (VGSC) Na v 1.8 is expressed predominantly by damage-sensing primary afferent nerves and is important for the development and maintenance of persistent pain states. Here we demonstrate that μO-conotoxin MrVIB from Conus marmoreus displays substantial selectivity for Na v 1.8 and inhibits pain behavior in models of persistent pain. In rat sensory neurons, submicromolar concentrations of MrVIB blocked tetrodotoxin-resistant current characteristic of Na v 1.8 but not Na v 1.9 or tetrodotoxin-sensitive VGSC currents. MrVIB blocked human Na v 1.8 expressed in Xenopus oocytes with selectivity at least 10-fold greater than other VGSCs. In neuropathic and chronic inflammatory pain models, allodynia and hyperalgesia were both reduced by intrathecal infusion of MrVIB (0.03–3 nmol), whereas motor side effects occurred only at 30-fold higher doses. In contrast, the nonselective VGSC blocker lignocaine displayed no selectivity for allodynia and hyperalgesia versus motor side effects. The actions of MrVIB reveal that VGSC antagonists displaying selectivity toward Na v 1.8 can alleviate chronic pain behavior with a greater therapeutic index than nonselective antagonists.
Publisher: Springer Science and Business Media LLC
Date: 09-11-2021
DOI: 10.1038/S41598-021-01277-4
Abstract: α-Conotoxins are small disulfide-rich peptides targeting nicotinic acetylcholine receptors (nAChRs) characterised by a C I C II -X m -C III -X n -C IV framework that invariably adopt the native globular conformations which is typically most potent. α-Conotoxins are ided into several structural subgroups based on the number of residues within the two loops braced by the disulfide bonds (m/n), with the 4/7 and 4/3 subgroups dominating. AusIA is a relatively rare α5/5-conotoxin isolated from the venom of Conus australis. Surprisingly, the ribbon isomer displayed equipotency to the wild-type globular AusIA at human α7-containing nAChR. To understand the molecular basis for equipotency, we determined the co-crystal structures of both isomers at Lymnea stagnalis acetylcholine binding protein. The additional residue in the first loop of AusIA was found to be a critical determinant of equipotency, with 11-fold and 86-fold shifts in potency in favour of globular AusIA over ribbon AusIA observed following deletion of Ala4 or Arg5, respectively. This ergence in the potency between globular AusIA and ribbon AusIA was further enhanced upon truncation of the non-conserved Val at the C-termini. Conversely, equipotency could be replicated in LsIA and TxIA [A10L] following insertion of an Ala in the first loop. These findings provide a new understanding of the role the first loop in ribbon and globular α-conotoxins can play in directing α-conotoxin nAChR pharmacology.
Publisher: SAGE Publications
Date: 2016
Publisher: Royal Society of Chemistry
Date: 2015
Publisher: Public Library of Science (PLoS)
Date: 25-03-2013
Publisher: Elsevier BV
Date: 12-2015
DOI: 10.1016/J.TOXICON.2015.09.012
Abstract: Transcriptome sequencing is now widely adopted as an efficient means to study the chemical ersity of venoms. To improve the efficiency of analysis of these large datasets, we have optimised an analysis pipeline for cone snail venom gland transcriptomes. The pipeline combines ConoSorter with sequence architecture-based elimination and similarity searching using BLAST to improve the accuracy of sequence identification and classification, while reducing requirements for manual intervention. As a proof-of-concept, we used this approach reanalysed three previously published cone snail transcriptomes from erse dietary groups. Our pipeline method generated similar results to the published studies with significantly less manual intervention. We additionally found undiscovered sequences in the piscovorous Conus geographus and vermivorous Conus miles and identified sequences in incorrect superfamilies in the molluscivorus Conus marmoreus and C. geographus transcriptomes. Our results indicate that this method can improve toxin detection without extending analysis time. While this method was evaluated on cone snail transcriptomes it can be easily optimised to retrieve toxins from other venomous animals.
Publisher: Elsevier BV
Date: 02-2003
Publisher: Frontiers Media SA
Date: 08-04-2022
Publisher: Bentham Science Publishers Ltd.
Date: 10-2011
DOI: 10.2174/187152811797200687
Abstract: Venomous animals produce a erse range of peptides and small molecules that are of both therapeutic and pharmacologic value. One such animal, the cone snail, produces peptides known as conotoxins, which may be of interest to those studying the mammalian immune system. Conotoxins are a family of venom peptides that display extraordinary ersity and often exquisite specificity for membrane protein targets, especially voltage and ligand activated ion channels. Conopeptides are proving to be important pharmacological tools to probe human physiology, with some showing promise as therapeutics for conditions such as neuropathic pain. The potential of these peptides to interact and modulate the human immune system has not been investigated despite literature suggesting that conotoxins could be valuable research tools and potential therapeutics in area of immunology. Known pharmacological targets of conopeptides expressed by immunocompetent cells include voltage-gated potassium channel (Kv), voltage-gated calcium channel (Cav), nicotinic and acetylcholine receptors. In addition, the 5-HT3, GABAB and NMDA receptors that are not considered classic immunomodulators but may play a secondary role in modulating immune responses. This review highlights venom peptides with potential to act at immunological targets within the mammalian immune system.
Publisher: American Chemical Society (ACS)
Date: 30-11-2010
DOI: 10.1021/JM100989W
Abstract: Disulfide bond engineering is an important approach to improve the metabolic half-life of cysteine-containing peptides. Eleven analogues of oxytocin were synthesized including disulfide bond replacements by thioether, selenylsulfide, diselenide, and ditelluride bridges, and their stabilities in human plasma and activity at the human oxytocin receptor were assessed. The cystathionine (K(i) = 1.5 nM, and EC₅₀ = 32 nM), selenylsulfide (K(i) = 0.29/0.72 nM, and EC₅₀ = 2.6/154 nM), diselenide (K(i) = 11.8 nM, and EC₅₀ = 18 nM), and ditelluride analogues (K(i) = 7.6 nM, and EC₅₀ = 27.3 nM) retained considerable affinity and functional potency as compared to oxytocin (K(i) = 0.79 nM, and EC₅₀ = 15 nM), while shortening the disulfide bridge abolished binding and functional activity. The mimetics showed a 1.5-3-fold enhancement of plasma stability as compared to oxytocin (t(½) = 12 h). By contrast, the all-D-oxytocin and head to tail cyclic oxytocin analogues, while significantly more stable with half-lives greater than 48 h, had little or no detectable binding or functional activity.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 02-2000
DOI: 10.1097/00007691-200002000-00013
Abstract: Ion channels are intimately linked to all neurotransmission and neurotransmitter release processes, but in disease states often contribute adversely to disease pathology. The ersity and distribution of ion channel types and subtypes being uncovered through the use of molecular biology and toxin probes present an exciting opportunity for the discovery of new, more selective drugs. Among ion channels targeted by cone shell venom peptides (conotoxins) are the voltage-sensitive sodium, calcium, and potassium channels which open and then close (inactivate) in response to membrane depolarization, and thus regulate neurotransmission and the neurotransmitter release process. Conotoxins also target ligand-gated ion channels, including the NMDA-glutamate channel and the nicotinic acetylcholine receptor channel. The ersity of subtypes, especially those subtypes upregulated in disease states, makes ion channels a rapidly expanding therapeutic area. Conotoxins represent some of the most selective inhibitors of ion channel subtypes and have often been used as the defining ligand. In this overview, the structures and therapeutic potential of conotoxins active at ion channels are highlighted. The activity and structures are then contrasted with ciguatoxins, which are responsible for the food poisoning known as ciguatera. A universal liquid chromatography/mass spectrometry approach to the detection of these classes of toxins is briefly discussed.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 05-11-2010
Abstract: Neuronal (N)-type Ca(2+) channel-selective omega-conotoxins have emerged as potential new drugs for the treatment of chronic pain. In this study, two new omega-conotoxins, CVIE and CVIF, were discovered from a Conus catus cDNA library. Both conopeptides potently displaced (125)I-GVIA binding to rat brain membranes. In Xenopus laevis oocytes, CVIE and CVIF potently and selectively inhibited depolarization-activated Ba(2+) currents through recombinant N-type (alpha1(B-b)/alpha(2)delta1/beta(3)) Ca(2+) channels. Recovery from block increased with membrane hyperpolarization, indicating that CVIE and CVIF have a higher affinity for channels in the inactivated state. The link between inactivation and the reversibility of omega-conotoxin action was investigated by creating molecular ersity in beta subunits: N-type channels with beta(2a) subunits almost completely recovered from CVIE or CVIF block, whereas those with beta(3) subunits exhibited weak recovery, suggesting that reversibility of the omega-conotoxin block may depend on the type of beta-subunit isoform. In rat dorsal root ganglion sensory neurons, neither peptide had an effect on low-voltage-activated T-type channels but potently and selectively inhibited high voltage-activated N-type Ca(2+) channels in a voltage-dependent manner. In rat spinal cord slices, both peptides reversibly inhibited excitatory monosynaptic transmission between primary afferents and dorsal horn superficial lamina neurons. Homology models of CVIE and CVIF suggest that omega-conotoxin/voltage-gated Ca(2+) channel interaction is dominated by ionic/electrostatic interactions. In the rat partial sciatic nerve ligation model of neuropathic pain, CVIE and CVIF (1 nM) significantly reduced allodynic behavior. These N-type Ca(2+) channel-selective omega-conotoxins are therefore useful as neurophysiological tools and as potential therapeutic agents to inhibit nociceptive pain pathways.
Publisher: SAGE Publications
Date: 2013
Abstract: Antagonists of N-type voltage-gated calcium channels (VGCC), Ca v 2.2, can manage severe chronic pain with intrathecal use and may be effective systemically. A series of novel ω-conotoxins that selectively inhibit N-type VGCCs was isolated from Conus catus. In the present study, the potency and reversibility of ω-conotoxins CVID, CVIE and CVIF to inhibit N-type calcium currents were investigated in mouse isolated dorsal root ganglion (DRG) neurons. The systemic potency of each ω-conotoxin to reverse signs of mouse chronic inflammatory pain was also compared. In DRG neurons, the rank order of potency to inhibit N-type calcium currents was CVIE CVIF CVID. After subcutaneous administration, CVID and CVIE, but not CVIF, partially reversed impaired weight bearing in mice injected with Freund's complete adjuvant (CFA) three days prior to testing. No side-effects associated with systemic administration of ω-conotoxins were observed. The present study indicates a potential for CVID and CVIE to be developed as systemically active analgesics with no accompanying neurological side-effects.
Publisher: Wiley
Date: 02-09-2018
DOI: 10.1111/BPH.13962
Publisher: Elsevier BV
Date: 09-2003
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 21-10-2022
DOI: 10.1097/J.PAIN.0000000000002795
Abstract: The bladder wall is innervated by a complex network of afferent nerves that detect bladder stretch during filling. Sensory signals, generated in response to distension, are relayed to the spinal cord and brain to evoke physiological and painful sensations and regulate urine storage and voiding. Hyperexcitability of these sensory pathways is a key component in the development of chronic bladder hypersensitivity disorders including interstitial cystitis/bladder pain syndrome and overactive bladder syndrome. Despite this, the full array of ion channels that regulate bladder afferent responses to mechanical stimuli have yet to be determined. Here, we investigated the role of low-voltage-activated T-type calcium (Ca V 3) channels in regulating bladder afferent responses to distension. Using single-cell reverse-transcription polymerase chain reaction and immunofluorescence, we revealed ubiquitous expression of Ca V 3.2, but not Ca V 3.1 or Ca V 3.3, in in idual bladder-innervating dorsal root ganglia neurons. Pharmacological inhibition of Ca V 3.2 with TTA-A2 and ABT-639, selective blockers of T-type calcium channels, dose-dependently attenuated ex-vivo bladder afferent responses to distension in the absence of changes to muscle compliance. Further evaluation revealed that Ca V 3.2 blockers significantly inhibited both low- and high-threshold afferents, decreasing peak responses to distension, and delayed activation thresholds, thereby attenuating bladder afferent responses to both physiological and noxious distension. Nocifensive visceromotor responses to noxious bladder distension in vivo were also significantly reduced by inhibition of Ca V 3 with TTA-A2. Together, these data provide evidence of a major role for Ca V 3.2 in regulating bladder afferent responses to bladder distension and nociceptive signalling to the spinal cord.
Publisher: Elsevier BV
Date: 12-2007
DOI: 10.1016/J.SBI.2007.08.004
Abstract: Bacteria, irrespective of natural habitat, are exposed to constant fluctuations in their growth conditions. Consequently they have developed sophisticated responses, modulated by the re-modelling of protein complexes and by phosphorylation-dependent signal transduction systems, to adapt to and to survive a variety of insults. Ultimately these signalling systems affect transcriptional regulons either by activating an alternative sigma factor subunit of RNA polymerase, for ex le, sigma E (sigma(E)) of Escherichia coli and sigma B (sigma(B)) and sigma F (sigma(F)) in Bacillus subtilis or by activating DNA-binding two-component response regulators. Recent structure determinations, and systems biology analysis of key regulators in well-characterised stress-responsive pathways, illustrate conserved and novel mechanisms in these representative model bacteria.
Publisher: CSIRO Publishing
Date: 2008
DOI: 10.1071/CH07327
Abstract: A simple and efficient method has been developed for the synthesis of two anthranilamide-based non-peptide mimetics of ω-conotoxin GVIA. These anthranilamide derivatives aim to mimic the K2, R17, and Y13 residues of the peptide. The synthetic route described enables the rapid synthesis of anthranilamide analogues with identical alkyl chain lengths. The target compounds show affinity to rat N-type voltage gated calcium channels (Cav2.2) with EC50 values of 42 and 75 μM.
Publisher: MDPI AG
Date: 28-01-2020
Abstract: Slow lorises are enigmatic animal that represent the only venomous primate lineage. Their defensive secretions have received little attention. In this study we determined the full length sequence of the protein secreted by their unique brachial glands. The full length sequences displayed homology to the main allergenic protein present in cat dander. We thus compared the molecular features of the slow loris brachial gland protein and the cat dander allergen protein, showing remarkable similarities between them. Thus we postulate that allergenic proteins play a role in the slow loris defensive arsenal. These results shed light on these neglected, novel animals.
Publisher: MDPI AG
Date: 29-10-2019
Abstract: Voltage-gated sodium channels (NaVs) are a key determinant of neuronal signalling. Neurotoxins from erse taxa that selectively activate or inhibit NaV channels have helped unravel the role of NaV channels in diseases, including chronic pain. Spider venoms contain the most erse array of inhibitor cystine knot (ICK) toxins (knottins). This review provides an overview on how spider knottins modulate NaV channels and describes the structural features and molecular determinants that influence their affinity and subtype selectivity. Genetic and functional evidence support a major involvement of NaV subtypes in various chronic pain conditions. The exquisite inhibitory properties of spider knottins over key NaV subtypes make them the best lead molecules for the development of novel analgesics to treat chronic pain.
Publisher: Springer Science and Business Media LLC
Date: 2005
DOI: 10.1007/BF03033782
Publisher: Elsevier BV
Date: 06-2002
DOI: 10.1016/S0041-0101(01)00259-8
Abstract: We report the isolation and initial characterisation of Indian Ocean ciguatoxin (I-CTX) present in toxic lipid soluble extracts isolated from ciguateric fishes collected off the Republic of Mauritius in the Indian Ocean. Following i.p. injection of this extract, mice displayed symptoms that were similar, though not identical, to those produced by Pacific and Caribbean ciguatoxins (P-CTXs and C-CTXs). Using a radiolabelled brevetoxin (PbTx) binding assay and mouse bioassay guided fractionation, I-CTX was purified by Florisil, Sephadex LH-20 and TSK HW-40S chromatography with good recovery. Isolation to purity was not possible by preparative reversed phase high-performance liquid chromatography (HPLC) due to significant losses of toxicity. However, analytical reversed phase HPLC coupled to an electrospray mass spectrometry detector identified a [M + H](+) ion at m/z 1141.58 which co-eluted with activity that displaced [3H]-PbTx binding to rat brain. This mass corresponded to C-CTX-1, but the fragmentation pattern of I-CTX showed a different ratio of pseudo molecular and product ions. I-CTX was found to elute later than P-CTX-1 but was practically indistinguishable from C-CTX-1 on reversed phase HPLC, while the TSK HW-40S column chromatography differentiated I-CTX from the later eluting C-CTX-1. Taken together, these results indicate that I-CTX is a new ciguatoxin (CTX) responsible for ciguatera caused by reef fish in the Indian Ocean.
Publisher: Wiley
Date: 27-04-2018
Publisher: Wiley
Date: 07-05-2002
DOI: 10.1046/J.1471-4159.2002.00872.X
Abstract: The pharmacology of the N -methyl-d-aspartate (NMDA) receptor site was examined in pathologically affected and relatively spared regions of cerebral cortex tissue obtained at autopsy from Alzheimer's disease cases and matched controls. The affinity and density of the [(3)H]MK-801 binding site were delineated along with the enhancement of [(3)H]MK-801 binding by glutamate and spermine. Maximal enhancement induced by either ligand was regionally variable glutamate-mediated maximal enhancement was higher in controls than in Alzheimer's cases in pathologically spared regions, whereas spermine-mediated maximal enhancement was higher in controls in areas susceptible to pathological damage. These and other data suggest that the subunit composition of NMDA receptors may be locally variable. Studies with modified conantokin-G (con-G) peptides showed that Ala(7)-con-G had higher affinity than Lys(7)-con-G, and also defined two distinct binding sites in controls. Nevertheless, the affinity for Lys(7)-con-G was higher overall in Alzheimer's brain than in control brain, whereas the reverse was true for Ala(7)-con-G. Over-excitation mediated by specific NMDA receptors might contribute to localized brain damage in Alzheimer's disease. Modified conantokins are useful for identifying the NMDA receptors involved, and may have potential as protective agents.
Publisher: Wiley
Date: 08-2000
DOI: 10.1046/J.1432-1327.2000.01508.X
Abstract: A novel conotoxin belonging to the 'four-loop' structural class has been isolated from the venom of the piscivorous cone snail Conus tulipa. It was identified using a chemical-directed strategy based largely on mass spectrometric techniques. The new toxin, conotoxin TVIIA, consists of 30 amino-acid residues and contains three disulfide bonds. The amino-acid sequence was determined by Edman analysis as SCSGRDSRCOOVCCMGLMCSRGKCVSIYGE where O = 4-transL-hydroxyproline. Two under-hydroxylated analogues, [Pro10]TVIIA and [Pro10,11]TVIIA, were also identified in the venom of C. tulipa. The sequences of TVIIA and [Pro10]TVIIA were further verified by chemical synthesis and coelution studies with native material. Conotoxin TVIIA has a six cysteine/four-loop structural framework common to many peptides from Conus venoms including the omega-, delta- and kappa-conotoxins. However, TVIIA displays little sequence homology with these well-characterized pharmacological classes of peptides, but displays striking sequence homology with conotoxin GS, a peptide from Conus geographus that blocks skeletal muscle sodium channels. These new toxins and GS share several biochemical features and represent a distinct subgroup of the four-loop conotoxins.
Publisher: Springer Science and Business Media LLC
Date: 26-05-2017
DOI: 10.1038/SREP46816
Abstract: Scientific Reports 7: Article number: 40883 published online: 20 January 2017 updated: 26 May 2017 In this Article, Affiliation 6 is incorrectly listed as ‘Venomtech, Sophie-Antipolis, 06560, Valbonne, France’. The correct affiliation is listed below: VenomeTech, Sophie-Antipolis, 06560, Valbonne,France.
Publisher: Elsevier BV
Date: 08-2012
DOI: 10.1016/J.BCP.2012.05.008
Abstract: The μO-conotoxins are notable for their unique selectivity for Na(v)1.8 over other sodium channel isoforms, making them attractive drug leads for the treatment of neuropathic pain. We describe the discovery of a novel μO-conotoxin, MfVIA, from the venom of Conus magnificus using high-throughput screening approaches. MfVIA was found to be a hydrophobic 32-residue peptide (amino acid sequence RDCQEKWEYCIVPILGFVYCCPGLICGPFVCV) with highest sequence homology to μO-conotoxin MrVIB. To overcome the synthetic challenges posed by μO-conotoxins due to their hydrophobic nature and difficult folding, we developed a novel regioselective approach for the synthesis of μO-conotoxins. Performing selective oxidative deprotections of the cysteine side-chain protecting groups of the fully protected peptide allowed manipulations in organic solvents with no chromatography required between steps. Using this approach, we obtained correctly folded MfVIA with increased synthetic yields. Biological activity of MfVIA was assessed using membrane potential-sensitive dyes and electrophysiological recording techniques. MfVIA preferentially inhibits Na(v)1.8 (IC₅₀ 95.9±74.3 nM) and Na(v)1.4 (IC₅₀ 81±16 nM), with significantly lower affinity for other Na(v) subtypes (IC₅₀ 431-6203 nM Na(v)1.5>1.6∼1.7∼1.3∼1.1∼1.2). This improved approach to μO-conotoxin synthesis will facilitate the optimization of μO-conotoxins as novel analgesic molecules to improve pain management.
Publisher: MDPI AG
Date: 15-03-2022
DOI: 10.3390/MD20030209
Abstract: Cone snail venom bio ersity reflects dietary preference and predatory and defensive envenomation strategies across the ≈900 species of Conidae. To better understand the mechanisms of adaptive radiations in closely related species, we investigated the venom of two phylogenetically and spatially related species, C. flavidus and C. frigidus of the Virgiconus clade. Transcriptomic analysis revealed that the major superfamily profiles were conserved between the two species, including 68 shared conotoxin transcripts. These shared transcripts contributed 90% of the conotoxin expression in C. frigidus and only 49% in C. flavidus, which showed greater toxin ersification in the dominant O1, I2, A, O2, O3, and M superfamilies compared to C. frigidus. On the basis of morphology, two additional sub-groups closely resembling C. flavidus were also identified from One Tree Island Reef. Despite the morphological resemblance, the venom duct proteomes of these cryptic sub-groups were distinct from C. flavidus. We suggest rapid conotoxin sequence ergence may have facilitated adaptive radiation and the establishment of new species and the regulatory mechanisms facilitating species-specific venom evolution.
Publisher: Springer Science and Business Media LLC
Date: 10-2003
DOI: 10.1038/NRD1197
Publisher: MDPI AG
Date: 21-01-2019
DOI: 10.3390/MD17010071
Abstract: The piscivorous cone snail Conus tulipa has evolved a net-hunting strategy, akin to the deadly Conus geographus, and is considered the second most dangerous cone snail to humans. Here, we present the first venomics study of C. tulipa venom using integrated transcriptomic and proteomic approaches. Parallel transcriptomic analysis of two C. tulipa specimens revealed striking differences in conopeptide expression levels (2.5-fold) between in iduals, identifying 522 and 328 conotoxin precursors from 18 known gene superfamilies. Despite broad overlap at the superfamily level, only 86 precursors (11%) were common to both specimens. Conantokins (NMDA antagonists) from the superfamily B1 dominated the transcriptome and proteome of C. tulipa venom, along with superfamilies B2, A, O1, O3, con-ikot-ikot and conopressins, plus novel putative conotoxins precursors T1.3, T6.2, T6.3, T6.4 and T8.1. Thus, C. tulipa venom comprised both paralytic (putative ion channel modulating α-, ω-, μ-, δ-) and non-paralytic (conantokins, con-ikot-ikots, conopressins) conotoxins. This venomic study confirms the potential for non-paralytic conotoxins to contribute to the net-hunting strategy of C. tulipa.
Publisher: MDPI AG
Date: 09-03-2018
DOI: 10.3390/IJMS19030788
Abstract: Cone snail venoms are considered a treasure trove of bioactive peptides. Despite over 800 species of cone snails being known, each producing over 1000 venom peptides, only about 150 unique venom peptides are structurally and functionally characterized. To overcome the limitations of the traditional low-throughput bio-discovery approaches, multi-omics systems approaches have been introduced to accelerate venom peptide discovery and characterisation. This “venomic” approach is starting to unravel the full complexity of cone snail venoms and to provide new insights into their biology and evolution. The main challenge for venomics is the effective integration of transcriptomics, proteomics, and pharmacological data and the efficient analysis of big datasets. Novel database search tools and visualisation techniques are now being introduced that facilitate data exploration, with ongoing advances in related omics fields being expected to further enhance venomics studies. Despite these challenges and future opportunities, cone snail venomics has already exponentially expanded the number of novel venom peptide sequences identified from the species investigated, although most novel conotoxins remain to be pharmacologically characterised. Therefore, efficient high-throughput peptide production systems and/or banks of miniaturized discovery assays are required to overcome this bottleneck and thus enhance cone snail venom bioprospecting and accelerate the identification of novel drug leads.
Publisher: Royal Society of Chemistry (RSC)
Date: 2008
DOI: 10.1039/B808866G
Abstract: Fractionation of a cytotoxic extract obtained from a southern Australian marine sponge, Phorbas sp., yielded the known diterpenes phorbasins B-F () together with five new members of the phorbasin family, phorbasins G-K (). Structures were assigned to the new phorbasins based on detailed spectroscopic analysis. A preliminary structure activity relationship (SAR) evaluation based on the co-metabolites phorbasins B-K () revealed aspects of the phorbasin pharmacophore.
Publisher: Elsevier BV
Date: 05-1993
DOI: 10.1016/0041-0101(93)90118-3
Abstract: Ciguatoxin-2, a major ciguatoxin present in the flesh and viscera of ciguateric fishes, has been shown by 1H nuclear magnetic resonance studies (2-dimensional homonuclear Hartman Hahn, nuclear Overhauser effect and decoupling difference experiments) to be a diastereomer of ciguatoxin-3, differing only in stereochemistry at carbon 52 (a quaternary carbon). This difference accounts for the significant changes in the chemical shift of resonances for protons in this region of ciguatoxin-2. Differences between ciguatoxin-1, -2 and -3 involve modifications at only one end of the ciguatoxins (ring M) and modest differences in potency, indicating that this ring contributes to, but is not critical for, high affinity binding of the ciguatoxins to voltage-dependent sodium channels. It is proposed that ciguatoxin-2 originates from a different precursor to the precursor (presumably gambiertoxin-4b) for ciguatoxin-1 and -3, and that both precursors are produced by a common biosynthetic pathway in Gambierdiscus toxicus.
Publisher: Springer Science and Business Media LLC
Date: 20-04-2017
DOI: 10.1038/S41598-017-01129-0
Abstract: Voltage-gated sodium (Na V ) channels are essential for the transmission of pain signals in humans making them prime targets for the development of new analgesics. Spider venoms are a rich source of peptide modulators useful to study ion channel structure and function. Here we describe β/δ-TRTX-Pre1a, a 35-residue tarantula peptide that selectively interacts with neuronal Na V channels inhibiting peak current of hNa V 1.1, rNa V 1.2, hNa V 1.6, and hNa V 1.7 while concurrently inhibiting fast inactivation of hNa V 1.1 and rNa V 1.3. The DII and DIV S3-S4 loops of Na V channel voltage sensors are important for the interaction of Pre1a with Na V channels but cannot account for its unique subtype selectivity. Through analysis of the binding regions we ascertained that the variability of the S1-S2 loops between Na V channels contributes substantially to the selectivity profile observed for Pre1a, particularly with regards to fast inactivation. A serine residue on the DIV S2 helix was found to be sufficient to explain Pre1a’s potent and selective inhibitory effect on the fast inactivation process of Na V 1.1 and 1.3. This work highlights that interactions with both S1-S2 and S3-S4 of Na V channels may be necessary for functional modulation, and that targeting the erse S1-S2 region within voltage-sensing domains provides an avenue to develop subtype selective tools.
Publisher: American Chemical Society (ACS)
Date: 03-03-2015
DOI: 10.1021/JACS.5B00244
Abstract: Covalently attached peptide dendrimers can enhance binding affinity and functional activity. Homogenous di- and tetravalent dendrimers incorporating the α7-nicotinic receptor blocker α-conotoxin ImI (α-ImI) with polyethylene glycol spacers were designed and synthesized via a copper-catalyzed azide-alkyne cycloaddition of azide-modified α-ImI to an alkyne-modified polylysine dendron. NMR and CD structural analysis confirmed that each α-ImI moiety in the dendrimers had the same 3D structure as native α-ImI. The binding of the α-ImI dendrimers to binding protein Ac-AChBP was measured by surface plasmon resonance and revealed enhanced affinity. Quantitative electrophysiology showed that α-ImI dendrimers had ∼100-fold enhanced potency at hα7 nAChRs (IC50 = 4 nM) compared to native α-ImI (IC50 = 440 nM). In contrast, no significant potency enhancement was observed at heteromeric hα3β2 and hα9α10 nAChRs. These findings indicate that multimeric ligands can significantly enhance conotoxin potency and selectivity at homomeric nicotinic ion channels.
Publisher: Elsevier BV
Date: 12-1999
Publisher: Elsevier BV
Date: 04-1993
DOI: 10.1016/0041-0101(93)90179-M
Abstract: The actions of pure ciguatoxin-1, ciguatoxin-2 and ciguatoxin-3 were assessed on the contractile activity of isolated guinea-pig left atria and ilea. Low concentrations of each ciguatoxin caused transient positive inotropy, whereas moderate concentrations induced transient and sustained positive inotropic phases. The transient positive inotropic phase was inhibited by tetrodotoxin or atenolol, indicating this phase stems from indirect effects of the ciguatoxins via the stimulation of intrinsic adrenergic nerves. On atria pretreated with atropine and alpha- and beta-adrenoceptor antagonists to block neural actions of the ciguatoxins, moderate concentrations of each ciguatoxin induced only slowly developing, sustained positive inotropy. ED50s for the indirect positive inotropic phase were 2.7 x 10(-11), 1.6 x 10(-10) and 1.4 x 10(-11) M and for the direct positive inotropic phase were 1.6 x 10(-10), 1.4 x 10(-9) and 1.5 x 10(-9) M for ciguatoxin-1, -2 and -3, respectively, indicating that their effects on neurons are 10-fold (ciguatoxin-1 and -2) to 100-fold (ciguatoxin-3) more potent than those directly on the myocardium. High concentrations of each ciguatoxin additionally induced sustained negative inotropy which could be reversed by lidocaine. On guinea-pig ilea, each ciguatoxin induced a transient contracture which could be abolished by atropine. Each ciguatoxin significantly reduced the contractile response of ilea to nicotine, without affecting the contractile response to acetylcholine. We conclude that ciguatoxin-1, -2 and -3 activate similarly the voltage-dependent Na+ channels in neuronal and myocardial tissues, but vary in their relative affinity for the Na+ channels in these tissues.
Publisher: Elsevier BV
Date: 08-1992
DOI: 10.1016/0041-0101(92)90389-M
Abstract: This report describes the action of ciguatoxin-1, the major ciguatoxin present in fishes that cause ciguatera, on the contractile activity of human cardiac musculature. Ciguatoxin-1 caused a large, sustained and concentration-dependent positive inotropy in human atrial trabeculae that were obtained during coronary artery bypass surgery from otherwise healthy hearts. Atenolol (a beta 1-adrenoceptor selective antagonist without local anaesthetic-type activity) or low concentrations of tetrodotoxin abolished the positive inotropy caused by ciguatoxin-1, indicating that ciguatoxin-1 stimulated neural elements present in this tissue to release noradrenaline. The positive inotropic action of ciguatoxin-1 did not stem from a significant direct action on myocardial voltage-dependent sodium channels, nor did it stem from significant alpha 1- or beta 2-adrenoreceptor stimulation. Ciguatoxin-1 caused positive inotropy in preparations stimulated at between 0.02 and 2.0 Hz. Mannitol, currently the treatment of choice for ciguatera, did not significantly reverse the positive inotropy induced by ciguatoxin-1 in human atrial trabeculae.
Publisher: Elsevier BV
Date: 10-2005
DOI: 10.1016/J.NEUROPHARM.2005.04.024
Abstract: The basis for the neuroprotectant effect of D-mannitol in reducing the sensory neurological disturbances seen in ciguatera poisoning, is unclear. Pacific ciguatoxin-1 (P-CTX-1), at a concentration 10 nM, caused a statistically significant swelling of rat sensory dorsal root ganglia (DRG) neurons that was reversed by hyperosmolar 50 mM D-mannitol. However, using electron paramagnetic resonance (EPR) spectroscopy, it was found that P-CTX-1 failed to generate hydroxyl free radicals at concentrations of toxin that caused profound effects on neuronal excitability. Whole-cell patch-cl recordings from DRG neurons revealed that both hyper- and iso-osmolar 50 mM D-mannitol prevented the membrane depolarisation and repetitive firing of action potentials induced by P-CTX-1. In addition, both hyper- and iso-osmolar 50 mM D-mannitol prevented the hyperpolarising shift in steady-state inactivation and the rise in leakage current through tetrodotoxin (TTX)-sensitive Na(v) channels, as well as the increased rate of recovery from inactivation of TTX-resistant Na(v) channels induced by P-CTX-1. D-Mannitol also reduced, but did not prevent, the inhibition of peak TTX-sensitive and TTX-resistant I(Na) litude by P-CTX-1. Additional experiments using hyper- and iso-osmolar D-sorbitol, hyperosmolar sucrose and the free radical scavenging agents Trolox and L-ascorbic acid showed that these agents, unlike D-mannitol, failed to prevent the effects of P-CTX-1 on spike electrogenesis and Na(v) channel gating. These selective actions of D-mannitol indicate that it does not act purely as an osmotic agent to reduce swelling of nerves, but involves a more complex action dependent on the Na(v) channel subtype, possibly to alter or reduce toxin association.
Publisher: Informa UK Limited
Date: 1993
Publisher: Wiley
Date: 23-01-2014
DOI: 10.1002/BIP.22368
Abstract: Voltage-gated sodium (Nav) channels are responsible for generation and propagation of action potentials throughout the nervous system. Their malfunction causes several disorders and chronic conditions including neuropathic pain. Potent subtype specific ligands are essential for deciphering the molecular mechanisms of Nav channel function and development of effective therapeutics. µ-Conotoxin SIIIA is a potent mammalian Nav 1.2 channel blocker that exhibits analgesic activity in rodents. We undertook to reengineer loop 1 through a strategy involving charge alterations and truncations which led to the development of µ-SIIIA mimetics with novel selectivity profiles. A novel [N5K/D15A]SIIIA(3-20) mutant with enhanced net positive charge showed a dramatic increase in its Nav 1.2 potency (IC50 of 0.5 nM vs. 9.6 nM for native SIIIA) though further truncations led to loss of potency. Unexpectedly, it appears that SIIIA loop 1 significantly influences its Nav channel interactions despite loop 2 and 3 residues constituting the pharmacophore. This minimal functional conotoxin scaffold may allow further development of selective NaV blockers.
Publisher: Public Library of Science (PLoS)
Date: 16-03-2017
Publisher: Elsevier BV
Date: 2004
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 10-1998
DOI: 10.1097/00007691-199810000-00010
Abstract: Environmental poisoning is most commonly associated with chronic long-term exposure to toxins rather than to acute exposure. Such repeated exposure to sublethal doses of compounds and elements presents problems in risk assessment. This is primarily because the data are unavailable to describe relationships between dose and effect at lower levels of exposure to toxins. Bioavailability of toxins also presents a problem because the data on bioavailability are sparse and seldom as high as the default of 100% bioavailability commonly used in risk assessment. Ex les are presented of two toxins: arsenic as an elemental anthropogenic and geologic poison and ciguatoxin, a polyether ladder compound, as a toxin produced naturally by dinoflagellates. Bioavailability drives the toxicity of arsenic from contaminated sites, whereas tissue accumulation drives the toxicity of ciguatoxin. Considerable benefit is derived from the harmonization of regulatory processes where there is linkage of health and environmental factors in the derivation of credible risk assessment.
Publisher: The Royal Society
Date: 22-07-2015
Abstract: Some venomous cone snails feed on small fishes using an immobilizing combination of synergistic venom peptides that target K v and Na v channels. As part of this envenomation strategy, δ-conotoxins are potent ichtyotoxins that enhance Na v channel function. δ-Conotoxins belong to an ancient and widely distributed gene superfamily, but any evolutionary link from ancestral worm-eating cone snails to modern piscivorous species has not been elucidated. Here, we report the discovery of SuVIA, a potent vertebrate-active δ-conotoxin characterized from a vermivorous cone snail ( Conus suturatus ). SuVIA is equipotent at hNa V 1.3, hNa V 1.4 and hNa V 1.6 with EC 50 s in the low nanomolar range. SuVIA also increased peak hNa V 1.7 current by approximately 75% and shifted the voltage-dependence of activation to more hyperpolarized potentials from –15 mV to –25 mV, with little effect on the voltage-dependence of inactivation. Interestingly, the proximal venom gland expression and pain-inducing effect of SuVIA in mammals suggest that δ-conotoxins in vermivorous cone snails play a defensive role against higher order vertebrates. We propose that δ-conotoxins originally evolved in ancestral vermivorous cones to defend against larger predators including fishes have been repurposed to facilitate a shift to piscivorous behaviour, suggesting an unexpected underlying mechanism for this remarkable evolutionary transition.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 10-2013
DOI: 10.1016/J.PAIN.2013.06.015
Abstract: Ciguatera, the most common form of nonbacterial ichthyosarcotoxism, is caused by consumption of fish that have bioaccumulated the polyether sodium channel activator ciguatoxin. The neurological symptoms of ciguatera include distressing, often persistent sensory disturbances such as paraesthesias and the pathognomonic symptom of cold allodynia. We show that intracutaneous administration of ciguatoxin in humans elicits a pronounced axon-reflex flare and replicates cold allodynia. To identify compounds able to inhibit ciguatoxin-induced Nav responses, we developed a novel in vitro ciguatoxin assay using the human neuroblastoma cell line SH-SY5Y. Pharmacological characterisation of this assay demonstrated a major contribution of Nav1.2 and Nav1.3, but not Nav1.7, to ciguatoxin-induced Ca2+ responses. Clinically available Nav inhibitors, as well as the Kv7 agonist flupirtine, inhibited tetrodotoxin-sensitive ciguatoxin-evoked responses. To establish their in vivo efficacy, we used a novel animal model of ciguatoxin-induced cold allodynia. However, differences in the efficacy of these compounds to reverse ciguatoxin-induced cold allodynia did not correlate with their potency to inhibit ciguatoxin-induced responses in SH-SY5Y cells or at heterologously expressed Nav1.3, Nav1.6, Nav1.7, or Nav1.8, indicating cold allodynia might be more complex than simple activation of Nav channels. These findings highlight the need for suitable animal models to guide the empiric choice of analgesics, and suggest that lamotrigine and flupirtine could be potentially useful for the treatment of ciguatera.
Publisher: American Chemical Society (ACS)
Date: 25-07-1998
DOI: 10.1021/BI9806549
Publisher: Public Library of Science (PLoS)
Date: 28-07-2016
Publisher: Elsevier BV
Date: 1988
DOI: 10.1016/0041-0101(88)90246-2
Abstract: Ciguatoxin, the toxin present in fish responsible for ciguatera, at doses equal or above the maximum positive inotropic dose in atria (greater than 0.15 mouse units/ml) induced arrhythmias in atria and papillary muscles stimulated at 1 Hz and dose-dependent negative inotropy in atria. Negative inotropy was enhanced by ouabain or by an increase in stimulation to 3 Hz, little affected by procaine or increasing Ringer [Ca2+] and reversed by lidocaine and tetrodotoxin (TTX). Ciguatoxin caused negative inotropy associated with cell depolarisation in 1.2 mM Ca2+-Ringer and additionally caused signs of Ca overload in 3.2 mM Ca2+-Ringer. Ciguatoxin induced transient after-contractions and contracture in atria which were common in 3.2 mM but not 1.2 mM Ca2+-Ringer and which were enhanced by ouabain. TTX and lidocaine abolished after-contractions and contracture while procaine was less effective. Extrasystoles consisting of short bursts of 1-2 extra contractions per sec were seen in atria and papillary muscles within 45 min of ciguatoxin being added. The effect was observed in 3.2 mM but seldom in 1.2 mM Ca2+-Ringer and was absent when low doses of propranolol or TTX were added prior to ciguatoxin. Flutter was observed in a few papillary muscles after ciguatoxin. These results suggest that the toxic effects of ciguatoxin stem from its direct action of opening myocardial Na+ channels. Extrasystoles appeared to result mainly from its effect on neural Na+ channels causing an increased release of noradrenaline from the nerves associated with the myocardium.
Publisher: MDPI AG
Date: 30-07-2022
DOI: 10.3390/MEMBRANES12080749
Abstract: The natural product indole-3-carbinol (I3C) and its major digestive product 3,3′-diindolylmethane (DIM) have shown clinical promise in multiple forms of cancer including breast cancer. In this study, we explored the calcium channel activity of DIM, its synthetic derivative 3,3′-Diindolylmethanone (DIM-one) and related I3C and DIM-one analogs. For the first time, DIM, DIM-one and analog IX were identified as selective blockers for T-type CaV3.3 (IC50s DIM 2.09 µM DIM-one 9.07 µM) while compound IX inhibited both CaV3.2 (6.68 µM) and CaV3.3 (IC50 = 3.05 µM) using a FLIPR cell-based assay to measure inhibition of T-type calcium channel window current. Further characterization of DIM by electrophysiology revealed it inhibited inward Ca2+ current through CaV3.1 (IC50 = 8.32 µM) and CaV3.3 (IC50 = 9.63 µM), while IX partially blocked CaV3.2 and CaV3.3 inward Ca2+ current. In contrast, DIM-one preferentially blocked CaV3.1 inward Ca2+ current (IC50 = 1.53 µM). The anti-proliferative activities of these compounds revealed that oxidation of the methylene group of DIM shifted the selectivity of DIMs from breast cancer cell line MCF-7 to colon cancer cell line HT-29.
Publisher: Wiley
Date: 05-12-2015
Abstract: The design of disulfide bond mimetics is an important strategy for optimising cysteine-rich peptides in drug development. Mimetics of the drug lead conotoxin MrIA, in which one disulfide bond is selectively replaced of by a 1,4-disubstituted-1,2,3-triazole bridge, are described. Sequential copper-catalyzed azide-alkyne cycloaddition (CuAAC click reaction) followed by disulfide formation resulted in the regioselective syntheses of triazole-disulfide hybrid MrIA analogues. Mimetics with a triazole replacing the Cys4-Cys13 disulfide bond retained tertiary structure and full in vitro and in vivo activity as norepinephrine reuptake inhibitors. Importantly, these mimetics are resistant to reduction in the presence of glutathione, thus resulting in improved plasma stability and increased suitability for drug development.
Publisher: Proceedings of the National Academy of Sciences
Date: 21-09-2020
Abstract: The venom of Australian funnel-web spiders contains δ-hexatoxins (δ-HXTXs) that exert fatal neurotoxic effects in humans by inhibiting inactivation of voltage-gated sodium channels, but their precise ecological role remains unclear. Sequencing of venom-gland transcriptomes from 10 funnel-web species uncovered 22 δ-HXTXs. Evolutionary analysis revealed extreme conservation of these toxins, despite their ancient origin. We isolated the lethal δ-HXTX from venom of the Sydney funnel-web spider and showed that it induces pain in mice, suggesting a role in predator deterrence. Although humans are not the target of δ-HXTXs, these toxins likely evolved to deter vertebrate predators commonly encountered by these spiders, such as bandicoots, birds, and lizards. Thus, the lethal potency of δ-HXTXs against humans is an unfortunate evolutionary coincidence.
Publisher: The Royal Society of Chemistry
Date: 18-09-2014
DOI: 10.1039/9781849735087-00297
Abstract: Ion channels are important drug targets for a range of diseases including pain, epilepsy and addiction. However, progress towards the development of more selective inhibitors that generate fewer dose-limiting side effects, or open up new therapeutic opportunities, has been slow. Due to the potentially higher selectivity offered by venom peptides, many pharmaceutical companies are embracing biological-based approaches to the identification of novel ion channel modulators. This will help overcome some of the limitations of low molecular weight modulators, whose affinity is often driven by factors such as lipid solubility and interactions with more conserved transmembrane domains. This chapter will cover this rapidly emerging field, providing ex les of venom peptide and small molecule approaches towards the development of Cav2.2, Nav1.7 and Kv1.3 inhibitors for the treatment of pain and autoimmune diseases.
Publisher: Informa UK Limited
Date: 10-2010
Publisher: Wiley
Date: 03-2004
Publisher: Elsevier BV
Date: 07-2009
DOI: 10.1016/J.TOXICON.2009.03.013
Abstract: Ciguatera is a significant food borne disease caused by potent polyether toxins known as ciguatoxins, which accumulate in the flesh of ciguateric fish at risk levels above 0.1 ppb. The management of ciguatera has been hindered by the lack of analytical methods to detect and quantify clinically relevant levels of ciguatoxin in easily prepared crude extracts of fish. Here we report a ciguatoxin rapid extraction method (CREM) that allows the rapid preparation of fish flesh extracts for the detection and quantification of ciguatoxin by gradient reversed-phase liquid chromatography-tandem mass spectrometry (LC/MS/MS). CREM-LC/MS/MS delivers a linear response to P-CTX-1 spiked into fish prior to extraction. A similar response was obtained for P-CTX-1 spiked after extraction, indicating >95% extraction efficiency was achieved overall and 85% at the limit of quantification (0.1 ppb). Using this approach, levels >or=0.1 ppb P-CTX-1 could be detected and quantified from an extract of 2g fish flesh, making it suitable as a confirmatory assay for suspect ciguateric carnivorous fish in the Pacific Ocean. The approach is designed to simplify the extraction and analysis of multiple s les per day.
Publisher: Elsevier BV
Date: 12-2006
DOI: 10.1016/J.TOXICON.2006.07.019
Abstract: Ciguatera is a global disease caused by the consumption of certain warm-water fish that have accumulated orally effective levels of sodium channel activator toxins (ciguatoxins) through the marine food chain. Symptoms of ciguatera arising from the consumption of ciguateric fish include a range of gastrointestinal, neurological and cardiovascular disturbances. This review examines progress in our understanding of ciguatera from an Australian perspective, especially the laboratory-based research into the problem that was initiated by the late "Bob" Endean at the University of Queensland.
Publisher: Springer New York
Date: 2009
DOI: 10.1007/978-1-4419-1132-2_5
Abstract: Venom peptides offer enormous opportunity for the discovery of peptide drug leads. This review focusses on the potential of cone snails that have developed arrays of small peptides as part of highly evolved venoms used for prey capture and defence. Many of these peptides selectively modulate ion channels and transporters, making them a valuable source of new ligands for studying the role these targets play in normal and disease physiology. A number of these conopeptides reduce pain in animals models and several are now in preclinical and clinical development for the treatment of severe pain often associated with diseases such as cancer.
Publisher: Elsevier BV
Date: 04-2010
DOI: 10.1016/J.BCP.2009.11.019
Abstract: Monoamine transporters are a group of transmembrane neurotransmitter sodium symporter (NSS) transporters that play a crucial role in regulating biogenic monoamine concentrations at peripheral and central synapses. Given the key role played by serotonin, dopamine and noradrenaline in addictive and disease states, structure-function studies have been conducted to help guide the development of improved central nervous system therapeutics. Extensive pharmacological, immunological and biochemical studies, in conjunction with three-dimensional homology modeling, have been performed to structurally and functionally characterise the monoamine transporter substrate permeation pathway, substrate selectivity, and binding sites for ions, substrates and inhibitors at the molecular level. However, only recently has it been possible to start to construct an accurate molecular interaction network for the monoamine transporters and their corresponding substrates and inhibitors. Crystal structures of Aquifex aeolicus leucine transporter (LeuT(Aa)), a homologous protein to monoamine transporters that has been experimentally demonstrated to share similar structural folds with monoamine transporters, have been determined in complex with amino acids and inhibitors. The molecular interactions of leucine and tricyclic antidepressants (TCA) has supported many of the predictions based on the mutational studies. Models constructed from LeuT(Aa) are now allowing a rational approach to further clarify the molecular determinants of NSS transporter-ligand complexes, and potentially the ability to better manipulate drug specificity and affinity. In this review, we compare the structure-function relationships of other SLC6 NSS family transporters with monoamine transporters, and discuss possible mechanisms involved in substrate binding and transport, and modes of inhibition by TCAs.
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C7NJ04969B
Abstract: Novel mixed opioid agonist/N-VGCC blocker peptides, design, synthesis and biological profile.
Publisher: MDPI AG
Date: 26-01-2021
DOI: 10.3390/MD19020060
Abstract: Conotoxins are disulfide-rich peptides found in the venom of cone snails. Due to their exquisite potency and high selectivity for a wide range of voltage and ligand gated ion channels they are attractive drug leads in neuropharmacology. Recently, cone snails were found to have the capability to rapidly switch between venom types with different proteome profiles in response to predatory or defensive stimuli. A novel conotoxin, GXIA (original name G117), belonging to the I3-subfamily was identified as the major component of the predatory venom of piscivorous Conus geographus. Using 2D solution NMR spectroscopy techniques, we resolved the 3D structure for GXIA, the first structure reported for the I3-subfamily and framework XI family. The 32 amino acid peptide is comprised of eight cysteine residues with the resultant disulfide connectivity forming an ICK+1 motif. With a triple stranded β-sheet, the GXIA backbone shows striking similarity to several tarantula toxins targeting the voltage sensor of voltage gated potassium and sodium channels. Supported by an hipathic surface, the structural evidence suggests that GXIA is able to embed in the membrane and bind to the voltage sensor domain of a putative ion channel target.
Publisher: Elsevier BV
Date: 12-2017
Publisher: Elsevier BV
Date: 1983
Publisher: Wiley
Date: 24-10-2017
Publisher: CSIRO Publishing
Date: 2020
DOI: 10.1071/CH19588
Abstract: Given the complexity of cone snail venoms, high throughput venomics approaches are required to fully investigate venom composition, envenomation strategies, and evolutionary trajectories. This study describes 158 conotoxins in the venom transcriptome of the little studied C. striolatus from the fish hunting clade Pionoconus. Despite similar gene superfamily distributions along the venom duct, only 18 common transcripts were identified between distal, central, and proximal venom duct transcriptomes. Proteomic analysis of the injected predatory venom collected from the same in idual revealed an ~18-fold enhanced complexity at the proteomic level, consistent with complex post-translational modifications and variable venom peptide processing occurring in the venom duct. Overall, C. striolatus venom was dominated by M, O1, O2, and A gene superfamily conotoxins and conkunitzins, which are potential modulators of sodium, calcium, and potassium channels. Conkunitzins and gene superfamily A peptides dominated the proximal over the distal duct, the M and O1 gene superfamily peptides were distributed along the full length of the duct, while the O2 gene superfamily peptides dominated the distal duct. Interestingly, the predatory injected venom of C. striolatus was dominated by peptides from gene superfamilies M, O1, O2, A, and conkunitzins, suggesting the predatory venom of C. striolatus may arise at multiple sites along the venom duct.
Publisher: Springer Science and Business Media LLC
Date: 18-11-2014
DOI: 10.1038/NCOMMS6421
Abstract: Bacterial cell ision is facilitated by a molecular machine—the isome—that assembles at mid-cell in iding cells. The formation of the cytokinetic Z-ring by the tubulin homologue FtsZ is regulated by several factors, including the isome component EzrA. Here we describe the structure of the 60-kDa cytoplasmic domain of EzrA, which comprises five linear repeats of an unusual triple helical bundle. The EzrA structure is bent into a semicircle, providing the protein with the potential to interact at both N- and C-termini with adjacent membrane-bound isome components. We also identify at least two binding sites for FtsZ on EzrA and map regions of EzrA that are responsible for regulating FtsZ assembly. The in idual repeats, and their linear organization, are homologous to the spectrin proteins that connect actin filaments to the membrane in eukaryotes, and we thus propose that EzrA is the founding member of the bacterial spectrin family.
Publisher: Frontiers Media SA
Date: 24-12-2021
DOI: 10.3389/FPHAR.2021.795455
Abstract: Given the important role of voltage-gated sodium (Na V ) channel-modulating spider toxins in elucidating the function, pharmacology, and mechanism of action of therapeutically relevant Na V channels, we screened the venom from Australian theraphosid species against the human pain target hNa V 1.7. Using assay-guided fractionation, we isolated a 33-residue inhibitor cystine knot (ICK) peptide (Ssp1a) belonging to the NaSpTx1 family. Recombinant Ssp1a (rSsp1a) inhibited neuronal hNa V subtypes with a rank order of potency hNa V 1.7 & 1.6 & 1.2 & 1.3 & 1.1. rSsp1a inhibited hNa V 1.7, hNa V 1.2 and hNa V 1.3 without significantly altering the voltage-dependence of activation, inactivation, or delay in recovery from inactivation. However, rSsp1a demonstrated voltage-dependent inhibition at hNa V 1.7 and rSsp1a-bound hNa V 1.7 opened at extreme depolarizations, suggesting rSsp1a likely interacted with voltage-sensing domain II (VSD II) of hNa V 1.7 to trap the channel in its resting state. Nuclear magnetic resonance spectroscopy revealed key structural features of Ssp1a, including an hipathic surface with hydrophobic and charged patches shown by docking studies to comprise the interacting surface. This study provides the basis for future structure-function studies to guide the development of subtype selective inhibitors.
Publisher: Elsevier BV
Date: 08-2018
DOI: 10.1016/J.IJMM.2018.01.008
Abstract: Lipoproteins are attached to the outer leaflet of the membrane by a di- or tri-acylglyceryl moiety and are thus positioned in the membrane-cell wall interface. Consequently, lipoproteins are involved in many surface associated functions, including cell wall synthesis, electron transport, uptake of nutrients, surface stress response, signal transduction, and they represent a reservoir of bacterial virulence factors. Inspection of 123 annotated Staphylococcus aureus genome sequences in the public domain revealed that this organism devotes about 2-3% of its coding capacity to lipoproteins, corresponding to about 70 lipoproteins per genome. 60 of these lipoproteins were identified in 95% of the genomes analyzed, which thus constitute the core lipoproteome of S. aureus. 30% of the conserved staphylococcal lipoproteins are substrate-binding proteins of ABC transporters with roles in nutrient transport. With a few exceptions, much less is known about the function of the remaining lipoproteins, representing a large gap in our knowledge of this functionally important group of proteins. Here, we summarize current knowledge, and integrate information from genetic context analysis, expression and regulatory data, domain architecture, sequence and structural information, and phylogenetic distribution to provide potential starting points for experimental evaluation of the biological function of the poorly or uncharacterized lipoproteome of S. aureus.
Publisher: Wiley
Date: 02-2004
DOI: 10.1080/15216540410001668055
Abstract: Cone snails have evolved a vast array of peptide toxins for prey capture and defence. These peptides are directed against a wide variety of pharmacological targets, making them an invaluable source of ligands for studying the properties of these targets in normal and diseased states. A number of these peptides have shown efficacy in vivo, including inhibitors of calcium channels, the norepinephrine transporter, nicotinic acetylcholine receptors, NMDA receptors and neurotensin receptors, with several having undergone pre-clinical or clinical development for the treatment of pain.
Publisher: Proceedings of the National Academy of Sciences
Date: 13-05-2013
Abstract: We recently reported the isolation of a scorpion toxin named U 1 -liotoxin-Lw1a (U 1 -LITX-Lw1a) that adopts an unusual 3D fold termed the disulfide-directed hairpin (DDH) motif, which is the proposed evolutionary structural precursor of the three-disulfide-containing inhibitor cystine knot (ICK) motif found widely in animals and plants. Here we reveal that U 1 -LITX-Lw1a targets and activates the mammalian ryanodine receptor intracellular calcium release channel (RyR) with high (fM) potency and provides a functional link between DDH and ICK scorpion toxins. Moreover, U 1 -LITX-Lw1a, now described as φ-liotoxin-Lw1a (φ-LITX-Lw1a), has a similar mode of action on RyRs as scorpion calcines, although with significantly greater potency, inducing full channel openings at lower (fM) toxin concentrations whereas at higher pM concentrations increasing the frequency and duration of channel openings to a submaximal state. In addition, we show that the C-terminal residue of φ-LITX-Lw1a is crucial for the increase in full receptor openings but not for the increase in receptor subconductance opening, thereby supporting the two-binding-site hypothesis of scorpion toxins on RyRs. φ-LITX-Lw1a has potential both as a pharmacological tool and as a lead molecule for the treatment of human diseases that involve RyRs, such as malignant hyperthermia and polymorphic ventricular tachycardia.
Publisher: Wiley
Date: 08-2004
Publisher: Public Library of Science (PLoS)
Date: 05-03-2021
DOI: 10.1371/JOURNAL.PONE.0243645
Abstract: Chemical transfection is broadly used to transiently transfect mammalian cells, although often associated with cellular stress and membrane instability, which imposes challenges for most cellular assays, including high-throughput (HT) assays. In the current study, we compared the effectiveness of calcium phosphate, FuGENE and Lipofectamine 3000 to transiently express two key voltage-gated ion channels critical in pain pathways, Ca V 2.2 and Na V 1.7. The expression and function of these channels were validated using two HT platforms, the Fluorescence Imaging Plate Reader FLIPR Tetra and the automated patch cl QPatch 16X. We found that all transfection methods tested demonstrated similar effectiveness when applied to FLIPR Tetra assays. Lipofectamine 3000-mediated transfection produced the largest peak currents for automated patch cl QPatch assays. However, the FuGENE-mediated transfection was the most effective for QPatch assays as indicated by the superior number of cells displaying GΩ seal formation in whole-cell patch cl configuration, medium to large peak currents, and higher rates of accomplished assays for both Ca V 2.2 and Na V 1.7 channels. Our findings can facilitate the development of HT automated patch cl assays for the discovery and characterization of novel analgesics and modulators of pain pathways, as well as assisting studies examining the pharmacology of mutated channels.
Publisher: Wiley
Date: 10-1995
DOI: 10.1111/J.1476-5381.1995.TB15056.X
Abstract: 1. The effects of ciguatoxin-1 (CTX-1) on the membrane potential of smooth muscle cells have been examined in rat proximal tail arteries isolated in vitro. 2. CTX-1 (> or = 10 pM) increased the frequency of spontaneous excitatory junction potentials (s.e.j.ps). At 100-400 pM, there was also a marked and maintained depolarization (19.7 +/- 1.4 mV, n = 14, at 400 pM). 3. In 20-400 pM CTX-1, perivascular stimuli evoked excitatory junction potentials (e.j.ps) which were prolonged in time course relative to control. 4. Although threshold and latency of the e.j.p. were not affected by CTX-1 ( or = 100 pM. 5. The spontaneous activity and the depolarization produced by CTX-1 were reduced in the presence of Ca2+ (0.1 mM)/Mg2+ (25 mM), omega-conotoxin (0.1 microM) or Cd2+ (50-100 microM). 6. All effects of CTX-1 were abolished by tetrodotoxin (0.3 microM). 7. Raised Ca2+ (6 mM) reduced the depolarization and spontaneous activity produced by CTX-1. 8. In 400 pM CTX-1, the membrane repolarized (17 +/- 3.2 mV, n = 4) following the addition of phentolamine (1 microM). S.e.j.ps and e.j.ps were selectively abolished by suramin (1 mM), and the membrane repolarized by 1.3 +/- 1.6 mV (n = 4). 9. We conclude that CTX-1 releases noradrenaline and ATP by initiating asynchronous discharge of postganglionic perivascular axons. In 100-400 pM CTX-1, the smooth muscle was depolarized to levels resembling those recorded in this artery during ongoing vasoconstrictor discharge in vivo.
Publisher: Elsevier BV
Date: 02-1997
DOI: 10.1016/S0041-0101(96)00132-8
Abstract: Reverse-phase high-performance liquid chromatography/mass spectrometry (HPLC/MS) was used to identify Pacific ciguatoxins (P-CTX) and P-CTX congeners present in a highly purified extract from the viscera of ciguateric moray eels (Lycodontis javanicus) collected in the central Pacific Ocean. Fourteen P-CTX or P-CTX congeners were identified with protonated molecular ions [M + H]+ m/z 1095.7 (two), 1111.6 (six) or 1127.7 (six), including dominant ions for P-CTX-1, -2 and -3. In addition to the protonated species, each of these ciguatoxins gave rise to prominent [M + NH4]+ and [M + Na]+ ions. The 11 new P-CTX congeners, not readily detected by mouse bioassay, were present in trace amounts (2-13% of P-CTX-2 levels) and identified as several oxidized P-CTX-1, -2 and -3, and a possible diasteriomer of P-CTX-1. Acetonitrile-water gradients buffered with 1 mM ammonium acetate improved the separation and detection of the minor ciguatoxins compared with an acetonitrile-water gradient modified with 0.1% TFA. Turbo-assisted HPLC/MS had sufficient sensitivity to detect P-CTX-1 in a crude extract from the flesh of an Australian ciguateric fish. Compounds with masses equivalent to other isolated ciguatoxins, including Caribbean-CTX-1, gambiertoxin-4A and P-CTX-3C, were not detected in these s les. HPLC/MS can readily identify multiple ciguatoxins accumulated by fish and has the potential to be used as a confirmatory analytical method for characterizing the low levels of ciguatoxins contaminating ciguateric fish.
Publisher: Elsevier BV
Date: 10-2010
DOI: 10.1016/J.TOXICON.2009.08.002
Abstract: Ciguatoxin (P-CTX-1B) from the dinoflagellate Gambierdiscus toxicus, belongs to the family of polyether neurotoxins responsible for the neurological poisoning disorder ciguatera. Although it is the most widespread marine-borne disease affecting humans, there is no current FDA-approved treatment available except for symptomatic therapies. In this paper, we report that P-CTX-1B promotes catecholamine secretion from bovine chromaffin cells, an effect that is insensitive to concomitant activation of capacitative Ca(2+) entry. Moreover, we confirm that brevenal, a polyether from the dinoflagellate Karenia brevis, blocks P-CTX-1B-induced catecholamine secretion. This effect is partially reversible. Our results therefore raise the prospect of finding functional antagonists for P-CTX-1B that could be useful for the treatment of ciguatera.
Publisher: Wiley
Date: 2007
DOI: 10.1111/J.1460-9568.2006.05299.X
Abstract: Omega-conotoxins are routinely used as selective inhibitors of different classes of voltage-gated calcium channels (VGCCs) in excitable cells. In the present study, we examined the potent N-type VGCC antagonist omega-conotoxin CVID and non-selective N- and P/Q-type antagonist CVIB for their ability to block native VGCCs in rat dorsal root ganglion (DRG) neurons and recombinant VGCCs expressed in Xenopus oocytes. Omega-conotoxins CVID and CVIB inhibited depolarization-activated whole-cell VGCC currents in DRG neurons with pIC50 values of 8.12 +/- 0.05 and 7.64 +/- 0.08, respectively. Inhibition of Ba2+ currents in DRG neurons by CVID (approximately 66% of total) appeared to be irreversible for > 30 min washout, whereas Ba2+ currents exhibited rapid recovery from block by CVIB (> or = 80% within 3 min). The recoverable component of the Ba2+ current inhibited by CVIB was mediated by the N-type VGCC, whereas the irreversibly blocked current (approximately 22% of total) was attributable to P/Q-type VGCCs. Omega-conotoxin CVIB reversibly inhibited Ba2+ currents mediated by N- (Ca(V)2.2) and P/Q- (Ca(V)2.1), but not R- (Ca(V)2.3) type VGCCs expressed in Xenopus oocytes. The alpha2delta1 auxiliary subunit co-expressed with Ca(V)2.2 and Ca(V)2.1 reduced the sensitivity of VGCCs to CVIB but had no effect on reversibility of block. Determination of the NMR structure of CVIB identified structural differences to CVID that may underlie differences in selectivity of these closely related conotoxins. Omega-conotoxins CVIB and CVID may be useful as antagonists of N- and P/Q-type VGCCs, particularly in sensory neurons involved in processing primary nociceptive information.
Publisher: MDPI AG
Date: 13-06-2018
DOI: 10.3390/MD16060208
Publisher: Frontiers Media SA
Date: 13-04-2023
DOI: 10.3389/FPHAR.2023.1170514
Abstract: αD-conotoxins are 11 kDa homodimers that potently inhibit nicotinic acetylcholine receptors (nAChRs) through a non-competitive (allosteric) mechanism. In this study, we describe the allosteric binding mode of the granulin-like C-terminal (CTD) of VxXXB bound to Lymnea stagnalis acetylcholine binding protein ( Ls -AChBP), a soluble homologue of the extracellular ligand-binding domain of nAChRs. This co-crystal complex revealed a novel allosteric binding site for nAChR antagonists outside the C-loop that caps the orthosteric site defined by the nAChR agonist nicotine and the antagonist epibatidine. Mutational and docking studies on Ls -AChBP supported a two-site binding mode for full-length VxXXB, with the first CTD binding site located outside the C-loop as seen in the co-crystal complex, with a second CTD binding site located near the N-terminal end of the adjacent subunit of AChBP. These results provide new structural insight into a novel allosteric mechanism of nAChR inhibition and define the cooperative binding mode of the N-terminal domain linked granulin core domains of αD-conotoxins.
Publisher: MDPI AG
Date: 03-08-2022
Abstract: To begin to understand the impact of food chain dynamics on ciguatera risk, we used published data to model the transfer of ciguatoxins across four trophic levels of a marine food chain in Platypus Bay, Australia. The data to support this first attempt to conceptualize the scale of each trophic transfer step was limited, resulting in broad estimates. The hypothetical scenario we explored generated a low-toxicity 10 kg Spanish mackerel (Scomberomorus commerson) with a flesh concentration of 0.1 µg/kg of Pacific-ciguatoxin-1 (P-CTX-1, also known as CTX1B) from 19.5–78.1 µg of P-CTX-1 equivalents (eq.) that enter the marine food chain from a population of 12–49 million benthic dinoflagellates (Gambierdiscus sp.) producing 1.6 × 10−12 g/cell of the P-CTX-1 precursor, P-CTX-4B. This number of Gambierdiscus could be epiphytic on 22–88 kg of the benthic macroalgae (Cladophora) that carpets the bottom of much of Platypus Bay, with the toxin transferred to an estimated 40,000–160,000 alpheid shrimps in the second trophic level. This large number of shrimps appears unrealistic, but toxic shrimps would likely be consumed by a school of small, blotched javelin fish (Pomadasys maculatus) at the third trophic level, reducing the number of shrimps consumed by each fish. The Spanish mackerel would accumulate a flesh concentration of 0.1 µg/kg P-CTX-1 eq. by preying upon the school of blotched javelin and consuming 3.6–14.4 µg of P-CTX-1 eq. However, published data indicate this burden of toxin could be accumulated by a 10 kg Spanish mackerel from as few as one to three blotched javelin fish, suggesting that much greater amounts of toxin than modelled here must at certain times be produced and transferred through Platypus Bay food chains. This modelling highlights the need for better quantitative estimates of ciguatoxin production, biotransformation, and depuration through marine food chains to improve our understanding and management of ciguatera risk.
Publisher: Elsevier BV
Date: 10-1996
Abstract: The omega-conotoxins are a set of structurally related peptides that have a wide range of specificities for different subtypes of the voltage-sensitive calcium channel (VSCC). To understand their VSCC subtype differentiation we studied the structure of two naturally occurring omega-conotoxins, MVIIA (specific to N-type) and SVIB (specific to P/Q-type) and a synthetic hybrid, SNX-202, which has altered specificities to both VSCC subtypes. The secondary structures of the three peptides are almost identical, consisting of a triple-stranded beta-sheet and several turns. A comparison of NMR data emphasizes the structural similarities between the peptides and highlights some minor structural differences. In the three-dimensional structures of SVIB and MVIIA these are manifested as orientational differences between two key loops. The structural rigidity of MVIIA was also examined. H alpha shifts are similar in a range of solvents, indicating that there are no solvent-induced changes in structure. The omega-conotoxins form a consensus structure despite differences in sequence and VSCC subtype specificity. This indicates that the omega-conotoxin macrosites for the N/P/Q-subfamily of VSCCs are related, with specificity for receptor targets being conferred by the positions of functional side-chains on the surface of the peptides.
Publisher: Elsevier BV
Date: 04-2010
Publisher: AMPCo
Date: 05-1997
DOI: 10.5694/J.1326-5377.1997.TB123219.X
Abstract: Ciguatera (poisoning caused by eating fish contaminated with algal toxins) is usually diagnosed clinically. We describe a Queensland family of four (including a pregnant woman) with ciguatera, confirmed by bioassay of the implicated fish for ciguatoxin. All four recovered, illustrating the effectiveness of treatment with intravenous mannitol. At birth, the infant appeared to suffer no adverse effects attributable to ciguatera to our knowledge, this is the first report of the effect of severe ciguatera in the first trimester of pregnancy.
Publisher: Elsevier BV
Date: 06-1999
Publisher: Elsevier BV
Date: 06-1998
Publisher: Wiley
Date: 29-07-2022
DOI: 10.1111/BPH.15923
Abstract: Over past decades, targeted therapies and immunotherapy have improved survival and reduced the morbidity of patients with BRAF‐mutated melanoma. However, drug resistance and relapse hinder overall success. Therefore, there is an urgent need for novel compounds with therapeutic efficacy against BRAF‐melanoma. This prompted us to investigate the antiproliferative profile of a tachykinin‐peptide from the Octopus kaurna , Octpep‐1 in melanoma. We evaluated the cytotoxicity of Octpep‐1 by MTT assay. Mechanistic insights on viability and cellular damage caused by Octpep‐1 were gained via flow cytometry and bioenergetics. Structural and pharmacological characterization was conducted by molecular modelling, molecular biology, CRISPR/Cas9 technology, high‐throughput mRNA and calcium flux analysis. In vivo efficacy was validated in two independent xerograph animal models (mice and zebrafish). Octpep‐1 selectively reduced the proliferative capacity of human melanoma BRAF V600E ‐mutated cells with minimal effects on fibroblasts. In melanoma‐treated cells, Octpep‐1 increased ROS with unaltered mitochondrial membrane potential and promoted non‐mitochondrial and mitochondrial respiration with inefficient ATP coupling. Molecular modelling revealed that the cytotoxicity of Octpep‐1 depends upon the α‐helix and polyproline conformation in the C‐terminal region of the peptide. A truncated form of the C‐terminal end of Octpep‐1 displayed enhanced potency and efficacy against melanoma. Octpep‐1 reduced the progression of tumours in xenograft melanoma mice and zebrafish. We unravel the intrinsic anti‐tumoural properties of a tachykinin peptide. This peptide mediates the selective cytotoxicity in BRAF‐mutated melanoma in vitro and prevents tumour progression in vivo, providing a foundation for a therapy against melanoma.
Publisher: Wiley
Date: 27-06-2017
DOI: 10.1111/BPH.13865
Publisher: Springer Science and Business Media LLC
Date: 09-03-2007
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 08-03-2012
Abstract: Conopeptides are a erse group of recently evolved venom peptides used for prey capture and/or defense. Each species of cone snails produces in excess of 1000 conopeptides, with those pharmacologically characterized (≈ 0.1%) targeting a erse range of membrane proteins typically with high potency and specificity. The majority of conopeptides inhibit voltage- or ligand-gated ion channels, providing valuable research tools for the dissection of the role played by specific ion channels in excitable cells. It is noteworthy that many of these targets are found to be expressed in pain pathways, with several conopeptides having entered the clinic as potential treatments for pain [e.g., pyroglutamate1-MrIA (Xen2174)] and one now marketed for intrathecal treatment of severe pain [ziconotide (Prialt)]. This review discusses the ersity, pharmacology, structure-activity relationships, and therapeutic potential of cone snail venom peptide families acting at voltage-gated ion channels (ω-, μ-, μO-, δ-, ι-, and κ-conotoxins), ligand-gated ion channels (α-conotoxins, σ-conotoxin, ikot-ikot, and conantokins), G-protein-coupled receptors (ρ-conopeptides, conopressins, and contulakins), and neurotransmitter transporters (χ-conopeptides), with expanded discussion on the clinical potential of sodium and calcium channel inhibitors and α-conotoxins. Expanding the discovery of new bioactives using proteomic/transcriptomic approaches combined with high-throughput platforms and better defining conopeptide structure-activity relationships using relevant membrane protein crystal structures are expected to grow the already significant impact conopeptides have had as both research probes and leads to new therapies.
Publisher: CSIRO Publishing
Date: 2020
DOI: 10.1071/CH19456
Abstract: In-solution conjugation is the most commonly used strategy to label peptides and proteins with fluorophores. However, lack of site-specific control and high costs of fluorophores are recognised limitations of this approach. Here, we established facile access to grams of Cy5-COOH via a two-step synthetic route, demonstrated that Cy5 is stable to HF treatment and therefore compatible with tert-butyloxycarbonyl solid phase peptide synthesis (Boc-SPPS), and coupled Cy5 to the N-terminus of α-conotoxin RgIA while still attached to the resin. Folding of the two-disulfide containing Cy5-RgIA benefitted from the hydrophobic nature of Cy5, resulting in only the globular disulfide bond isomer. In contrast, wild-type α-RgIA folded into the inactive ribbon and bioactive globular isomer under the same conditions. Labelled α-RgIA retained its ability to inhibit acetylcholine (100µM)-evoked current reversibly with an IC50 of 5.0nM (Hill coefficient=1.7) for Cy5-RgIA and an IC50 of 1.6 (Hill coefficient=1.2) for α-RgIA at the α9α10 nicotinic acetylcholine receptor (nAChR) heterologously expressed in Xenopus oocytes. Cy5-RgIA was then used to successfully visualise nAChRs in the RAW264.7 mouse macrophage cell line. This work introduced not only a new and valuable nAChR probe, but also a new versatile synthetic strategy that facilitates production of milligram to gram quantities of fluorophore-labelled peptides at low cost, which is often required for invivo experiments. The strategy is compatible with Boc- and 9-fluorenylmethoxycarbonyl (Fmoc)-chemistry, allows site-specific labelling of free amines anywhere in the peptide sequence, and can also be used for the introduction of Cy3/Cy5 fluorescence resonance energy transfer (FRET) pairs.
Publisher: Elsevier BV
Date: 07-2009
DOI: 10.1016/J.PEPTIDES.2009.03.019
Abstract: Cone snails have evolved an assortment of venom peptides as an evolutionary strategy for rapid prey immobilization and defence. Earlier studies estimated approximately 100 conopeptides per species. In this study we optimized liquid chromatography and electrospray ionization mass spectrometry for the detection of conopeptides in crude venom to characterize conopeptides present in the venom of in idual specimens of Conus textile, C. imperialis and C. marmoreus. Using this approach, we have expanded the predicted number of venom peptides 10-fold to an estimate of 1000-1900 conopeptides per species. Our investigation has also revealed a surprisingly high level of intra-species variation that distinguishes cone snails from other venomous species including spiders and scorpions. Given this inherent ersity and variability, more sensitive bioassays and sequencing techniques will be required to fully explore conotoxin bioactivity.
Publisher: Wiley
Date: 17-10-2003
DOI: 10.1016/S0014-5793(03)01161-X
Abstract: The Xenopus laevis oocyte expression system was used to determine the activities of alpha-conotoxins EpI and the ribbon isomer of AuIB, on defined nicotinic acetylcholine receptors (nAChRs). In contrast to previous findings on intracardiac ganglion neurones, alpha-EpI showed no significant activity on oocyte-expressed alpha3beta4 and alpha3beta2 nAChRs but blocked the alpha7 nAChR with an IC50 value of 30 nM. A similar IC50 value (103 nM) was obtained on the alpha7/5HT3 chimeric receptor stably expressed in mammalian cells. Ribbon AuIB maintained its selectivity on oocyte-expressed alpha3beta4 receptors but unlike in native cells, where it was 10-fold more potent than native alpha-AuIB, had 25-fold lower activity. These results indicate that as yet unidentified factors influence alpha-conotoxin pharmacology at native versus oocyte-expressed nAChRs.
Publisher: American Chemical Society (ACS)
Date: 03-04-2013
DOI: 10.1021/CB400012K
Abstract: Scorpion α-toxins are invaluable pharmacological tools for studying voltage-gated sodium channels, but few structure-function studies have been undertaken due to their challenging synthesis. To address this deficiency, we report a chemical engineering strategy based upon native chemical ligation. The chemical synthesis of α-toxin OD1 was achieved by chemical ligation of three unprotected peptide segments. A high resolution X-ray structure (1.8 Å) of synthetic OD1 showed the typical βαββ α-toxin fold and revealed important conformational differences in the pharmacophore region when compared with other α-toxin structures. Pharmacological analysis of synthetic OD1 revealed potent α-toxin activity (inhibition of fast inactivation) at Nav1.7, as well as Nav1.4 and Nav1.6. In addition, OD1 also produced potent β-toxin activity at Nav1.4 and Nav1.6 (shift of channel activation in the hyperpolarizing direction), indicating that OD1 might interact at more than one site with Nav1.4 and Nav1.6. Investigation of nine OD1 mutants revealed that three residues in the reverse turn contributed significantly to selectivity, with the triple OD1 mutant (D9K, D10P, K11H) being 40-fold more selective for Nav1.7 over Nav1.6, while OD1 K11V was 5-fold more selective for Nav1.6 than Nav1.7. This switch in selectivity highlights the importance of the reverse turn for engineering α-toxins with altered selectivity at Nav subtypes.
Publisher: Elsevier BV
Date: 1991
DOI: 10.1016/0041-0101(91)90209-A
Abstract: Viscera (48.3 kg) from moray eels (Lycodontis javanicus) collected in a ciguatera endemic area were extracted and the ciguatoxins characterized. Three major ciguatoxins, CTX-1, CTX-2 and CTX-3, were isolated and purified to homogeneity on reverse phase high performance liquid chromatography. Several minor toxins were also detected. CTX-1 (490 micrograms) was comparable by both 1H nuclear magnetic resonance (1H NMR) and mass spectroscopy (MH+ m/z = 1111) to ciguatoxin isolated previously from moray eels. CTX-2 (280 micrograms) and CTX-3 (100 micrograms) were less polar ciguatoxins not previously characterized. CTX-2 and CTX-3 differed from CTX-1 by 16 mass units, suggesting that they were less oxygenated analogues. 1H NMR revealed that the hydroxyl at C54 in CTX-1 was absent in CTX-2 and CTX-3. An additional change in the chemistry of CTX-2 compared to CTX-1 and CTX-3 was also suggested on the basis of 1H NMR, indicating that CTX-2 may arise from a different precursor to CTX-1. CTX-3 is likely to be an intermediate in the oxidation of a gambiertoxin (sodium channel toxins from Gambierdiscus toxicus) to CTX-1. The i.p. LD50 values for CTX-1, CTX-2 and CTX-3 were 0.25, 2.3 and 0.9 micrograms/kg, respectively. The signs induced in mice by the ciguatoxins were similar, except that CTX-2 and CTX-3 induced hind-limb paralysis that was absent with CTX-1. Each ciguatoxin was potent orally. CTX-1, CTX-2 and CTX-3 competitively inhibited the binding of [3H]brevetoxin-3 to voltage-dependent sodium channels with relative potencies qualitatively (but not quantitatively) comparable to mouse lethality. This study reveals that the relatively small chemical differences between CTX-1, CTX-2 and CTX-3 give rise to significant structure-activity and pharmacokinetic differences.
Publisher: MDPI AG
Date: 21-03-2023
Abstract: Published data were used to model the transfer of ciguatoxins (CTX) across three trophic levels of a marine food chain on the Great Barrier Reef (GBR), Australia, to produce a mildly toxic common coral trout (Plectropomus leopardus), one of the most targeted food fishes on the GBR. Our model generated a 1.6 kg grouper with a flesh concentration of 0.1 µg/kg of Pacific-ciguatoxin-1 (P-CTX-1 = CTX1B) from 1.1 to 4.3 µg of P-CTX-1 equivalents (eq.) entering the food chain from 0.7 to 2.7 million benthic dinoflagellates (Gambierdiscus sp.) producing 1.6 pg/cell of the P-CTX-1 precursor, P-CTX-4B (CTX4B). We simulated the food chain transfer of ciguatoxins via surgeonfishes by modelling Ctenochaetus striatus feeding on turf algae. A C. striatus feeding on ≥1000 Gambierdiscus/cm2 of turf algae accumulates sufficient toxin in days that when preyed on, produces a 1.6 kg common coral trout with a flesh concentration of 0.1 µg/kg P-CTX-1. Our model shows that even transient blooms of highly ciguatoxic Gambierdiscus can generate ciguateric fishes. In contrast, sparse cell densities of ≤10 Gambierdiscus/cm2 are unlikely to pose a significant risk, at least in areas where the P-CTX-1 family of ciguatoxins predominate. The ciguatera risk from intermediate Gambierdiscus densities (~100 cells/cm2) is more difficult to assess, as it requires feeding times for surgeonfish (~4–14 days) that overlap with turnover rates of turf algae that are grazed by herbivorous fishes, at least in regions such as the GBR, where stocks of herbivorous fishes are not impacted by fishing. We use our model to explore how the duration of ciguatoxic Gambierdiscus blooms, the type of ciguatoxins they produce, and fish feeding behaviours can produce differences in relative toxicities between trophic levels. Our simple model indicates thresholds for the design of risk and mitigation strategies for ciguatera and the variables that can be manipulated to explore alternate scenarios for the accumulation and transfer of P-CTX-1 analogues through marine food chains and, potentially, for other ciguatoxins in other regions, as more data become available.
Publisher: Elsevier BV
Date: 08-1992
DOI: 10.1016/0041-0101(92)90390-Q
Abstract: Most cases of ciguatera (fish poisoning) result from consumption of the flesh of fishes contaminated with ciguatoxin(s) however, the relatively low toxicity of ciguateric fish flesh has hindered attempts to identify these ciguatoxin(s). Utilising high performance liquid chromatography, mass spectroscopy and mouse bioassay signs we have determined that ciguatoxin-1 (MH+ m/z = 1112), ciguatoxin-2 and ciguatoxin-3 are the major ciguatoxins present in the flesh of ciguateric fish. Ciguatoxin-1, -2 and -3 were present in yields of 0.19, 0.09 and 0.02 microgram/kg flesh, respectively, in Scomberomorus commersoni 0.08, 0.09 and 0.07 microgram/kg flesh, respectively, in Plectropomus spp. and 0.67, 0.61 and 0.06 microgram/kg flesh, respectively, in Pomadasys maculatus. Two minor toxins, which may be further oxidised analogues of ciguatoxin-1 and ciguatoxin-2, were also identified. The presence of multiple ciguatoxins in fish flesh has important consequences for the detection of ciguateric fish and may be a contributing factor to the observed variability in the symptoms of ciguatera.
Publisher: American Chemical Society (ACS)
Date: 10-1999
DOI: 10.1021/JO990389C
Publisher: Elsevier BV
Date: 05-1997
DOI: 10.1016/S0041-0101(96)00166-3
Abstract: On 24 February 1995, six U.S. soldiers serving with the Multinational Force in Haiti became ill after eating a locally caught fish identified as the greater amberjack Seriola dumerili. The victims presented with nausea, vomiting, watery diarrhea and abdominal cr s 5-8 hr after consumption. Also present in some victims were numbness in the extremities or perioral region, bradycardia and scalp paresthesia. Patients were treated with i.v. hydration therapy and antiemetics. All recovered without sequelae over the course of 1-3 months. A portion of the cooked fish was obtained for analysis. A semipurified lipid extract was prepared according to standard methods and analyzed for the presence of Na+ channel site 5 binding activity using a brevetoxin receptor binding assay. By this assay, the fish s le contained the equivalent of approximately 20 ng Caribbean ciguatoxin/g flesh. The presence of the major Caribbean ciguatoxin (C-CTX-1) was confirmed by liquid chromatography-mass spectrometry. Using the receptor binding assay to monitor activity in TSK and PRP-1 column fractions, two minor toxins were detected in addition to C-CTX-1. One of these minor toxins was more polar, and the other less polar, than C-CTX-1. These data provide firm evidence that a family of C-CTX-1 is responsible for ciguatera in the Caribbean.
Publisher: Elsevier BV
Date: 08-2004
Publisher: Elsevier BV
Date: 11-1993
DOI: 10.1016/0742-8413(93)90217-9
Abstract: 1. Ciguatera is a disease caused by sodium channel activator toxins and results from the consumption of warm water fish contaminated by the ciguatoxin class of polyether toxins. 2. Other toxins, including okadaic acid and maitotoxin, have no proven role in causing human illness associated with ciguatera. 3. Ciguatera often affects only a discrete region of a reef, with flare-ups of ciguatera being both temporally and spatially unpredictable. 4. The ciguatoxins likely arise through the biotransformation and acid-catalysed spiroisomerisation of gambiertoxin-4A produced by Gambierdiscus toxicus and it is unlikely that other toxic benthic dinoflagellates are involved. 5. Events leading to a ciguatera outbreak are initiated by environmental and genetic factors that favour the proliferation of gambiertoxins, with an apparent role for anthropomorphic effects however, the precise factors involved are yet to be determined. 6. The gambiertoxins and/or ciguatoxins are transferred from the benthos to herbivorous species (fish, invertebrates etc) and then to carnivorous fish via marine food chains. 7. Factors influencing the concentration of ciguatoxins that accumulate in fish include the rate of dietary intake, the efficiency of assimilation, the degree and nature of any toxin biotransformation, the rate of depuration, and the rate of growth of fish.
Publisher: Elsevier BV
Date: 02-2013
Publisher: Elsevier BV
Date: 07-2010
Publisher: Elsevier BV
Date: 04-2010
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.
Publisher: American Chemical Society (ACS)
Date: 22-02-2016
DOI: 10.1021/ACS.JMEDCHEM.5B00911
Abstract: Opioid receptor screening of a conopeptide library led to a novel selective κ-opioid agonist peptide (conorphin T). Intensive medicinal chemistry, guided by potency, selectivity, and stability assays generated a pharmacophore model supporting rational design of highly potent and selective κ-opioid receptor (KOR) agonists (conorphins) with exceptional plasma stability. Conorphins are defined by a hydrophobic benzoprolyl moiety, a double arginine sequence, a spacer amino acid followed by a hydrophobic residue and a C-terminal vicinal disulfide moiety. The pharmacophore model was supported by computational docking studies, revealing receptor-ligand interactions similar to KOR agonist dynorphin A (1-8). A conorphin agonist inhibited colonic nociceptors in a mouse tissue model of chronic visceral hypersensitivity, suggesting the potential of KOR agonists for the treatment of chronic abdominal pain. This new conorphine KOR agonist class and pharmacophore model provide opportunities for future rational drug development and probes for exploring the role of the κ-opioid receptor.
Publisher: Frontiers Media SA
Date: 08-12-2021
DOI: 10.3389/FPHAR.2021.803397
Abstract: OmIA, isolated from Conus omaria venom, is a potent antagonist at α7 nAChRs. We determined the co-crystal structure of OmIA with Lymnae stagnalis acetylcholine binding protein ( Ls -AChBP) that identified His5, Val10 and Asn11 as key determinants for the high potency of OmIA at α7 nAChRs. Remarkably, despite a competitive binding mode observed in the co-crystal structure, OmIA and analogues displayed functional insurmountable antagonism at α7 and α3β4 nAChRs, except OmIA analogues having long side chain at position 10 ([V10Q]OmIA and [V10L]OmIA), which were partial insurmountable antagonist at α7 nAChRs in the presence of type II positive allosteric modulators (PAMs). A “two-state, two-step” model was used to explain these observations, with [V10Q]OmIA and [V10L]OmIA co-existing in a fast reversible/surmountable as well as a tight binding/insurmountable state. OmIA and analogues also showed biphasic-inhibition at α7 nAChRs in the presence of PNU120596, with a preference for the high-affinity binding site following prolonged exposure. The molecular basis of binding and complex pharmacological profile of OmIA at α7 nAChRs presented in here expands on the potential of α-conotoxins to probe the pharmacological properties of nAChRs and may help guide the development novel α7 modulators.
Publisher: Elsevier BV
Date: 08-2004
Publisher: MDPI AG
Date: 28-10-2022
Abstract: Australian funnel-web spiders are amongst the most dangerous venomous animals. Their venoms induce potentially deadly symptoms, including hyper- and hypotension, tachycardia, bradycardia and pulmonary oedema. Human envenomation is more frequent with the ground-dwelling species, including the infamous Sydney funnel-web spider (Atrax robustus) although, only two tree-dwelling species induce more severe envenomation. To unravel the mechanisms that lead to this stark difference in clinical outcomes, we investigated the venom transcriptome and proteome of arboreal Hadronyche cerberea and H. formidabilis. Overall, Hadronyche venoms comprised 44 toxin superfamilies, with 12 being exclusive to tree-dwellers. Surprisingly, the major venom components were neprilysins and uncharacterized peptides, in addition to the well-known ω- and δ-hexatoxins and double-knot peptides. The insecticidal effects of Hadronyche venom on sheep blowflies were more potent than Atrax venom, and the venom of both tree- and ground-dwelling species potently modulated human voltage-gated sodium channels, particularly NaV1.2. Only the venom of tree-dwellers exhibited potent modulation of voltage-gated calcium channels. H. formidabilis appeared to be under less ersifying selection pressure compared to the newly adapted tree-dweller, H. cerberea. Thus, this study contributes to unravelling the fascinating molecular and pharmacological basis for the severe envenomation caused by the Australian tree-dwelling funnel-web spiders.
Publisher: Springer Science and Business Media LLC
Date: 22-02-2017
DOI: 10.1038/SREP42810
Abstract: Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (Na V ), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on Na V 1.1–1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective Na V toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all Na V subtypes, an increase in the inactivation time constant was observed only at Na V 1.8, while the slope factor of the conductance-voltage curves was significantly increased for Na V 1.7 and peak current was significantly increased for Na V 1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of Na V 1.8 and the tetrodotoxin-sensitive isoforms Na V 1.7 and Na V 1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of Na V isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation.
Publisher: Wiley
Date: 16-10-2019
DOI: 10.1111/PRE.12349
Publisher: Royal Society of Chemistry (RSC)
Date: 2013
DOI: 10.1039/C3CC40537K
Abstract: We report the total chemical synthesis of human C3a by one-pot native chemical ligation of three unprotected peptide segments, followed by efficient in vitro folding that yielded the anaphylatoxin C3a in high yield and excellent purity. Synthetic C3a was fully active and its crystal structure at 2.1 Å resolution showed 3 helices and a C-terminal turn motif.
Publisher: Elsevier BV
Date: 2015
DOI: 10.1016/J.BCP.2014.09.024
Abstract: Cold pain is a frequent symptom in neuropathic pain. Compared to other pain modalities, such as heat pain, the mechanisms behind physiological and pathological cold pain remain elusive. Moreover, it is becoming increasingly evident that cold pain pharmacology differs between various neuropathic pain conditions, making mechanism-directed treatment based on an understanding of the underlying pathophysiological mechanisms imperative to achieving clinical success. Here we review the processes of physiological and abnormal cold sensing, the pharmacology of cold nociception, cold hyperalgesia and cold allodynia, and provide an overview of cold pain syndromes and their current and potential treatments.
Publisher: Wiley
Date: 09-07-2012
Publisher: American Chemical Society (ACS)
Date: 06-06-2014
DOI: 10.1021/BI5004475
Abstract: We isolated a novel, atypical long-chain three-finger toxin (TFT), α-elapitoxin-Dpp2d (α-EPTX-Dpp2d), from black mamba (Dendroaspis polylepis polylepis) venom. Proteolytic digestion with trypsin and V8 protease, together with MS/MS de novo sequencing, indicated that the mature toxin has an amidated C-terminal arginine, a posttranslational modification rarely observed for snake TFTs. α-EPTX-Dpp2d was found to potently inhibit α7 neuronal nicotinic acetylcholine receptors (nAChR IC₅₀, 58 ± 24 nM) and muscle-type nAChR (IC₅₀, 114 ± 37 nM) but did not affect α3β2 and α3β4 nAChR isoforms at 1 μM concentrations. Competitive radioligand binding assays demonstrated that α-EPTX-Dpp2d competes with epibatidine binding to the Lymnea stagnalis acetylcholine-binding protein (Ls-AChBP IC₅₀, 4.9 ± 2.3 nM). The activity profile and binding data are reminiscent of classical long-chain TFTs with a free carboxyl termini, suggesting that amidation does not significantly affect toxin selectivity. The crystal structure of α-EPTX-Dpp2d was determined at 1.7 Å resolution and displayed a dimeric toxin assembly with each monomer positioned in an antiparallel orientation. The dimeric structure is stabilized by extensive intermolecular hydrogen bonds and electrostatic interactions, which raised the possibility that the toxin may exist as a noncovalent homodimer in solution. However, chemical cross-linking and size-exclusion chromatography coupled with multiangle laser light scattering (MALLS) data indicated that the toxin is predominantly monomeric under physiological conditions. Because of its high potency and selectivity, we expect this toxin to be a valuable pharmacological tool for studying the structure and function of nAChRs.
Publisher: Frontiers Media SA
Date: 05-03-2020
Publisher: Wiley
Date: 15-01-1996
DOI: 10.1002/(SICI)1097-0231(19960115)10:1<138::AID-RCM442>3.0.CO;2-3
Publisher: Frontiers Media SA
Date: 11-04-2019
Publisher: Elsevier BV
Date: 09-2006
DOI: 10.1016/J.BCP.2006.03.027
Abstract: Venomous species have evolved cocktails of bioactive peptides to facilitate prey capture. Given their often exquisite potency and target selectivity, venom peptides provide unique biochemical tools for probing the function of membrane proteins at the molecular level. In the field of the nicotinic acetylcholine receptors (nAChRs), the subtype specific snake alpha-neurotoxins and cone snail alpha-conotoxins have been widely used to probe receptor structure and function in native tissues and recombinant systems. However, only recently has it been possible to generate an accurate molecular view of these nAChR-toxin interactions. Crystal structures of AChBP, a homologue of the nAChR ligand binding domain, have now been solved in complex with alpha-cobratoxin, alpha-conotoxin PnIA and alpha-conotoxin ImI. The orientation of all three toxins in the ACh binding site confirms many of the predictions obtained from mutagenesis and docking simulations on homology models of mammalian nAChR. The precise understanding of the molecular determinants of these complexes is expected to contribute to the development of more selective nAChR modulators. In this commentary, we review the structural data on nAChR-toxin interactions and discuss their implications for the design of novel ligands acting at the nAChR.
Publisher: American Chemical Society (ACS)
Date: 21-10-2019
DOI: 10.1021/ACS.CHEMREV.9B00207
Abstract: The venom of the marine predatory cone snails (genus
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 20-05-2004
Publisher: MDPI AG
Date: 13-04-2015
DOI: 10.3390/MD13042030
Publisher: American Chemical Society (ACS)
Date: 26-08-2023
Publisher: Wiley
Date: 03-1994
Abstract: A range of marine polyether toxins from dinoflagellates were analysed by ionspray mass spectrometry. Ciguatoxin-1 ([M+H]+ m/z = 1,111.8) purified from several fish species yielded singly charged ions corresponding to the parent ion, sodium and H2O adducts and ions for the loss of up to five H2O molecules. Ciguatoxin-1 was detected to 1 ng however, interference from fish lipids precluded direct detection of ciguatoxin-1 in crude extracts from fish flesh spiked with ciguatoxin-1 at a level equivalent to 1.5 ng ciguatoxin-1/g of extracted flesh. Maitotoxin-2 yielded doubly and triply charged ions for sodium and potassium salts and likely possessed only one sulphate ester (M(r) = 3,298 for the mono-sodium salt). Maitotoxin-3, a recently isolated small maitotoxin, yielded singly charged ions including ions for the loss of one sulphate and up to four H2O molecules. Maitotoxin-3 is proposed to be a polyether compound possessing two sulphate esters (M(r) = 1,060.5 for the disodium salt). Brevetoxin-A ([M+H]+ m/z = 867.5) and brevetoxin-B ([M+H]+ m/z = 895.5) yielded singly charged ions corresponding to the parent ion, Na+ adducts and the loss of up to four H2O molecules. Okadaic acid ([M+H]+ m/z = 805.5) yielded singly charged ions corresponding to the parent ion and ions for the loss of up to three H2O molecules. A signal for M + 18 Da species that may represent [M+NH4]+ was observed for ciguatoxin-1, brevetoxin-A and -B, and okadaic acid. For all polyethers examined, the orifice potential influenced the relative intensity of the ions detected in a predictable manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 17-08-2021
Publisher: Springer Science and Business Media LLC
Date: 10-01-2018
DOI: 10.1038/S41598-017-17422-X
Abstract: Cone snail venoms have separately evolved for predation and defense. Despite remarkable inter- and intra-species variability, defined sets of synergistic venom peptides (cabals) are considered essential for prey capture by cone snails. To better understand the role of predatory cabals in cone snails, we used a high-throughput proteomic data mining and visualisation approach. Using this approach, the relationship between the predatory venom peptides from nine C. purpurascens was systematically analysed. Surprisingly, potentially synergistic levels of κ-PVIIA and δ-PVIA were only identified in five of nine specimens. In contrast, the remaining four specimens lacked significant levels of these known excitotoxins and instead contained high levels of the muscle nAChR blockers ψ-PIIIE and αA-PIVA. Interestingly, one of nine specimens expressed both cabals, suggesting that these sub-groups might represent inter-breeding sub-species of C. purpurascens . High throughput cluster analysis also revealed these two cabals clustered with distinct groups of venom peptides that are presently uncharacterised. This is the first report showing that the cone snails of the same species can deploy two separate and distinct predatory cabals for prey capture and shows that the cabals deployed by this species can be more complex than presently realized. Our semi-automated proteomic analysis facilitates the deconvolution of complex venoms to identify co-evolved families of peptides and help unravel their evolutionary relationships in complex venoms.
Publisher: Wiley
Date: 03-1994
Abstract: Three strains of cultured Gambierdiscus toxicus yielded distinct maitotoxins (maitotoxin-1, -2, and -3) which were purified to homogeneity by high pressure liquid chromatography. Maitotoxins-1 and -2 are large toxins (molecular weights for the sodium salts = 3,422 and 3,298, respectively), whereas maitotoxin-3 is relatively small (molecular weight = 1,060 for the sodium salt). The contractile actions on isolated guinea-pig left atria, vas deferens and ilea of maitotoxins-1 and -2 were compared with those of the small maitotoxin, maitotoxin-3. Maitotoxin-1, -2 and -3 each produced quantitatively similar, concentration-dependent patterns of positive and negative inotropy in atria when compared on a mouse unit/ml basis (one mouse unit is the intraperitoneal LD50 dose for a 20 g mouse the LD50 for maitotoxin-2 = 0.08 microgram/kg). Concentrations of maitotoxin-2 greater than 5 x 10(-13) M caused positive inotropy. The three maitotoxins produced patterns of contractions in vas deferens and ilea that were qualitatively similar, including a period of prominent spike activity in vas deferens. On a mouse unit/ml basis, the order of potency on smooth muscle was maitotoxin-1 > maitotoxin-3 > maitotoxin-2. The contractile responses of smooth muscle to the maitotoxins were followed by an inhibitory phase where control agonist responses could not be elicited. The maitotoxin-induced contractile responses of vas deferens were inhibited by nicardipine but not phentolamine, indicating that in this tissue, each maitotoxin has mainly a direct contractile effect that requires calcium influx through voltage-sensitive calcium channels.(ABSTRACT TRUNCATED AT 250 WORDS)
Publisher: Elsevier BV
Date: 1991
DOI: 10.1016/0041-0101(91)90068-3
Abstract: Thirteen strains of Gambierdiscus toxicus isolated from Queensland (Australia), Hawaii, French Polynesia and the Virgin Islands were mass cultured and extracted for ciguatoxin. A biodetrital s le containing wild G. toxicus collected from the Republic of Kiribati was also extracted for ciguatoxin. Ciguatoxin, as characterized from moray eels, was not detected in any of the strains examined. Two Queensland strains and the wild G. toxicus produced putative ciguatoxin precursors named gambiertoxins. These gambiertoxins were less polar than ciguatoxin but produced bioassay signs in mice and in-vitro responses in isolated guinea pig atria and vas deferens which were similar (but not identical) to those produced by ciguatoxin. The gambiertoxins from cultured cells were also shown to competitively inhibit the binding of [3H]brevetoxin-3 to rat brain membranes in a dose-dependent manner. The gambiertoxins were more potent than ciguatoxin (on a per mouse unit basis) at stimulating neural elements of guinea pig atria. The two culture strains produced similar amounts of gambiertoxins, even when grown in nutrient media made from different seawater containing different concentrations of nutrients. Changes in nutrient media did not induce the other strains of G. toxicus to produce gambiertoxins. The production of these ciguatoxin precursors appears to be limited to only certain genetic strains of G. toxicus, with the majority of strains not producing these toxins. We propose that ciguatera occurs when blooms of G. toxicus strains genetically capable of producing these ciguatoxin precursors enter the marine food chain. These toxins could then become oxidatively metabolized in fishes to the major polar ciguatoxin. Wild cells produced approximately 100-fold greater quantities of gambiertoxins per cell than did the two culture strains indicating that there is considerable potential for increased production of these ciguatoxin precursors from G. toxicus in culture.
Publisher: Springer Science and Business Media LLC
Date: 20-01-2017
DOI: 10.1038/SREP40883
Abstract: Human genetic studies have implicated the voltage-gated sodium channel Na V 1.7 as a therapeutic target for the treatment of pain. A novel peptide, μ-theraphotoxin-Pn3a, isolated from venom of the tarantula P hobeteus nigricolor, potently inhibits Na V 1.7 (IC 50 0.9 nM) with at least 40–1000-fold selectivity over all other Na V subtypes. Despite on-target activity in small-diameter dorsal root ganglia, spinal slices, and in a mouse model of pain induced by Na V 1.7 activation, Pn3a alone displayed no analgesic activity in formalin-, carrageenan- or FCA-induced pain in rodents when administered systemically. A broad lack of analgesic activity was also found for the selective Na V 1.7 inhibitors PF-04856264 and phlotoxin 1. However, when administered with subtherapeutic doses of opioids or the enkephalinase inhibitor thiorphan, these subtype-selective Na V 1.7 inhibitors produced profound analgesia. Our results suggest that in these inflammatory models, acute administration of peripherally restricted Na V 1.7 inhibitors can only produce analgesia when administered in combination with an opioid.
Publisher: Springer Berlin Heidelberg
Date: 2009
DOI: 10.1007/978-3-540-87895-7_2
Abstract: Marine molluscs known as cone snails produce beautiful shells and a complex array of over 50,000 venom peptides evolved for prey capture and defence. Many of these peptides selectively modulate ion channels and transporters, making them a valuable source of new ligands for studying the role these targets play in normal and disease physiology. A number of conopeptides reduce pain in animal models, and several are now in pre-clinical and clinical development for the treatment of severe pain often associated with diseases such as cancer. Less than 1% of cone snail venom peptides are pharmacologically characterised.
Publisher: Elsevier BV
Date: 02-1992
DOI: 10.1016/0041-0101(92)90474-J
Abstract: The ciguatoxins are lipid soluble polyether compounds which have structural and biochemical features in common with the brevetoxins. Pure ciguatoxin-1, ciguatoxin-2 or brevetoxin-2 added to water containing Gambusia affinis induced similar signs, including pronounced opercular movement and uncoordinated swimming preceding death. The estimated LD50s (48 hr) to G. affinis for ciguatoxin-1, ciguatoxin-2 and brevetoxin-2 were 0.5, 2.1 and 10 nmoles/litre, respectively, indicating that the ciguatoxins were up to 20-fold more potent than the brevetoxins in this assay. Previous studies reveal that the ciguatoxins are more potent than the brevetoxins in both i.p. lethality to mammals and affinity for voltage-dependent sodium channels. However, relative to their affinity for the voltage-dependent sodium channel, brevetoxin-2 is 4-fold more potent to fish than the ciguatoxins, whereas the ciguatoxins are up to 11-fold more potent to mice than brevetoxin-2. This study found that only 3.4% of administered ciguatoxin-1 was accumulated by G. affinis. Ciguatoxin-1 may be biotransformed by G. affinis. The lethal effects of the ciguatoxins in fish may impose an upper limit on the levels of ciguatoxin carried by fish, which could contribute to the low incidence of human fatality associated with ciguatera.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 12-2006
Abstract: Mu-conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels during prey capture. Using polymerase chain reaction techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new mu-conotoxin-TIIIA (TIIIA). A 125I-TIIIA binding assay was established to isolate native TIIIA from the crude venom of Conus striatus. The isolated peptide had three post-translational modifications, including two hydroxyproline residues and C-terminal amidation, and <35% homology to other mu-conotoxins. TIIIA potently displaced [3H]saxitoxin and 125I-TIIIA from rat brain (Nav1.2) and skeletal muscle (Nav1.4) membranes. Alanine and glutamine scans of TIIIA revealed several residues, including Arg14, that were critical for high-affinity binding to tetrodotoxin (TTX)-sensitive Na+ channels. We were surprised to find that [E15A]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC50, 15 vs. 148 pM at rat brain membrane). TIIIA was selective for Nav1.2 and -1.4 over Nav1.3, -1.5, -1.7, and -1.8 expressed in Xenopus laevis oocytes and had no effect on rat dorsal root ganglion neuron Na+ current. 1H NMR studies revealed that TIIIA adopted a single conformation in solution that was similar to the major conformation described previously for mu-conotoxin PIIIA. TIIIA and analogs provide new biochemical probes as well as insights into the structure-activity of mu-conotoxins.
Publisher: Wiley
Date: 14-07-2017
Publisher: Wiley
Date: 30-09-2019
DOI: 10.1002/PRO.3722
Publisher: CRC Press
Date: 12-03-2014
DOI: 10.1201/B16662-32
Publisher: Elsevier BV
Date: 07-2002
Publisher: Royal Society of Chemistry (RSC)
Date: 2022
DOI: 10.1039/D2MD00188H
Abstract: αD-VxXXB is a homodimer of paired 50-residue peptide with ten conserved cysteines. Here we reported the first synthetic approach to full-length VxXXB via α-ketohydroxylamine ligation that enables further characterisation of this novel class of allosteric nAChR inhibitors.
Publisher: CSIRO Publishing
Date: 2017
DOI: 10.1071/CH16407
Abstract: Peptide dendrimers are a novel class of precisely defined macromolecules of emerging interest. Here, we describe the synthesis, structure, binding affinity, receptor selectivity, functional activity, and antinociceptive properties of oxytocin-related dendrimers containing up to 16 copies of [Lys8]-oxytocin or LVT. These were generated using a copper(i)-catalyzed azide–alkyne cycloaddition (CuAAc) reaction with azido-pegylated LVT peptides on an alkyne–polylysine scaffold. 2D NMR analysis demonstrated that each attached LVT ligand was freely rotating and maintained identical 3D structures in each dendrimeric macromolecule. The binding affinity Ki at the oxytocin receptor increased approximately 17-, 12-, 3-, and 1.5-fold respectively for the 2-, 4-, 8-, and 16-mer dendrimeric LVT conjugates, compared with monomer azido-pegylated LVT (Ki = 9.5 nM), consistent with a multivalency effect. A similar trend in affinity was also observed at the related human V1a, V1b, and V2 receptors, with no significant selectivity change observed across this family of receptors. All LVT dendrimers were functionally active in vitro on human oxytocin receptors and inhibited colonic nociceptors potently in a mouse model of chronic abdominal pain.
Publisher: Springer Science and Business Media LLC
Date: 2004
DOI: 10.1023/B:MODI.0000025656.79632.86
Abstract: Cone snails (Conidae) are marine predators with some extraordinary features. Their venom contains a hundred or more peptides that target numerous ion channels and receptors in mammals, including several that are involved in disease. omega-Conotoxins from fish hunting snails are 24-27 residue peptides with a rigid 4-loop cysteine framework that target the N-type voltage-gated calcium channel (VGCC). Two omega-conotoxins, MVIIA and CVID are currently in clinical development for chronic pain management (Ziconotide or Prialt, and AM336, respectively). In an attempt to develop small molecule equivalents of CVID, we defined the Calpha-Cbeta vectors of the residues believed to be important for binding to the N-type VGCC. Using these vectors, we undertook a virtual screening of virtual libraries approach to identify compounds that matched the pharmacophore. Cyclic pentapeptides containing residues of loop 2 of CVID, with one or more being a D-amino acid were designed and synthesised and were found to be active at the N-type VGCC (IC50 approximately 20 microM). Agreeing with the specificity profile of CVID, molecules were inactive at the P/Q-type VGCC.
Publisher: Elsevier BV
Date: 03-2008
Publisher: Elsevier BV
Date: 03-2010
DOI: 10.1016/J.BCP.2009.10.020
Abstract: An increasing number of putative therapeutic targets have been identified in recent years for the treatment of neuronal pathophysiologies including pain, epilepsy, stroke and schizophrenia. Many of these targets signal through calcium (Ca(2+)), either by directly facilitating Ca(2+) influx through an ion channel, or through activation of G proteins that couple to intracellular Ca(2+) stores or voltage-gated Ca(2+) channels. Immortalized neuronal cell lines are widely used models to study neuropharmacology. However, systematic pharmacological characterization of the receptors and ion channels expressed in these cell lines is lacking. In this study, we systematically assessed endogenous Ca(2+) signaling in response to addition of agonists at potential therapeutic targets in a range of cell lines of neuronal origin (ND7/23, SH-SY5Y, 50B11, F11 and Neuro2A cells) as well as HEK293 cells, a cell line commonly used for over-expression of receptors and ion channels. This study revealed a remarkable ersity of endogenous Ca(2+) responses in these cell lines, with one or more cell lines responding to addition of trypsin, bradykinin, ATP, nicotine, acetylcholine, histamine and neurotensin. Subtype specificity of these responses was inferred from agonist potency and the effect of receptor subtype specific antagonist. Surprisingly, HEK293 and SH-SY5Y cells responded to the largest number of agonists with potential roles in neuronal signaling. These findings have implications for the heterologous expression of neuronal receptors and ion channels in these cell lines, and highlight the potential of neuron-derived cell lines for the study of a range of endogenously expressed receptors and ion channels that signal through Ca(2+).
Publisher: Elsevier BV
Date: 2005
DOI: 10.1016/J.EJPHAR.2004.12.011
Abstract: The ability of the conotoxin rho-TIA, a 19-amino acid peptide isolated from the marine snail Conus tulipa, to antagonize contractions induced by noradrenaline through activation of alpha1A-adrenoceptors in rat vas deferens, alpha1B-adrenoceptors in rat spleen and alpha1D-adrenoceptors in rat aorta, and to inhibit the binding of [125I]HEAT (2-[[beta-(4-hydroxyphenyl)ethyl]aminomethyl]-1-tetralone) to membranes of human embryonic kidney (HEK) 293 cells expressing each of the recombinant rat alpha1-adrenoceptors was investigated. rho-TIA (100 nM to 1 microM) antagonized the contractions of vas deferens and aorta in response to noradrenaline without affecting maximal effects and with similar potencies (pA2 approximately 7.2, n=4). This suggests that rho-TIA is a competitive antagonist of alpha1A- and alpha1D-adrenoceptors with no selectivity between these subtypes. Incubation of rho-TIA (30 to 300 nM) with rat spleen caused a significant reduction of the maximal response to noradrenaline, suggesting that rho-TIA is a non-competitive antagonist at alpha1B-adrenoceptors. After receptor inactivation with phenoxybenzamine, the potency of rho-TIA in inhibiting contractions was examined with similar occupancies (approximately 25%) at each subtype. Its potency (pIC50) was 12 times higher in spleen (8.3+/-0.1, n=4) than in vas deferens (7.2+/-0.1, n=4) or aorta (7.2+/-0.1, n=4). In radioligand binding assays, rho-TIA decreased the number of binding sites (B(max)) in membranes from HEK293 cells expressing the rat alpha1B-adrenoceptors without affecting affinity (K(D)). In contrast, in HEK293 cells expressing rat alpha1A- or alpha1D-adrenoceptors, rho-TIA decreased the K(D) without affecting the B(max). It is concluded that rho-TIA will be useful for distinguishing the role of particular alpha1-adrenoceptor subtypes in native tissues.
Publisher: Elsevier BV
Date: 03-2012
DOI: 10.1016/J.TOXICON.2011.07.012
Abstract: Cone snail venoms continue to provide a rich source of bioactive peptides useful as research tools and leads to new therapeutics. We isolated two closely related conopeptides, MrIA and MrIB, which defined the χ-conopeptide class of bioactive peptides based on their unique ability to highly selectively and non-competitively inhibit the norepinephrine transporter (NET). An alanine scan of χ-MrIA revealed this class of peptides had a unusual cysteine-stabilised scaffold that presented a γ-turn in an optimised conformation for high affinity interactions with NET. χ-MrIA reversed the behavioural signs of mechanical allodynia in a chronic constriction injury rat model but its chemically unstable N-terminal asparagine precluded long-term use in implanted pumps. An extensive analoguing program identified Xen2174 to have improved stability and extended duration of analgaesia, without compromising efficacy versus side effects window observed forχ-MrIA. An open label, single IT bolus, dose-escalating study in cancer patients suffering severe chronic pain found Xen2174 relieved pain quickly over an extended period across a wide range of well-tolerated doses. Currently, Xen2174 is entering a Phase IIb double-blind study to determine safety and efficacy in bunionectomy pain.
Publisher: Wiley
Date: 2017
DOI: 10.1002/ANA.24841
Abstract: Cold allodynia occurs as a major symptom of neuropathic pain states. It remains poorly treated with current analgesics. Ciguatoxins (CTXs), ichthyosarcotoxins that cause ciguatera, produce a large peripheral sensitization to dynamic cold stimuli in Aδ-fibers by activating sodium channels without producing heat or mechanical allodynia. We used CTXs as a surrogate model of cold allodynia to dissect the framework of cold allodynia-activated central pain pathways. Reversible cold allodynia was induced in healthy male volunteers by shallow intracutaneous injection of low millimolar concentrations of CTX into the dorsal skin of the forefoot. Cold and warm stimuli were delivered to the treated and the control site using a Peltier-driven thermotest device. Functional magnetic resonance imaging (fMRI) scans were acquired with a 3T MRI scanner using a blood oxygen level-dependent (BOLD) protocol. The CTX-induced substantial peripheral sensitization to cooling stimuli in Aδ-fibers is particularly retrieved in BOLD changes due to dynamic temperature changes and less during constant cooling. Brain areas that responded during cold allodynia were almost always located bilaterally and appeared in the medial insula, medial cingulate cortex, secondary somatosensory cortex, frontal areas, and cerebellum. Whereas these areas also produced changes in BOLD signal during the dynamic warming stimulus on the control site, they remained silent during the warming stimuli on the injected site. We describe the defining feature of the cold allodynia pain percept in the human brain and illustrate why ciguatera sufferers often report a perceptual temperature reversal. ANN NEUROL 2017 :104-116.
Publisher: Elsevier BV
Date: 08-0005
Publisher: Public Library of Science (PLoS)
Date: 13-12-2011
Publisher: Elsevier BV
Date: 08-2010
Publisher: Elsevier BV
Date: 04-2005
DOI: 10.1016/J.TAAP.2004.08.020
Abstract: The present study investigated the actions of the polyether marine toxin Pacific ciguatoxin-1 (P-CTX-1) on neuronal excitability in rat dorsal root ganglion (DRG) neurons using patch-cl recording techniques. Under current-cl conditions, bath application of 2-20 nM P-CTX-1 caused a rapid, concentration-dependent depolarization of the resting membrane potential in neurons expressing tetrodotoxin (TTX)-sensitive voltage-gated sodium (Nav) channels. This action was completely suppressed by the addition of 200 nM TTX to the external solution, indicating that this effect was mediated through TTX-sensitive Nav channels. In addition, P-CTX-1 also prolonged action potential and afterhyperpolarization (AHP) duration. In a subpopulation of neurons, P-CTX-1 also produced tonic action potential firing, an effect that was not accompanied by significant oscillation of the resting membrane potential. Conversely, in neurons expressing TTX-resistant Nav currents, P-CTX-1 failed to alter any parameter of neuronal excitability examined in this study. Under voltage-cl conditions in rat DRG neurons, P-CTX-1 inhibited both delayed-rectifier and 'A-type' potassium currents in a dose-dependent manner, actions that occurred in the absence of alterations to the voltage dependence of activation. These actions appear to underlie the prolongation of the action potential and AHP, and contribute to repetitive firing. These data indicate that a block of potassium channels contributes to the increase in neuronal excitability, associated with a modulation of Nav channel gating, observed clinically in response to ciguatera poisoning.
Publisher: Elsevier BV
Date: 05-2020
Publisher: MDPI AG
Date: 17-03-2016
Publisher: Springer Science and Business Media LLC
Date: 06-08-2001
DOI: 10.1038/NN0901-902
Publisher: Elsevier BV
Date: 06-2007
Publisher: Public Library of Science (PLoS)
Date: 07-09-2017
Publisher: Elsevier BV
Date: 06-1997
DOI: 10.1016/S0041-0101(96)00191-2
Abstract: The toxins involved in ciguatera (fish poisoning) in the Caribbean Sea were isolated from Caranx latus, a pelagic fish often implicated in ciguatera in the Caribbean region, and purified by mouse bioassay directed fractionation. Five toxins were separated by reverse-phase high-performance liquid chromatography (HPLC). In order of increasing hydrophobicity, these toxins included a sleep-inducing fraction (< 1% of total toxicity), a major Caribbean ciguatoxin (C-CTX-1, 65% of toxicity), a minor Caribbean ciguatoxin (C-CTX-2, 13% of toxicity), a minor toxin (approximately 1% of toxicity) and a hydrophobic, fast-acting toxin (approximately 19% of toxicity). The i.p. injection into mice of each toxin induced signs typical of site-5 sodium channel activator toxins such as the Pacific ciguatoxins and brevetoxins. C-CTX-1 and C-CTX-2 were purified to homogeneity (LD50 = 3.6 and approximately 1 microgram/kg, respectively) and subjected to ion spray mass spectrometry. Both lost up to five H2O molecules and each had a [M+H]+ ion, m/z 1141.7, suggesting that C-CTX-1 and -2 are diastereomers that differ from the Pacific family of ciguatoxins. Turbo-assisted HPLC-mass spectrometry identified C-CTX-1, C-CTX-2 and three C-CTX-1-related compounds in an enriched fraction but no Pacific ciguatoxins were detected. The presence of different families of ciguatoxins in ciguateric fish from the Caribbean Sea and Pacific Ocean probably underlies the clinical differences in the ciguatera syndrome reported in these two regions. A Caribbean strain of the benthic dinoflagellate, Gambierdiscus toxicus, is suspected as source of these ciguatoxins. The extent to which these toxins are biotransformed as they pass through the marine food chain remains to be determined.
Publisher: Elsevier BV
Date: 04-2020
DOI: 10.1016/J.EJPHAR.2020.172947
Abstract: Previously, we showed that no two of seven opioids administered by the intracerebroventricular route had the same potency rank order for evoking antinociception, constipation and respiratory depression in rats. To gain insight at the cellular level, this study was designed to systematically investigate the activity profiles of six commonly used opioid ligands using the forskolin-stimulated cAMP assay and a β-arrestin2 recruitment assay in cultured HEK-293 cells transfected with MOP(μ), DOP(δ) or KOP(κ) receptors(-r). Morphine was a potent agonist at the MOP-r in the cAMP assay whereas it was a weak agonist at the KOP-r and DOP-r. Oxycodone had moderate efficacy and low potency at the MOP-r. Buprenorphine was a potent MOP-r and DOP-r agonist its efficacy rank order was DOP > MOP > KOP. Fentanyl was a potent agonist at the MOP-r its efficacy rank order was MOP > DOP > KOP. For DPDPE, its agonist efficacy was confined to the DOP-r, whereas for U69593, its efficacy rank order was KOP>> MOP. For the β-arrestin2 assay, fentanyl had full efficacy at the MOP-r whereas morphine and oxycodone were weak with insignificant efficacy at DOP and KOP receptors. Buprenorphine did not recruit β-arrestin2 at all three opioid-receptors. DPDPE and U69593 had full efficacy for β-arrestin2 recruitment to the DOP-r and KOP-r respectively. Despite the low efficacy and potency of morphine, oxycodone and buprenorphine in recruiting β-arrestin2 to the MOP-r herein, these opioids all evoked respiratory depression and constipation in rats. Together, our findings discount a key role for β-arrestin2 recruitment at the MOP-r in evoking opioid-related side-effects.
Publisher: American Chemical Society (ACS)
Date: 05-1999
DOI: 10.1021/BI982980U
Publisher: Elsevier BV
Date: 06-2004
Publisher: American Association for the Advancement of Science (AAAS)
Date: 05-12-2017
DOI: 10.1126/SCISIGNAL.AAN3398
Abstract: An oxytocin derivative that may not trigger adverse side effects has been generated.
Publisher: Elsevier BV
Date: 1982
DOI: 10.1016/0041-0101(82)90101-5
Abstract: The toxic skin tubercle gland secretion of the stonefish, Synanceia trachynis, contains a smooth muscle antispasmogen that is readily distinguished from papaverine, verapamil and both alpha and beta adrenoreceptor agonists. At 15 micrograms/ml, the toxin containing fraction markedly inhibited the phasic response and had a lesser inhibitory effect on the tonic response of acetylcholine-and KCl-induced contractures of guinea-pig ileum, while at 50 micrograms/ml, the toxin markedly inhibited both responses. Over the same dose range the toxin fraction markedly inhibited BaCl2-induced contractile responses but did not distinguish between the phasic and tonic components. Whereas the toxin fraction markedly reduced the tone of previously induced supramaximal KCl and acetylcholine responses of guinea-pig ileum, it had little effect on the tone of previously induced BaCl2 responses. The toxin non-competitively inhibited acetylcholine-induced contractions of guinea-pig ileum and similarly non-competitively inhibited the response induced by Ca2+ in high-[K+] Ringer. The toxin is not a chelating agent for Ca2+ or Mg2+ and it does not affect ATP-induced contracture of glycerinated guinea-pig ileum. The antispasmogenic effect of the toxin contained in the fraction does not result from inhibition of cAMP phosphodiesterase activity.
Publisher: CRC Press
Date: 12-03-2014
DOI: 10.1201/B16662-1
Publisher: MDPI AG
Date: 30-06-2020
DOI: 10.3390/MD18070343
Abstract: The 27-amino acid (aa)-long δ-conotoxin TxVIA, originally isolated from the mollusc-hunting cone snail Conus textile, slows voltage-gated sodium (NaV) channel inactivation in molluscan neurons, but its mammalian ion channel targets remain undetermined. In this study, we confirmed that TxVIA was inactive on mammalian NaV1.2 and NaV1.7 even at high concentrations (10 µM). Given the fact that invertebrate NaV channel and T-type calcium channels (CaV3.x) are evolutionarily related, we examined the possibility that TxVIA may act on CaV3.x. Electrophysiological characterisation of the native TxVIA on CaV3.1, 3.2 and 3.3 revealed that TxVIA preferentially inhibits CaV3.2 current (IC50 = 0.24 μM) and enhances CaV3.1 current at higher concentrations. In fish bioassays TxVIA showed little effect on zebrafish behaviours when injected intramuscular at 250 ng/100 mg fish. The binding sites for TxVIA at NaV1.7 and CaV3.1 revealed that their channel binding sites contained a common epitope.
Publisher: American Chemical Society (ACS)
Date: 31-03-2022
DOI: 10.1021/ACSCHEMNEURO.1C00857
Abstract: α-Conotoxins that target muscle nicotinic acetylcholine receptors (nAChRs) commonly fall into two structural classes, frameworks I and II containing two and three disulfide bonds, respectively. Conotoxin SII is the sole member of the cysteine-rich framework II with ill-defined interactions at the nAChRs. Following directed synthesis of α-SII, NMR analysis revealed a well-defined structure containing a 3
Publisher: Wiley
Date: 07-1997
DOI: 10.1002/19970504NT2
Publisher: Elsevier BV
Date: 12-2014
DOI: 10.1016/J.TOXICON.2014.09.011
Abstract: Conus geographus is the most dangerous cone snail species known, with reported human fatality rates as high as 65%. Crude venom gland extracts have been used to determine animal LD50 and to aid the isolation of several potent paralytic toxins. However, not only is the composition of injected venoms known to differ significantly from that in dissected venom glands, but also to vary according to predatory or defensive stimuli. Therefore, to study the venom that is directly relevant to human envenomation, the defense-evoked venom of several specimens of C. geographus was collected and analyzed by standard LC-MS methods. The molecular composition of in idual defense-evoked venom showed significant intraspecific variations, but a core of paralytic conotoxins including α-GI, α-GII, μ-GIIIA, ω-GVIA and ω-GVIIA was always present in large amounts, consistent with the symptomology and high fatality rate in humans. Differences between injected and dissected venoms obtained from the same specimen were also evident. Interestingly, an apparent linear correlation between the dry weight/volume of injected venom and the size of the shell allowed extrapolation to a human lethal dose (0.038-0.029 mg/kg) from an historic fatal case of C. geographus envenomation, which may help in the management of future victims.
Publisher: CSIRO Publishing
Date: 2009
DOI: 10.1071/CH09216
Abstract: Grafting different regions of related peptides together to form a single protein chimera is a valuable tool in rapidly elucidating regions of activity or selectivity in peptides and proteins. To conveniently evaluate the contributions of the N- and C-terminal segments of ω-conotoxins CVID and MVIIC to activity, we employed native chemical ligation in CVID-MVIIC chimera design. Assembly of these peptide segments via the ligation method improved overall yield and coupling efficiency, with no difficult sequences encountered in contrast to the traditional full-length chain assembly of CVID. Radio-ligand binding assays revealed regions of importance for receptor recognition.
Publisher: Springer Science and Business Media LLC
Date: 11-1986
DOI: 10.1007/BF00508787
Publisher: Elsevier BV
Date: 08-1993
DOI: 10.1016/0041-0101(93)90262-H
Abstract: Mannitol (1 g/kg i.v.) is currently the treatment of choice for acute ciguatera, but confirmation of this treatment's apparent efficacy awaits further experimental or controlled clinical evidence. In mice, mannitol (1 g/kg i.v.) administered before or after i.p. ciguatoxin did not influence the signs of intoxication or the time to death. The effects of oral ciguatoxin differed from those following i.p. ciguatoxin, but again i.v. mannitol provided no detectable benefit. Development of hypothermia was rapid in mice receiving i.p. or oral ciguatoxin and was unaffected by i.v. mannitol. A sublethal i.p. dose of ciguatoxin initially retarded (day 0-4) but then accelerated (day 4-12) the growth of mice. Mannitol (i.v.) had no influence on these effects of ciguatoxin on the growth of mice. Ciguatoxin inhibited responses of isolated diaphragms to nerve stimulation (ED50 = 9 x 10(-11) M), while directly stimulated diaphragms were inhibited by five-fold higher concentrations. Mannitol (50 mM) added to the organ bath did not influence the ciguatoxin-induced inhibition of diaphragm responses to nerve stimulation in vitro. Responses of isolated diaphragm to nerve stimulation were normal in preparations removed from ciguatoxin-treated mice displaying pronounced dyspnoea (gasping). However, responses to nerve stimulation were reduced in preparations removed from mice immediately following death from ciguatoxin. Mannitol (i.v.) partially protected the phrenic nerve-diaphragm from this effect of ciguatoxin in vivo. We conclude that the lethal effects of ciguatoxin in mice probably stem from a central action, and suggest that species differences may account for the absence of any marked beneficial effect of i.v. mannitol in the mouse model for ciguatera in humans.
Publisher: Royal Society of Chemistry
Date: 2015
Publisher: American Chemical Society (ACS)
Date: 15-09-2011
DOI: 10.1021/JA206408Q
Abstract: The two disulfide bonds of α-conotoxin ImI, a peptide antagonist of the α7 nicotinic acetylcholine receptor (nAChR), were systematically replaced with isosteric redox-stable cystathionine thioethers. Regioselective thioether formation was accomplished on solid support through substitution of a γ-chlorohomoalanine by an intramolecular cysteine thiol to produce hybrid thioether/disulfide analogues (2 and 3) as well as a dual cystathionine analogue (4) that were found to be structurally homologous to α-conotoxin ImI by (1)H NMR. The antagonistic activity at the α7 nAChR of cystathionine analogue 3 (pIC(50) = 6.41 ± 0.09) was identical to that of α-conotoxin ImI (1, pIC(50) = 6.41 ± 0.09), whereas those of 2 (pIC(50) = 5.96 ± 0.09) and 4 (pIC(50) = 5.89 ± 0.09) showed a modest decrease. The effect of oxidation of the thioethers to sulfoxides was also investigated, with significant changes in the biological activities observed ranging from a >30-fold reduction (2S═O) to a 3-fold increase (3S═O(B)) in potencies.
Publisher: Elsevier BV
Date: 06-2016
DOI: 10.1016/J.TOXICON.2015.11.004
Abstract: Ciguatoxins are the major toxins responsible for ciguatera fish poisoning, a disease dominated by muco-cutaneous sensory disorders including paresthesiae, cold dysesthesia and pruritus. While the ciguatoxins are well known to target voltage-gated sodium channels (VGSCs), the ensuing molecular mechanisms underlying these sensory disorders remain poorly understood. In this study, we propose a primary sensory neuron-keratinocyte co-culture as an appropriate model to study the neuro-cutaneous effects of ciguatoxins. Using this model, we show for the first time that nanomolar concentrations of Pacific ciguatoxin-2 (P-CTX-2) induced a VGSC-dependent release of substance P (SP) and calcitonin gene-related peptide (CGRP). As these neuropeptides are known mediators of pain and itch sensations, the ciguatoxin-induced sensory disturbances in ciguatera fish poisoning may involve the release of these neuropeptides. We further determined time- and P-CTX-2 concentration-dependence of the release of SP and CGRP from the co-culture model. Moreover, we highlighted the influence of extracellular calcium on the release of neuropeptides elicited by P-CTX-2. These findings underline the usefulness of this novel in vitro model for studying the cellular and molecular mechanisms of the neuro-cutaneous effects of ciguatoxins, which may assist with identifying potential therapeutics for ciguatera fish poisoning.
Publisher: Wiley
Date: 24-10-2017
Abstract: Conotoxins are a large family of disulfide-rich peptides that contain unique cysteine frameworks that target a broad range of ion channels and receptors. We recently discovered the 33-residue conotoxin Φ-MiXXVIIA from Conus miles with a novel cysteine framework comprising three consecutive cysteine residues and four disulfide bonds. Regioselective chemical synthesis helped decipher the disulfide bond connectivity and the structure of Φ-MiXXVIIA was determined by NMR spectroscopy. The 3D structure displays a unique topology containing two β-hairpins that resemble the N-terminal domain of granulin. Similar to granulin, Φ-MiXXVIIA promotes cell proliferation (EC
Publisher: Wiley
Date: 06-2004
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 15-05-2015
Abstract: Spider venoms are a rich source of ion channel modulators with therapeutic potential. Given the analgesic potential of subtype-selective inhibitors of voltage-gated sodium (NaV) channels, we screened spider venoms for inhibitors of human NaV1.7 (hNaV1.7) using a high-throughput fluorescent assay. Here, we describe the discovery of a novel NaV1.7 inhibitor, μ-TRTX-Tp1a (Tp1a), isolated from the venom of the Peruvian green-velvet tarantula Thrixopelma pruriens. Recombinant and synthetic forms of this 33-residue peptide preferentially inhibited hNaV1.7 > hNaV1.6 > hNaV1.2 > hNaV1.1 > hNaV1.3 channels in fluorescent assays. NaV1.7 inhibition was diminished (IC50 11.5 nM) and the association rate decreased for the C-terminal acid form of Tp1a compared with the native amidated form (IC50 2.1 nM), suggesting that the peptide C terminus contributes to its interaction with hNaV1.7. Tp1a had no effect on human voltage-gated calcium channels or nicotinic acetylcholine receptors at 5 μM. Unlike most spider toxins that modulate NaV channels, Tp1a inhibited hNaV1.7 without significantly altering the voltage dependence of activation or inactivation. Tp1a proved to be analgesic by reversing spontaneous pain induced in mice by intraplantar injection in OD1, a scorpion toxin that potentiates hNaV1.7. The structure of Tp1a as determined using NMR spectroscopy revealed a classic inhibitor cystine knot (ICK) motif. The molecular surface of Tp1a presents a hydrophobic patch surrounded by positively charged residues, with subtle differences from other ICK spider toxins that might contribute to its different pharmacological profile. Tp1a may help guide the development of more selective and potent hNaV1.7 inhibitors for treatment of chronic pain.
Publisher: Elsevier BV
Date: 08-2003
Publisher: Wiley
Date: 13-11-2015
DOI: 10.1111/EJN.13098
Publisher: American Chemical Society (ACS)
Date: 10-04-2014
DOI: 10.1021/CR400401E
Publisher: American Physiological Society
Date: 03-2015
Abstract: Changes in ion channel function and expression are characteristic of neuropathic pain. Voltage-gated calcium channels (VGCCs) are integral for neurotransmission and membrane excitability, but relatively little is known about changes in their expression after nerve injury. In this study, we investigate whether peripheral nerve ligation is followed by changes in the density and proportion of high-voltage-activated (HVA) VGCC current subtypes in dorsal root ganglion (DRG) neurons, the contribution of presynaptic N-type calcium channels in evoked excitatory postsynaptic currents (EPSCs) recorded from dorsal horn neurons in the spinal cord, and the changes in expression of mRNA encoding VGCC subunits in DRG neurons. Using C57BL/6 mice [8- to 11-wk-old males ( n = 91)] for partial sciatic nerve ligation or sham surgery, we performed whole cell patch-cl recordings on isolated DRG neurons and dorsal horn neurons and measured the expression of all VGCC subunits with RT-PCR in DRG neurons. After nerve injury, the density of P/Q-type current was reduced overall in DRG neurons. There was an increase in the percentage of N-type and a decrease in that of P/Q-type current in medium- to large-diameter neurons. No changes were found in the contribution of presynaptic N-type calcium channels in evoked EPSCs recorded from dorsal horn neurons. The α2δ-1 subunit was upregulated by 1.7-fold and γ-3, γ-2, and β-4 subunits were all downregulated 1.7-fold in injured neurons compared with sham-operated neurons. This comprehensive characterization of HVA VGCC subtypes in mouse DRG neurons after nerve injury revealed changes in N- and P/Q-type current proportions only in medium- to large-diameter neurons.
Publisher: MDPI AG
Date: 23-07-2021
Abstract: We review and develop conceptual models for the bio-transfer of ciguatoxins in food chains for Platypus Bay and the Great Barrier Reef on the east coast of Australia. Platypus Bay is unique in repeatedly producing ciguateric fishes in Australia, with ciguatoxins produced by benthic dinoflagellates (Gambierdiscus spp.) growing epiphytically on free-living, benthic macroalgae. The Gambierdiscus are consumed by invertebrates living within the macroalgae, which are preyed upon by small carnivorous fishes, which are then preyed upon by Spanish mackerel (Scomberomorus commerson). We hypothesise that Gambierdiscus and/or Fukuyoa species growing on turf algae are the main source of ciguatoxins entering marine food chains to cause ciguatera on the Great Barrier Reef. The abundance of surgeonfish that feed on turf algae may act as a feedback mechanism controlling the flow of ciguatoxins through this marine food chain. If this hypothesis is broadly applicable, then a reduction in herbivory from overharvesting of herbivores could lead to increases in ciguatera by concentrating ciguatoxins through the remaining, smaller population of herbivores. Modelling the dilution of ciguatoxins by somatic growth in Spanish mackerel and coral trout (Plectropomus leopardus) revealed that growth could not significantly reduce the toxicity of fish flesh, except in young fast-growing fishes or legal-sized fishes contaminated with low levels of ciguatoxins. If Spanish mackerel along the east coast of Australia can depurate ciguatoxins, it is most likely with a half-life of ≤1-year. Our review and conceptual models can aid management and research of ciguatera in Australia, and globally.
Publisher: Elsevier BV
Date: 12-2013
Publisher: Wiley
Date: 14-07-2017
Publisher: Springer US
Date: 2005
Publisher: Elsevier BV
Date: 06-2012
DOI: 10.1016/J.BCP.2012.02.022
Abstract: The human neuroblastoma cell line SH-SY5Y is a potentially useful model for the identification and characterisation of Na(v) modulators, but little is known about the pharmacology of their endogenously expressed Na(v)s. The aim of this study was to determine the expression of endogenous Na(v) α and β subunits in SH-SY5Y cells using PCR and immunohistochemical approaches, and pharmacologically characterise the Na(v) isoforms endogenously expressed in this cell line using electrophysiological and fluorescence approaches. SH-SY5Y human neuroblastoma cells were found to endogenously express several Na(v) isoforms including Na(v)1.2 and Na(v)1.7. Activation of endogenously expressed Na(v)s with veratridine or the scorpion toxin OD1 caused membrane depolarisation and subsequent Ca(2+) influx through voltage-gated L- and N-type calcium channels, allowing Na(v) activation to be detected with membrane potential and fluorescent Ca(2) dyes. μ-Conotoxin TIIIA and ProTxII identified Na(v)1.2 and Na(v)1.7 as the major contributors of this response. The Na(v)1.7-selective scorpion toxin OD1 in combination with veratridine produced a Na(v)1.7-selective response, confirming that endogenously expressed human Na(v)1.7 in SH-SY5Y cells is functional and can be synergistically activated, providing a new assay format for ligand screening.
Publisher: Elsevier BV
Date: 1983
Publisher: MDPI AG
Date: 10-07-2023
Abstract: Ciguatera is a major circumtropical poisoning caused by the consumption of marine fish and invertebrates contaminated with ciguatoxins (CTXs): neurotoxins produced by endemic and benthic dinoflagellates which are biotransformed in the fish food-web. We provide a history of ciguatera research conducted over the past 70 years on ciguatoxins from the Pacific Ocean (P-CTXs) and Caribbean Sea (C-CTXs) and describe their main chemical, biochemical, and toxicological properties. Currently, there is no official method for the extraction and quantification of ciguatoxins, regardless their origin, mainly due to limited CTX-certified reference materials. In this review, the extraction and purification procedures of C-CTXs are investigated, considering specific objectives such as isolating reference materials, analysing fish toxin profiles, or ensuring food safety control. Certain in vitro assays may provide sufficient sensitivity to detect C-CTXs at sub-ppb levels in fish, but they do not allow for in idual identification of CTXs. Recent advances in analysis using liquid chromatography coupled with low- or high-resolution mass spectrometry provide new opportunities to identify known C-CTXs, to gain structural insights into new analogues, and to quantify C-CTXs. Together, these methods reveal that ciguatera arises from a multiplicity of CTXs, although one major form (C-CTX-1) seems to dominate. However, questions arise regarding the abundance and instability of certain C-CTXs, which are further complicated by the wide array of CTX-producing dinoflagellates and fish vectors. Further research is needed to assess the toxic potential of the new C-CTX and their role in ciguatera fish poisoning. With the identification of C-CTXs in the coastal USA and Eastern Atlantic Ocean, the investigation of ciguatera fish poisoning is now a truly global effort.
Publisher: Frontiers Media SA
Date: 19-06-2019
Publisher: Wiley
Date: 26-07-2007
Publisher: Elsevier BV
Date: 11-2003
DOI: 10.1016/J.TOXICON.2003.09.004
Abstract: A barracuda implicated in ciguatera fish poisoning in Guadeloupe was estimated to have an overall flesh toxicity of 15 MUg/g using mouse bioassay. A lipid soluble extract was separated into two toxic fractions, FrA and FrB, on a LH20 Sephadex column eluted with dichloromethane/methanol (1:1). When intraperitoneal injected into mice, FrA provoked symptoms characteristic of slow-acting ciguatoxins, whereas FrB produced symptoms indicative of fast-acting toxins (FAT). High performance liquid chromatography/mass spectrometry/radio-ligand binding (HPLC/MS/RLB) analysis confirmed the two fractions were distinct, because only a weak overlap of some compounds was observed. HPLC/MS/RLB analysis revealed C-CTX-1 as the potent toxin present in FrA, and two coeluting active compounds at m/z 809.43 and 857.42 in FrB, all displaying the characteristic pattern of ion formation for hydroxy-polyethers. Other C-CTX congeners and putative hydroxy-polyether-like compounds were detected in both fractions, however, the RLB found them inactive. C-CTX-1 accounted for > 90% of total toxicity in this barracuda and was confirmed to be a competitive inhibitor of brevetoxin binding to voltage-sensitive sodium channels (VSSCs) with a potency two-times lower than P-CTX-1. However, FAT active on VSSCs and < 900 Da were suspected to contribute to the overall toxicity.
Publisher: Elsevier BV
Date: 10-2003
Publisher: Rockefeller University Press
Date: 15-03-2004
Abstract: It has been shown that β auxiliary subunits increase current litude in voltage-dependent calcium channels. In this study, however, we found a novel inhibitory effect of β3 subunit on macroscopic Ba2+ currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed β3 (12.5 ng/cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at “physiological” holding potentials (HPs) of −60 and −80 mV. At a HP of −80 mV, coinjection of various concentrations (0–12.5 ng) of the β3 with Cav2.2α1 and α2δ enhanced the maximum conductance of expressed channels at lower β3 concentrations but at higher concentrations (& .5 ng/cell) caused a marked inhibition. The β3-induced current suppression was reversed at a HP of −120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba2+ (40 mM) as a charge carrier also largely diminished the effect of β3 at −80 mV. Therefore, experimental conditions (HP, alent cation concentration, and β3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel β3 effect. Steady-state inactivation curves revealed that N-type channels exhibited “closed-state” inactivation without β3, and that β3 caused an ∼40-mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately −85 mV. The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of β3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at −80 mV. Similar “ultra-slow” inactivation was observed for N-type channels without β3. Thus, β3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by causing a hyperpolarizing shift of the inactivation without affecting “ultra-slow” and “closed-state” inactivation properties.
Publisher: MDPI AG
Date: 2018
DOI: 10.3390/MD16010007
Publisher: Future Science Ltd
Date: 10-2014
DOI: 10.4155/FMC.14.99
Abstract: Peptide neurotoxins from cone snails called conotoxins are renowned for their therapeutic potential to treat pain and several neurodegenerative diseases. Inefficient assay-guided discovery methods have been replaced by high-throughput bioassays integrated with advanced MS and next-generation sequencing, ushering in the era of ‘venomics’. In this review, we focus on the impact of venomics on the understanding of cone snail biology as well as the application of venomics to accelerate the discovery of new conotoxins. We also discuss the continued importance of medicinal chemistry approaches to optimize conotoxins for clinical use, with a descriptive case study of MrIA featured.
Publisher: Elsevier BV
Date: 11-2009
Publisher: Wiley
Date: 08-12-2014
DOI: 10.1111/CBDD.12479
Abstract: N-type voltage-dependent Ca(2+) channels (CaV 2.2) are located at nerve endings in the central and peripheral nervous systems and are strongly associated with the pathological processes of cerebral ischaemia and neuropathic pain. CaV 2.2 blockers such as the ω-conotoxin MVIIA (Prialt) are analgesic and have opioid-sparing effects. With the aim to develop new multitarget analgesic compounds, we designed the first ω-conotoxin/opioid peptidomimetics based on the enkephalin-like sequence Tyr-D-Ala-Gly-Phe (for the opioid portion) and two fragments derived from the loop-2 pharmacophore of ω-conotoxin MVIIA. Antinociceptive activity evaluated in vitro and in vivo revealed differential affinity for CaV 2.2 and opioid receptors and no significant synergistic activity.
Publisher: Bentham Science Publishers Ltd.
Date: 08-2012
DOI: 10.2174/156802612802652457
Abstract: Cone snails have evolved many 1000s of small, structurally stable venom peptides (conopeptides) for prey capture and defense. Whilst < 0.1% have been pharmacologically characterised, those with known function typically target membrane proteins of therapeutic importance, including ion channels, transporters and GPCRs. Several conopeptides reduce pain in animals models, with one in clinical development (χ-conopeptide analogue Xen2174) and one marketed (ω- conotoxin MVIIA or Prialt) for the treatment of severe pain. In addition to their therapeutic potential, conopeptides have been valuable probes for studying the role of a number of key membrane proteins in normal and disease physiology.
Publisher: Elsevier BV
Date: 1983
DOI: 10.1016/0041-0101(83)90045-4
Abstract: A lipid-soluble toxin, similar to ciguatoxin as isolated by Scheuer et al. (1967), has been found in the flesh of the Spanish mackerel, Scomberomorus commersoni, caught in Queensland. The ciguatoxin-like substance was experimentally characterized by examination of specific biological and chromatographic properties of the lipid-soluble extract from a pooled s le of flesh from Spanish mackerel. Flesh from specimens known to have caused S. commersoni poisoning in humans was confirmed as toxic by cat bioassay. A toxin was extracted from S. commersoni which yielded, on partial purification, a clear, oily substance with an LD50 i.p. to mice of 0.72 mg/kg, and which had chromatographic properties similar to those of classical ciguatoxin. However, the Rf value on thin-layer chromatography plates was lower for S. commersoni toxin than for classical ciguatoxin. This is the first record of a ciguatoxin-like substance experimentally identified in S. commersoni, a pelagic fish that occurs throughout Queensland coastal waters. The majority of toxic S. commersoni are caught between latitudes 24 degrees and 26 degrees S.
Publisher: Elsevier BV
Date: 10-2003
Publisher: Elsevier BV
Date: 2003
Publisher: Springer International Publishing
Date: 2022
Publisher: Elsevier BV
Date: 12-2013
DOI: 10.1016/J.MARPOLBUL.2013.10.003
Abstract: Ciguatera fish poisoning (CFP) is known to be caused by the ciguatoxins from the dinoflagellate genus Gambierdiscus, however, there is the potential for other toxins such as okadaic acid and dinophysistoxins from the genus Prorocentrum, and palytoxin from the genus Ostreopsis, to contaminate seafood. These genera may also be indicators of ecosystem health and potentially impact on coral reef ecosystems and the role they may play in the succession of coral to macroalgae dominated reefs has not been researched. Sixteen GBR field sites spanning inshore, mid-lagoon and outer lagoon (offshore) regions were studied. S les were collected from September 2006 to December 2007 and abundance of benthic dinoflagellates on different host macroalgae and concentration of nutrients present in the water column were determined. The maximum abundance of Prorocentrum, Ostreopsis and Gambierdiscus found was 112, 793 and 50 cells per gram wet weight of host macroalgae, respectively. The average level of Dissolved Inorganic Nitrogen (DIN) in the water column across all sites (0.03 mg/L) was found to be more than double the threshold critical value (0.013 mg/L) for healthy coral reefs. Compared to a previous study 1984, there is evidence of a major shift in the distribution and abundance of these dinoflagellates. Inshore reefs have either of Prorocentrum (as at Green Island) or Ostreopsis (as at Magnetic Island) dominating the macroalgal surface niche which was once dominated by Gambierdiscus, whilst at offshore regions Gambierdiscus is still dominant. This succession may be linked to the ongoing eutrophication of the GBR lagoon and have consequences for the sources of toxins for ongoing cases of ciguatera.
Publisher: American Chemical Society (ACS)
Date: 12-08-2015
DOI: 10.1021/ACSCHEMNEURO.5B00113
Abstract: Selective activation of peripheral κ opioid receptors (KORs) may overcome the dose-limiting adverse effects of conventional opioid analgesics. We recently developed a vicinal disulfide-stabilized class of peptides with subnanomolar potency at the KOR. The aim of this study was to assess the analgesic effects of one of these peptides, named conorphin-1, in comparison with the prototypical KOR-selective small molecule agonist U-50488, in several rodent pain models. Surprisingly, neither conorphin-1 nor U-50488 were analgesic when delivered peripherally by intraplantar injection at local concentrations expected to fully activate the KOR at cutaneous nerve endings. While U-50488 was analgesic when delivered at high local concentrations, this effect could not be reversed by coadministration with the selective KOR antagonist ML190 or the nonselective opioid antagonist naloxone. Instead, U-50488 likely mediated its peripheral analgesic effect through nonselective inhibition of voltage-gated sodium channels, including peripheral sensory neuron isoforms NaV1.8 and NaV1.7. Our study suggests that targeting the KOR in peripheral sensory nerve endings innervating the skin is not an alternative analgesic approach.
Publisher: Springer Science and Business Media LLC
Date: 13-06-2023
DOI: 10.1038/S41467-023-38924-5
Abstract: Marine cone snails have attracted researchers from all disciplines but early life stages have received limited attention due to difficulties accessing or rearing juvenile specimens. Here, we document the culture of Conus magus from eggs through metamorphosis to reveal dramatic shifts in predatory feeding behaviour between post-metamorphic juveniles and adult specimens. Adult C. magus capture fish using a set of paralytic venom peptides combined with a hooked radular tooth used to tether envenomed fish. In contrast, early juveniles feed exclusively on polychaete worms using a unique “sting-and-stalk” foraging behaviour facilitated by short, unbarbed radular teeth and a distinct venom repertoire that induces hypoactivity in prey. Our results demonstrate how coordinated morphological, behavioural and molecular changes facilitate the shift from worm- to fish-hunting in C. magus , and showcase juvenile cone snails as a rich and unexplored source of novel venom peptides for ecological, evolutionary and biodiscovery studies.
Publisher: Elsevier BV
Date: 11-2020
Publisher: MDPI AG
Date: 06-03-2020
DOI: 10.3390/MD18030150
Abstract: Cone snails produce a fast-acting and often paralyzing venom, largely dominated by disulfide-rich conotoxins targeting ion channels. Although disulfide-poor conopeptides are usually minor components of cone snail venoms, their ability to target key membrane receptors such as GPCRs make them highly valuable as drug lead compounds. From the venom gland transcriptome of Conus miliaris, we report here on the discovery and characterization of two conopressins, which are nonapeptide ligands of the vasopressin/oxytocin receptor family. These novel sequence variants show unusual features, including a charge inversion at the critical position 8, with an aspartate instead of a highly conserved lysine or arginine residue. Both the amidated and acid C-terminal analogues were synthesized, followed by pharmacological characterization on human and zebrafish receptors and structural investigation by NMR. Whereas conopressin-M1 showed weak and only partial agonist activity at hV1bR (amidated form only) and ZFV1a1R (both amidated and acid form), both conopressin-M2 analogues acted as full agonists at the ZFV2 receptor with low micromolar affinity. Together with the NMR structures of amidated conopressins-M1, -M2 and -G, this study provides novel structure-activity relationship information that may help in the design of more selective ligands.
Publisher: Wiley
Date: 24-12-2003
Publisher: Elsevier BV
Date: 07-2018
DOI: 10.1016/J.BMC.2018.03.031
Abstract: Both N- and T-type calcium ion channels have been implicated in pain transmission and the N-type channel is a well-validated target for the treatment of neuropathic pain. An SAR investigation of a series of substituted aminobenzothiazoles identified a subset of five compounds with comparable activity to the positive control Z160 in a FLIPR-based intracellular calcium response assay measuring potency at both Ca
Publisher: Elsevier BV
Date: 02-2018
DOI: 10.1016/J.MOLIMM.2017.12.016
Abstract: The complement system is an essential component of the innate immune response. The anaphylatoxins C3a and C5a are key drivers of the complement system, acting through the receptors C3aR, C5aR1 and C5aR2 to regulate inflammation. While a role for C5a activation of C5aR1 in inflammatory and neuropathic pain has been established, the role of the complement system in burn-induced pain has not been investigated. To address this gap, we assessed the role of complement receptors C3aR, C5aR1 and C5aR2 in a mouse model of acute burn-induced pain. Superficial burn injury was induced in C57BL/6 mice by firm application of left hind paw plantar surface to a hot plate set at 52.5 °C for 25 s. Development of burn-induced mechanical allodynia, thermal allodynia, weight bearing changes and edema was assessed in C3aR
Publisher: Springer Science and Business Media LLC
Date: 06-06-2015
DOI: 10.1007/S00216-015-8787-Y
Abstract: The venom of cone snails is composed of highly modified peptides (conopeptides) that target a variety of ion channels and receptors. The venom of these marine gastropods represents a largely untapped resource of bioactive compounds of potential pharmaceutical value. Here, we use a combination of bioanalytical techniques to uncover the extent of venom expression variability in Conus purpurascens, a fish-hunting cone snail species. The injected venom of nine specimens of C. purpurascens was separated by reversed-phase high-performance liquid chromatography (RP-HPLC), and fractions were analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) in parallel with liquid chromatography-electrospray ionization (LC-ESI)-TripleTOF-MS to compare standard analytical protocols used in preparative bioassay-guided fractionations with a deeper peptidomic analysis. Here, we show that C. purpurascens exhibits pronounced intraspecific venom variability. RP-HPLC fractionation followed by MALDI-TOF-MS analysis of the injected venom of these nine specimens identified 463 distinct masses, with none common to all specimens. Using LC-ESI-TripleTOF-MS, the injected venom of these nine specimens yielded a total of 5517 unique masses. We also compare the injected venom of two specimens with their corresponding dissected venom. We found 2566 and 1990 unique masses for the dissected venom compared to 941 and 1959 masses in their corresponding injected venom. Of these, 742 and 1004 masses overlapped between the dissected and injected venom, respectively. The results indicate that larger conopeptide libraries can be assessed by studying multiple in iduals of a given cone snail species. This expanded library of conopeptides enhances the opportunities for discovery of molecular modulators with direct relevance to human therapeutics. Graphical Abstract The venom of cone snails are extraordinarily complex mixtures of highly modified peptides. Venom analysis requires separation through RP-HPLC followed by MALDI-TOF mass spectrometry or direct analysis using LC-ESI-TripleTOF-MS. Using these techniques, venom intraspecific variability and comparison between injected and dissected were assessed.
Publisher: Elsevier BV
Date: 09-2012
DOI: 10.1016/J.TOXICON.2012.04.340
Abstract: Conopeptides and conotoxins are small peptides produced by cone snails as a part of their predatory/defense strategies that target key ion channels and receptors in the nervous system. Some of these peptides also potently target mammalian ion channels involved in pain pathways. As a result, these venoms are a source of valuable pharmacological and therapeutic agents. The traditional approach towards conopeptide discovery relied on activity-guided fractionation, which is time consuming and resource-intensive. In this review, we discuss the advances in the fields of transcriptomics, proteomics and bioinformatics that now allow researchers to integrate these three platforms towards a more efficient discovery strategy. In this review, we also highlight the challenges associated with the wealth of data generated with this integrated approach and briefly discuss the impact these methods could have on the field of toxinology.
Publisher: Wiley
Date: 2016
DOI: 10.1111/MEC.13504
Abstract: Venoms comprise of complex mixtures of peptides evolved for predation and defensive purposes. Remarkably, some carnivorous cone snails can inject two distinct venoms in response to predatory or defensive stimuli, providing a unique opportunity to study separately how different ecological pressures contribute to toxin ersification. Here, we report the extraordinary defensive strategy of the Rhizoconus subgenus of cone snails. The defensive venom from this worm-hunting subgenus is unusually simple, almost exclusively composed of αD-conotoxins instead of the ubiquitous αA-conotoxins found in the more complex defensive venom of mollusc- and fish-hunting cone snails. A similarly compartmentalized venom gland as those observed in the other dietary groups facilitates the deployment of this defensive venom. Transcriptomic analysis of a Conus vexillum venom gland revealed the αD-conotoxins as the major transcripts, with lower amounts of 15 known and four new conotoxin superfamilies also detected with likely roles in prey capture. Our phylogenetic and molecular evolution analysis of the αD-conotoxins from five subgenera of cone snails suggests they evolved episodically as part of a defensive strategy in the Rhizoconus subgenus. Thus, our results demonstrate an important role for defence in the evolution of conotoxins.
Publisher: MDPI AG
Date: 19-03-2019
DOI: 10.3390/MD17030177
Abstract: In idual variation in animal venom has been linked to geographical location, feeding habit, season, size, and gender. Uniquely, cone snails possess the remarkable ability to change venom composition in response to predatory or defensive stimuli. To date, correlations between the venom gland transcriptome and proteome within and between in idual cone snails have not been reported. In this study, we use 454 pyrosequencing and mass spectrometry to decipher the transcriptomes and proteomes of the venom gland and corresponding predation-evoked venom of two specimens of Conus imperialis. Transcriptomic analyses revealed 17 conotoxin gene superfamilies common to both animals, including 5 novel superfamilies and two novel cysteine frameworks. While highly expressed transcripts were common to both specimens, variation of moderately and weakly expressed precursor sequences was surprisingly erse, with one specimen expressing two unique gene superfamilies and consistently producing more paralogs within each conotoxin gene superfamily. Using a quantitative labelling method, conotoxin variability was compared quantitatively, with highly expressed peptides showing a strong correlation between transcription and translation, whereas peptides expressed at lower levels showed a poor correlation. These results suggest that major transcripts are subject to stabilizing selection, while minor transcripts are subject to ersifying selection.
Publisher: MDPI AG
Date: 06-04-2006
DOI: 10.3390/MD403193
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 06-2018
Publisher: Wiley
Date: 27-08-2013
DOI: 10.1111/BPH.12251
Publisher: American Chemical Society (ACS)
Date: 17-02-2010
DOI: 10.1021/JA910602H
Abstract: Alpha-conotoxins are tightly folded miniproteins that antagonize nicotinic acetylcholine receptors (nAChR) with high specificity for erse subtypes. Here we report the use of selenocysteine in a supported phase method to direct native folding and produce alpha-conotoxins efficiently with improved biophysical properties. By replacing complementary cysteine pairs with selenocysteine pairs on an hiphilic resin, we were able to chemically direct all five structural subclasses of alpha-conotoxins exclusively into their native folds. X-ray analysis at 1.4 A resolution of alpha-selenoconotoxin PnIA confirmed the isosteric character of the diselenide bond and the integrity of the alpha-conotoxin fold. The alpha-selenoconotoxins exhibited similar or improved potency at rat diaphragm muscle and alpha3beta4, alpha7, and alpha1beta1 deltagamma nAChRs expressed in Xenopus oocytes plus improved disulfide bond scrambling stability in plasma. Together, these results underpin the development of more stable and potent nicotinic antagonists suitable for new drug therapies, and highlight the application of selenocysteine technology more broadly to disulfide-bonded peptides and proteins.
Publisher: Springer Science and Business Media LLC
Date: 31-03-2017
DOI: 10.1038/SREP45466
Abstract: Nicotinic acetylcholine receptors (nAChR) are therapeutic targets for a range of human diseases. α-Conotoxins are naturally occurring peptide antagonists of nAChRs that have been used as pharmacological probes and investigated as drug leads for nAChR related disorders. However, α-conotoxin interactions have been mostly characterised at the α7 and α3β2 nAChRs, with interactions at other subtypes poorly understood. This study provides novel structural insights into the molecular basis for α-conotoxin activity at α3β4 nAChR, a therapeutic target where subtype specific antagonists have potential to treat nicotine addiction and lung cancer. A co-crystal structure of α-conotoxin LsIA with Lymnaea stagnalis acetylcholine binding protein guided the design and functional characterisations of LsIA analogues that identified the minimum pharmacophore regulating α3β4 antagonism. Interactions of the LsIA R10F with β4 K57 and the conserved –NN– α-conotoxin motif with β4 I77 and I109 conferred α3β4 activity to the otherwise inactive LsIA. Using these structural insights, we designed LsIA analogues with α3β4 activity. This new understanding of the structural basis of protein-protein interactions between α-conotoxins and α3β4 may help rationally guide the development of α3β4 selective antagonists with therapeutic potential.
Publisher: MDPI AG
Date: 04-02-2013
Publisher: Informa UK Limited
Date: 29-12-2020
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 11-2005
DOI: 10.1016/J.PAIN.2005.08.002
Abstract: Xen2174 is a structural analogue of Mr1A, a chi-conopeptide recently isolated from the venom of the marine cone snail, Conus marmoreus. Although both chi-conopeptides are highly selective inhibitors of the norepinephrine transporter (NET), Xen2174 has superior chemical stability relative to Mr1A. It is well-known that tricyclic antidepressants (TCAs) are also potent NET inhibitors, but their poor selectivity relative to other monoamine transporters and various G-protein-coupled receptors, results in dose-limiting side-effects in vivo. As TCAs and the alpha(2)-adrenoceptor agonist, clonidine, have established efficacy for the relief of neuropathic pain, this study examined whether intrathecal (i.t.) Xen2174 alleviated mechanical allodynia in rats with either a chronic constriction injury of the sciatic nerve (CCI-rats) or an L5/L6 spinal-nerve injury. The anti-allodynic responses of i.t. Mr1A and i.t. morphine were also investigated in CCI-rats. Paw withdrawal thresholds were assessed using calibrated von Frey filaments. Bolus doses of i.t. Xen2174 produced dose-dependent relief of mechanical allodynia in CCI-rats and in spinal nerve-ligated rats. Dose-dependent anti-allodynic effects were also produced by i.t. bolus doses of Mr1A and morphine in CCI-rats, but a pronounced 'ceiling' effect was observed for i.t. morphine. The side-effect profiles were mild for both chi-conopeptides with an absence of sedation. Confirming the noradrenergic mechanism of action, i.t. co-administration of yohimbine (100 nmol) with Xen2174 (10 nmol) abolished Xen2174s anti-allodynic actions. Xen2174 appears to be a promising candidate for development as a novel therapeutic for i.t. administration to patients with persistent neuropathic pain.
Publisher: MDPI AG
Date: 07-11-2022
Abstract: Cells in a clonal culture of the WC1/1 strain of Gambierdiscus that produced ciguatoxin and maitotoxin-3 were observed to spontaneously fuse during the light phase of culture growth. Cells in the process of fusion were indistinguishable from other cells under the light microscope, except that at least one (often both) of the fusing cells displayed an extendible, finger-like protrusion (presumed peduncle) arising from near the sulcul region. Fusion started with one of the cells turning 90° to place the planes of the girdles approximately at right angles to each other, and movement of the transverse flagella ceased in both cells, or in the cell seen in girdle (lateral) view. The cell in girdle view appeared to fuse into the theca of the other cell. The cell that had turned 90° often rounded up and become egg shaped (obovoid) during early fusion. Fusion can be quick ( min) or can take more than an hour. We saw no evidence of the theca being shed during fusion. Measurement of the dorsoventral and transdiameters revealed a wide range for cell sizes that were distributed as a bimodal population in the clonal culture. This bimodal cell population structure was maintained in clonal cultures reisolated from a small or large cell from the original WC1/1 culture. Cellular production of ciguatoxins by the WC1/1 clone increased during the first two years in culture with a corresponding decrease in production of maitotoxin-3, but this inverse relationship was not maintained over the following ~1.5 years.
Publisher: Cold Spring Harbor Laboratory
Date: 28-04-2023
DOI: 10.1101/2023.04.26.537867
Abstract: Lemurs are a well-known ex le of adaptive radiation. Since colonizing Madagascar, more than 100 extant lemur species have evolved to fill the variety of ecological niches on the island. However, recent work suggests that lemurs do not exhibit one of the hallmarks of adaptive radiations: explosive speciation rates that decline over time. We test this idea using a phylogenomic dataset with broad taxonomic s ling of lemurs and their sister group, the lorisiforms of Asia and continental Africa. We find higher rates of speciation in Madagascar’s lemurs compared to lorisiforms and we confirm that lemurs did not experience an “early burst” of speciation after colonizing Madagascar. Instead, we identify three independent bursts of speciation approximately 15 million years ago that underly much of today’s lemur ersity. We demonstrate that the lemur clades with exceptionally high ersification rates have higher rates of introgression. This suggests that hybridization in these primates is not an evolutionary dead- end, but a driving force for ersification. Considering the conservation crisis affecting strepsirrhine primates, with approximately 95% of species being threatened with extinction, this phylogenomic study offers a new perspective for explaining Madagascar’s exceptional primate ersity and reveals patterns of speciation, extinction, and gene flow that will help inform future conservation decisions.
Publisher: Elsevier BV
Date: 11-2005
DOI: 10.1016/J.TOXICON.2005.07.002
Abstract: The effects of 31 plant extracts, which most are traditionally used to treat ciguatera fish poisoning in the Pacific area, were studied on the cytotoxicity of mouse neuroblastoma cells produced by ouabain, veratridine and/or brevetoxin-3 or Pacific ciguatoxin-1. The cell viability was determined using a quantitative colorimetric method. A marked cytotoxicity of seven of the 31 plant extracts studied, was observed. Despite this, these plant extracts were suspected to contain active compound(s) against the cytotoxicity produced by brevetoxin (2 extracts), brevetoxin, ouabain and/or veratridine (3 extracts), or only against that of ouabain and/or veratridine (2 extracts). Among the 24 plant extracts that exhibited by themselves no cytotoxicity, 22 were active against the effect of brevetoxin or against that of both veratridine and brevetoxin. Similar results were obtained when the seven most active plant extracts were reassayed using ciguatoxin instead of brevetoxin. In conclusion, the present work reports the first activity assessment of some plant extracts, achieved in vitro on a quite large scale. The fact that 27 plant extracts were found to exert, in vitro, a protective effect against the action of ciguatoxin and/or brevetoxin, paves the way for finding new active compounds to treat ciguatera fish poisoning, provided these compounds also reverse the effects of sodium channel activators.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 12-03-2019
DOI: 10.1126/SCISIGNAL.AAS9485
Abstract: G protein-coupled receptors (GPCRs) convert extracellular stimuli to intracellular responses that regulate numerous physiological processes. Crystallographic and biophysical advances in GPCR structural analysis have aided investigations of structure-function relationships that clarify our understanding of these dynamic receptors, but the molecular mechanisms associated with activation and signaling for in idual GPCRs may be more complex than was previously appreciated. Here, we investigated the proposed water-mediated, hydrogen-bonded activation switch between the conserved NPxxY motif on transmembrane helix 7 (TMH7) and a conserved tyrosine in TMH5, which contributes to α
Publisher: Elsevier BV
Date: 07-1998
DOI: 10.1016/S0304-3940(98)00575-8
Abstract: The actions of the marine neurotoxin, ciguatoxin-1 (CTX-1), were investigated in isolated parasympathetic neurones from neonatal rat intracardiac ganglia using patch-cl recording techniques. Under current cl conditions, bath application of 1-10 nM CTX-1 caused gradual membrane depolarization and tonic action potential firing. Action potential firing ceased with depolarization beyond approximately -35 mV and application of 300 nM tetrodotoxin (TTX) repolarized the cell to its control resting potential. In cell-attached membrane patches, 1-10 nM CTX-1 in the patch pipette markedly increased the open probability of single TTX-sensitive Na+ channels in response to depolarizing voltage steps but did not alter the unitary conductance (10 pS) or reversal potential. Under steady-state conditions, CTX-1 caused spontaneous opening of single Na+ channels which did not inactivate at hyperpolarized membrane potentials. CTX-1 increases neuronal excitability by shifting the voltage of activation of TTX-sensitive Na+ channels to more negative potentials.
Publisher: MDPI AG
Date: 11-02-2021
DOI: 10.3390/MD19020106
Abstract: The peripheral effects of ω-conotoxins, selective blockers of N-type voltage-gated calcium channels (CaV2.2), have not been characterised across different clinically relevant pain models. This study examines the effects of locally administered ω-conotoxin MVIIA, GVIA, and CVIF on mechanical and thermal paw withdrawal threshold (PWT) in postsurgical pain (PSP), cisplatin-induced neuropathy (CisIPN), and oxaliplatin-induced neuropathy (OIPN) rodent models. Intraplantar injection of 300, 100 and 30 nM MVIIA significantly (p 0.0001, p 0.0001, and p 0.05, respectively) alleviated mechanical allodynia of mice in PSP model compared to vehicle control group. Similarly, intraplantar injection of 300, 100, and 30 nM MVIIA (p 0.0001, p 0.01, and p 0.05, respectively), and 300 nM and 100 nM GVIA (p 0.0001 and p 0.05, respectively) significantly increased mechanical thresholds of mice in OIPN model. The ED50 of GVIA and MVIIA in OIPN was found to be 1.8 pmol aw and 0.8 pmol aw, respectively. However, none of the ω-conotoxins were effective in a mouse model of CisIPN. The intraplantar administration of 300 nM GVIA, MVIIA, and CVIF did not cause any locomotor side effects. The intraplantar administration of MVIIA can alleviate incision-induced mechanical allodynia, and GVIA and MVIIA effectively reduce OIPN associated mechanical pain, without locomotor side effects, in rodent models. In contrast, CVIF was inactive in these pain models, suggesting it is unable to block a subset of N-type voltage-gated calcium channels associated with nociceptors in the skin.
Publisher: American Chemical Society (ACS)
Date: 28-08-2008
DOI: 10.1021/JM800278K
Abstract: Alpha-conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). The majority of currently characterized alpha-conotoxins have a 4/7 loop size, and the major features of neuronal alpha-conotoxins include a globular disulfide connectivity and a helical structure centered around the third of their four cysteine residues. In this study, a novel "molecular pruning" approach was undertaken to define the relationship between loop size, structure, and function of alpha-conotoxins. This involved the systematic truncation of the second loop in the alpha-conotoxin [A10L]PnIA [4/7], a potent antagonist of the alpha7 nAChR. The penalty for truncation was found to be decreased conformational stability and increased susceptibility to disulfide bond scrambling. Truncation down to 4/4[A10L]PnIA maintained helicity and did not significantly reduce electrophysiological activity at alpha7 nAChRs, whereas 4/3[A10L]PnIA lost both alpha7 nAChR activity and helicity. In contrast, all truncated analogues lost approximately 100-fold affinity at the AChBP, a model protein for the extracellular domain of the nAChR. Docking simulations identified several hydrogen bonds lost upon truncation that provide an explanation for the reduced affinities observed at the alpha7 nAChR and AChBP.
Publisher: Elsevier BV
Date: 08-2005
Publisher: Public Library of Science (PLoS)
Date: 20-10-2008
Publisher: American Chemical Society (ACS)
Date: 06-1998
DOI: 10.1021/JA980389E
Publisher: MDPI AG
Date: 06-04-2006
DOI: 10.3390/MD403082
Publisher: MDPI AG
Date: 28-11-2018
DOI: 10.3390/MD16120475
Abstract: T-type calcium channel (CaV3.x) blockers are receiving increasing attention as potential therapeutics for the treatment of pathophysiological disorders and diseases, including absence epilepsy, Parkinson’s disease (PD), hypertension, cardiovascular diseases, cancers, and pain. However, few clinically approved CaV3.x blockers are available, and selective pharmacological tools are needed to further unravel the roles of in idual CaV3.x subtypes. In this work, through an efficient synthetic route to the marine fungal product pseudellone C, we obtained bisindole alkaloid analogs of pseudellone C with a modified tryptophan moiety and identified two CaV3.2 (2, IC50 = 18.24 µM 3, IC50 = 6.59 µM) and CaV3.3 (2, IC50 = 7.71 µM 3, IC50 = 3.81 µM) selective blockers using a FLIPR cell-based assay measuring CaV3.x window currents. Further characterization by whole-cell patch-cl revealed a preferential block of CaV3.1 activated current (2, IC50 = 5.60 µM 3, IC50 = 9.91 µM), suggesting their state-dependent block is subtype specific.
Publisher: Elsevier BV
Date: 09-2020
Publisher: MDPI AG
Date: 14-03-2019
DOI: 10.3390/MD17030165
Abstract: Integrated venomics techniques have shown that variable processing of conotoxins from Conus marmoreus resulted in a dramatic expansion in the number of expressed conotoxins. One conotoxin from C. marmoreus, the χ-conotoxin MrIA, is a selective inhibitor of human norepinephrine transporters (hNET) and therefore a drug candidate for attenuating chronic neuropathic pain. It has been found that “messy” processing of the MrIA transcripts results in the expression of MrIA analogs with different truncations of the pro-peptide that contains portions of the MrIA molecule. The aim of this study was to investigate if variable processing of the expressed peptides results in modulation of the existing hNET pharmacology or creates new pharmacologies. To this end, a number of MrIA analogs found in C. marmoreus venom were synthesized and evaluated for their activity at hNET receptors. While several of the analogs exhibited norepinephrine transporter inhibitory activity comparable to that of MrIA, none significantly improved on the potency of conotoxin MrIA, and those analogs with disrupted pharmacophores produced greatly reduced NET inhibition, confirming previous structure-activity relationships seen on χ-class conopeptides. Additionally, analogs were screened for new activities on ion channels using calcium influx assays, although no major new pharmacology was revealed.
Publisher: Elsevier BV
Date: 05-2016
Publisher: Springer New York
Date: 2001
DOI: 10.1007/978-1-4613-0143-1_3
Abstract: Ciguatera fish poisoning (ciguatera), a common poisoning caused by fish ingestion, is reviewed in the Western Atlantic and the Caribbean waters. It is endemic from Florida coasts (northern limit) to Martinique Island (southern limit), with outbreaks occurring from time to time. In the Caribbean, ciguatera causes a polymorphic syndrome with gastrointestinal, cardiovascular, and neurological signs and symptoms. Neurological and muscular dysfunctions can be treated by intravenous injection of D-mannitol. The lipid-soluble toxins involved are ciguatoxins that are likely produced by the dinoflagellate Gambierdiscus toxicus. G. toxicus strains are endemic in the Caribbean Sea and in theWestern Atlantic. Although it is likely that blooms of G. toxicus are ingested by herbivorous fishes, they are not implicated in ciguatera in the Caribbean. Rather, large carnivores (barracudas, jacks, snappers, groupers), consumers of smaller benthic fish, are often involved in ciguatera. Fish toxicity depends on fishing area and depth, fish size and tissues, and climatic disturbances. Ciguatoxins have been isolated and purified from Caribbean fish species. The structure of two epimers, C-CTX-1 and C-CTX-2 from horse-eye jack, comprise 14 trans-fused ether-linked rings and a hemiketal in terminal ring. Caribbean ciguatoxins are mainly detected in the laboratory by chicken, mouse, mosquito, or cell bioassays, and by analytical HPLC/tandem mass spectrometry down to parts per billion (ppb). A ciguatera management plan that integrates epidemiology, treatment, and a simple method of detection is required to ensure the protection of consumers.
Publisher: Wiley
Date: 21-06-2004
Publisher: Wiley
Date: 30-08-2010
Abstract: Bv8, a 77-residue protein isolated from frogs, is the prototypic member of the prokineticin family of cytokines. Prokineticins (PKs) have only recently been identified in vertebrates (including humans), and they are believed to be involved in a number of key physiological processes, such as angiogenesis, neurogenesis, nociception, and tissue development. We used a combination of Boc solid-phase peptide synthesis, native chemical ligation, and in vitro protein folding to establish robust chemical access to this molecule. Synthetic Bv8 was obtained in good yield and exhibited full activity in a human neuroblastoma cell line and rat dorsal root ganglion (DRG) neurons. The 3D structure of the synthetic protein was determined by using NMR spectroscopy and it was found to be homologous with that of mamba intestinal toxin 1, which is the only other known prokineticin structure. Analysis of a truncated mutant lacking five residues at the N terminus that are critical for receptor binding and activation showed no perturbation to the core protein structure. Together with the functional data, this suggests that receptor binding is likely to be a highly cooperative process possibly involving major allosterically driven structural rearrangements. The facile and efficient synthesis presented here will enable preparation of unique chemical analogues of prokineticins, which should be powerful tools for modulating the structure and function of prokineticins and their receptors, and studying the many physiological processes that have been linked to them.
Publisher: Wiley
Date: 29-11-2005
DOI: 10.1111/J.1471-4159.2005.03574.X
Abstract: The modulation of recombinant NMDA receptors by conantokin-G (con-G) and Ala7-conantokin-G (Ala7-Con-G) was investigated in Xenopus oocytes injected with capped RNA coding for NR1 splice variants and NR2 subunits using the two-electrode voltage cl technique. Glutamate exhibited a marginally higher apparent affinity for NR2A-containing receptors than NR2B-containing receptors, regardless of the NR1 subunit present. Conantokins were bath applied to give cumulative concentration responses in the presence of 3 and 30 mum glutamate. Both contantokins exhibited biphasic concentration-response relationships at NR2A-containing NMDA receptors, producing potentiation at low conantokin concentrations and inhibition at high concentrations. These effects were stronger with glutamate concentrations near its EC50, and less marked at saturating concentrations. In contrast, the conantokin concentration-response relation was monophasic and inhibitory at NR2B-containing receptors. We conclude that the combinations of subunits that comprise the NMDA receptor complex influence conantokin and glutamate affinities and the nature of the responses to conantokins.
Publisher: Elsevier BV
Date: 07-2006
Publisher: Springer Science and Business Media LLC
Date: 07-09-2018
DOI: 10.1038/S41598-018-31245-4
Abstract: Cone snails are a erse group of predatory marine invertebrates that deploy remarkably complex venoms to rapidly paralyse worm, mollusc or fish prey. ω-Conotoxins are neurotoxic peptides from cone snail venoms that inhibit Ca v 2.2 voltage-gated calcium channel, demonstrating potential for pain management via intrathecal (IT) administration. Here, we isolated and characterized two novel ω-conotoxins, MoVIA and MoVIB from Conus moncuri , the first to be identified in vermivorous (worm-hunting) cone snails. MoVIA and MoVIB potently inhibited human Ca v 2.2 in fluorimetric assays and rat Ca v 2.2 in patch cl studies, and both potently displaced radiolabeled ω-conotoxin GVIA ( 125 I-GVIA) from human SH-SY5Y cells and fish brain membranes (IC 50 2–9 pM). Intriguingly, an arginine at position 13 in MoVIA and MoVIB replaced the functionally critical tyrosine found in piscivorous ω-conotoxins. To investigate its role, we synthesized MoVIB-[R13Y] and MVIIA-[Y13R]. Interestingly, MVIIA-[Y13R] completely lost Ca v 2.2 activity and MoVIB-[R13Y] had reduced activity, indicating that Arg at position 13 was preferred in these vermivorous ω-conotoxins whereas tyrosine 13 is preferred in piscivorous ω-conotoxins. MoVIB reversed pain behavior in a rat neuropathic pain model, confirming that vermivorous cone snails are a new source of analgesic ω-conotoxins. Given vermivorous cone snails are ancestral to piscivorous species, our findings support the repurposing of defensive venom peptides in the evolution of piscivorous Conidae .
Publisher: Wiley
Date: 07-2002
DOI: 10.1046/J.1460-9568.2002.02071.X
Abstract: The actions of ciguatoxins from the Pacific (P-CTX-1) and Caribbean (C-CTX-1) regions were investigated in isolated parasympathetic neurons from rat intracardiac ganglia using patch-cl recording techniques. Under current-cl conditions, bath application of P-CTX-1 (1-10 nm) or C-CTX-1 (10-30 nm) caused a gradual depolarization that was accompanied by oscillation of the membrane potential leading to tonic action potential firing. Membrane potential oscillations were observed between -45 and -60 mV and had an litude of 10-20 mV and a mean frequency of 10 Hz. Oscillation frequency was temperature-dependent with a Q10 of 2.0. Membrane oscillations were temporarily inhibited by hyperpolarizing current pulses and potentiated by weak depolarizing current pulses. The litude of oscillations was reduced upon lowering the external Na+ concentration and inhibited by tetrodotoxin (TTX), tetracaine or Zn2+. Tetraethylammonium, 4-aminopyridine, Cs+, Cd2+, Ba2+, 1,4,4'-diothiocyanato-2,2'-stilbenedisulphonic acid (DIDS) and ouabain had no effect on the CTX-1-induced membrane depolarization and oscillations. Brevetoxin (PbTx-3, 100 nm), in contrast to CTX-1, caused a membrane depolarization that was not associated with oscillation of the membrane potential. Under voltage-cl conditions, P-CTX-1 inhibited the peak litude of the voltage-dependent Na+ current and shifted the activation curve to more negative potentials, but membrane oscillations were not seen in this configuration. These results suggest that ciguatoxins cause oscillation of the membrane potential in mammalian autonomic neurons by modifying the activation and inactivation properties of a population of TTX-sensitive Na+ channels.
No related grants have been discovered for Richard J. Lewis.