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April 2006 "Pain Relief Drugs from the Sea"
Desley Blanch interviews Dr. Livett about his research and progress with the development of the cone shell analgesic, ACV1, from Conus victoriae. This interview was broadcast on Radio Australia's Innovations program, April 10, 2006. For a printable transcript click here

For further information see Bruce Livett's Research on cone shell venom peptides for treatment of chronic pain conditions at the Department of Biochemistry and Molecular Biology and the Bio21 Institute for Innovation and Entrepreureship at the University of Melbourne.

  • For a free article on theories about pain, click here: Encyclopedia Britannica.

    Analgesic Component of Venom (ACV1) from Cone Snails :
    see Nature Science Update "Snail toxin could ease chronic pain" by Ingrid Holmes

  • Snails venom signals a pain free future Roger Highfield, Telegraph 14 Nov 2006 reports on studies with conotoxin RgIA (Conus regius) and conotoxin Vc1.1 (Conus victoriae)

  • The Cone Snail site is maintained by the University of Utah laboratory that studies cone snail venom and contains a wealth of information about these venomous, predatory marine snails.
    An "Internet Interview" with Bruce Livett: conducted in February 2001 about his scientific work with cone shells and conotoxins (and his interaction with other malacologists and shell collectors), is now available as a downloadable Adobe pdf file. This extensive Intervista web "interview" conducted by Eduardo Moreira for Callostoma was subsequently published (in condensed form) in American Conchologist Volume 30, Number 1, 2002, pp. 5 & 14.

    For a one-page description of Cone Shells and their Conotoxins click here

    For a video simulation of cone shell envenomaton click here

    Bruce Livett's more recent publications (1998-2009)

    Site Map of Cone Shells and Conotoxins HomePage


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    Visit the  Cone shell Bookshop ( 9 titles)

    Please mail me suggestions at the following address: b.livett@unimelb.edu.au


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    Welcome to What's New in 2015

    After an absence of several years, I am now back to editing this web page. It won't happen overnight, but it WILL happen.

    Some exciting new developments have come from Prof. David Craik at IMB, University of Queensland. David has been developing a cyclotide version of Conotoxin Vc1.1 which he has named IMB007 (great name!) and has shown it retains its significant analgesic properties in animals when given orally. He plans to raise funds to take this new cyclic conotoxin through clinical trials as a potential replacement for morphine and opiates currently used for neuropathic pain.

    IMB007 shows comparable oral efficacy to gabappentin in a rat neuropathic pain model (see UNIQUEST: Cyclic Conotoxin Analogues)

    Click here to visit David Craik's webpage and view the 3 videos about his exciting cyclotide research research applications.


    Welcome to What's New in 2012

    For starters, take a look at this impressive You-Tube of cone shell envenomation.

    10 January 2013

    The Cone Shells of Florida (2013): An Illustrated Key and a Review of the Recent Species. by John K. Tucker , with a foreword by Dr Emilio F. García. MdM Publishing, Wellington Fl., ISBN 978-0-9847140-1-8, hardcover in case with stitched binding, about 7Ľ by 10 inch size (18.5 by23 cm), 155 pages, with 48 full color plates and numerous large color illustrations for steps in the identification key.

    Abstract: This is the first clearly written and fully illustrated guide to the marine Mollusks in the families Conidae and Conolithidae found in the waters off Florida from the shoreline to the continental shelf. Written and illustrated by long time cone collector and expert now retired from the Illinois Natural History Survey. It is a clear and concise guide to these shells with well written descriptions that include a listing the numerous synonyms for these shells and superb color photographs of usually several specimens of each species. Additionally there is an illustrated step by step key to narrow down the identification of each of these shells. In all there are thirty species (including one new species) in the book and three subspecies. Every one interested in Cone shells and Florida shells both amateur and professional will find this book a tremendous improvement over the sometimes spotty and often inaccurate accounts of these shells found in other books. We are the publishers of this book and we are justifiably proud of it. We are looking forward to publishing a complete guide to the worldwide shells in the Conidae and Conolithidae by John Tucker and Manuel Tenorio in the near future.

    23 December 2012

    New postage stamps featuring cone shells.

    Added images of the following new stamps to the list Cone Shells on Stamps.

    Conus abbas, Maldives, 2010
    Conus amadis, Maldives, 2010
    Conus bengalensis, Maldives, 2010
    Conus capitaneus, Norfolk Is, 2012
    Conus ebreus, Norfolk Is, 2012
    Conus geographus, Niue, 2012
    Conus gloriamaris, Solomon Islands, 2012
    Conus regius, Caribbean, 2011

    Thanks go to Tom Walker for providing me with these new entries.

    24 November 2012

    Review: Bioengineering of Conopeptides for drug leads

    Bingham J-P, Andrews EA, Kiyabu SM, Cabalteja CC (2012) Mini Review: Drugs from Slugs. Part II – Conopeptide bioengineering. Chemico-Biological Interactions 200: 92–113.

    Address: Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, HI 96822, USA

    Abstract: The biological transformation of toxins as research probes, or as pharmaceutical drug leads, is an onerous and drawn out process. Issues regarding changes to pharmacological specificity, desired potency, and bioavailability are compounded naturally by their inherent toxicity. These often scuttle their progress as they move up the narrowing drug development pipeline. Yet one class of peptide toxins, from the genus Conus, has in many ways spearheaded the expansion of new peptide bioengineering techniques to aid peptide toxin pharmaceutical development. What has now emerged is the sequential bioengineering of new research probes and drug leads that owe their lineage to these highly potent and isoform specific peptides. Here we discuss the progressive bioengineering steps that many conopeptides have transitioned through, and specifically illustrate some of the biochemical approaches that have been established to maximize their biological research potential and pharmaceutical worth.

    View here

    see also: Bingham JP, Mitsunaga E, Bergeron ZL (2010). Drugs from slugs--past, present and future perspectives of omega-conotoxin research. Chem Biol Interact. 183:1-18.

    Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, HI 96822, USA. jbingham@hawaii.edu

    Abstract: Peptides from the venom of carnivorous cone shells have provided six decades of intense research, which has led to the discovery and development of novel analgesic peptide therapeutics. Our understanding of this unique natural marine resource is however somewhat limited. Given the past pharmacological record, future investigations into the toxinology of these highly venomous tropical marine snails will undoubtedly yield other highly selective ion channel inhibitors and modulators. With over a thousand conotoxin-derived sequences identified to date, those identified as ion channel inhibitors represent only a small fraction of the total. Here we discuss our present understanding of conotoxins, focusing on the omega-conotoxin peptide family, and illustrate how such a seemingly simple snail has yielded a highly effective clinical drug.

    omega Conotoxin GVIA mimetics that both bind and inhibit ion channel function

    Tranberg CE, Yang A, Vette I, McArthur JR, Baell JB, Lewis RJ, Tuck KL, Duggan PJ. (2012) omega-Conotoxin GVIA mimetics that bind and inhibit neuronal Ca(v)2.2 Ion Channels. Mar Drugs 10: 2349-2368.

    CSIRO Materials Science and Engineering, Bag 10, Clayton South, Victoria 3169, Australia; Email: l.tranberg@deakin.edu.au.

    Abstract: The neuronal voltage-gated N-type calcium channel (Ca(v)2.2) is a validated target for the treatment of neuropathic pain. A small library of anthranilamide-derived omega-Conotoxin GVIA mimetics bearing the diphenylmethylpiperazine moiety were prepared and tested using three experimental measures of calcium channel blockade. These consisted of a (125)I-omega-conotoxin GVIA displacement assay, a fluorescence-based calcium response assay with SH-SY5Y neuroblastoma cells, and a whole-cell patch clamp electrophysiology assay with HEK293 cells stably expressing human Ca(v)2.2 channels. A subset of compounds were active in all three assays. This is the first time that compounds designed to be mimics of omega-conotoxin GVIA and found to be active in the (125)I-omega-conotoxin GVIA displacement assay have also been shown to block functional ion channels in a dose-dependent manner.



    Disulfide connectivity of mu-conotoxins in the venom of Conus kinoshitai

    Khoo KK, Gupta K, Green BR, Zhang MM, Watkins M, Olivera BM, Balaram P, Yoshikami D, Bulaj G, Norton RS. (2012) Distinct disulfide isomers of mu-conotoxins KIIIA and KIIIB block voltage-gated sodium channels. Biochemistry. 2012 Nov 20.

    Salt Lake City, Utah.

    Abstract: In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of mu-conotoxin KIIIA, which was predicted originally to have a [C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely-related mu-conotoxins. The two major isomers of synthetic mu-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric CID fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a [C1-C15,C2-C9,C4-C16] disulfide connectivity, while the minor product adopts a [C1-C16,C2-C9,C4-C15] connectivity. Both of these peptides were potent blockers of NaV1.2 (Kd 5 and 230 nM, respectively). The solution structure for mu-KIIIA based on NMR data was recalculated with the [C1-C15,C2-C9,C4-C16] disulfide pattern; its structure was very similar to the mu-KIIIA structure calculated with the incorrect [C1-C9,C2-C15,C4-C16] disulfide pattern, with an alpha-helix spanning residues 7-12. In addition, the major folding isomers of mu-KIIIB, an N-terminally extended isoform of mu-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as for mu-KIIIA, and both blocked NaV1.2 (Kd 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic mu-KIIIA/mu-KIIIB folded in vitro is 1-5/2-4/3-6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of mu-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means to further expand the snail's repertoire of active peptides.

    Mass spectrometry reveals upwards of 3000 peptides in the venom of Conus marmoreus

    Dutertre S, Jin AH, Kaas Q, Jones A, Alewood PF, Lewis RJ.(2012) Deep venomics reveals the mechanism for expanded peptide diversity in cone snail venom. Mol Cell Proteomics. 2012 Nov 14.

    The University of Queensland, Australia.

    Abstract: Cone snails produce highly complex venom comprising mostly small biologically active peptides known as conotoxins or conopeptides. Early estimates that suggested 50-200 venom peptides are produced per species have been recently increased at least 10-fold using advanced mass spectrometry. To uncover the mechanism(s) responsible for generating this impressive diversity, we used an integrated approach combining second-generation transcriptome sequencing with high sensitivity proteomics. From the venom gland transcriptome of Conus marmoreus, a total of 105 conopeptide precursor sequences from 13 gene superfamilies were identified. Over 60% of these precursors belonged to the three gene superfamilies O1, T and M, consistent with their high levels of expression, which suggests these conotoxins play an important role in prey capture and/or defense. Seven gene superfamilies not previously identified in C. marmoreus, including 5 novel superfamilies, were also discovered. To confirm the expression of toxins identified at the transcript level, the injected venom of C. marmoreus was comprehensively analyzed by mass spectrometry, revealing 2710 and 3172 peptides using MALDI and ESI-MS, respectively, and 6254 peptides using an ESI-MS TripleTOF 5600 instrument. All conopeptides derived from transcriptomic sequences could be matched to masses obtained on the TripleTOF within 100 ppm accuracy, with 66 (63%) providing MS/MS coverage that unambiguously confirmed these matches. Comprehensive integration of transcriptomic and proteomic data revealed for the first time that the vast majority of the conopeptide diversity arises from a more limited set of genes through a process of variable peptide processing, which generates conopeptides with alternative cleavage sites, heterogeneous post-translational modifications, and highly variable N- and C-terminal truncations. Variable peptide processing is expected to contribute to the evolution of venoms, and explains how a limited set of ~100 gene transcripts can generate thousands of conopeptides in a single species of cone snail.

    The accessory beta-subunit influences the binding of mu-conotoxin to the Na-channel

    Zhang MM, Wilson MJ, Azam L, Gajewiak J, Rivier JE, Bulaj G, Olivera BM, Yoshikami D. (2012) Co-expression of Na(V) beta-subunits alters the kinetics of inhibition of voltage-gated sodium channels by pore-blocking mu-Conotoxins. Br J Pharmacol. 2012 Nov 12.

    Department of Biology, University of Utah, Salt Lake City, Utah, 84112, U.S.A.

    Abstract: Voltage-gated sodium channels (VGSCs) are assembled from two classes of subunits, a pore-bearing alpha-subunit (Na(V) 1) and one or two accessory beta-subunits (Na(V) betas). Neurons in mammals can express one or more of seven isoforms of Na(V) 1 and one or more of four isoforms of Na(V) beta. The peptide mu-conotoxins, like the guanidinium alkaloids tetrodotoxin (TTX) and saxitoxin (STX), inhibit VGSCs by blocking the pore in Na(V) 1. Hitherto, the effects of Na(V) beta-subunit co-expression on the activity of these toxins have not been comprehensively assessed. EXPERIMENTAL APPROACH: Four mu-conotoxins (mu-TIIIA, mu-PIIIA, mu-SmIIIA, and mu-KIIIA), TTX, and STX were tested against Na(V) 1.1, 1.2, 1.6 or 1.7, each co-expressed in Xenopus laevis oocytes with one of Na(V) beta1, beta2, beta3, or beta4 and, for Na(V) 1.7, binary combinations of thereof. KEY RESULTS: Co-expression of Na(V) beta-subunits modifies the block by mu-conotoxins: in general, Na(V) beta1 or beta3 co-expression tended to increase k(on) (in the most extreme instance by 9-fold) whereas Na(V) beta2 or beta4 co-expression decreased k(on) (in the most extreme instance by 240-fold). In contrast, the block by TTX and STX was only minimally, if at all, affected by Na(V) beta-subunit co-expression. Tests of Na(V) beta1:beta2 chimeras co-expressed with Na(V) 1.7 suggest that the extracellular portion of the Na(V) beta-subunit is largely responsible for altering mu-conotoxin kinetics. CONCLUSIONS AND IMPLICATIONS: These results are the first indication that Na(V) beta-subunit co-expression can markedly influence mu-conotoxin binding and, by extension, the outer vestibule of the pore of VGSCs. mu-Conotoxins could, in principle, be used to pharmacologically probe the Na(V) beta-subunit composition of endogenously-expressed VGSCs.

    Conotoxin expression along the venom duct in Conus textile

    Dobson R, Collodoro M, Gilles N, Turtoi A, De Pauw E, Quinton L. (2012) Secretion and maturation of conotoxins in the venom ducts of Conus textile. Toxicon 60: 1370-1379.

    Laboratory of Mass Spectrometry, Department of Chemistry, University of Liege, Liege, Belgium.

    Abstract: The 700 or more species of cone snail attack prey by employing complex venom which can vary considerably both within species and from one species to another. Cone snail venom is remarkable for the high proportion of conotoxins with varied post-translational modifications (PTMs) and for the production of more diverse toxin scaffolds than any other known venomous animal. The venom gland, which is several times longer than its shell, is also unique in being tubular. These unusual characteristics both raise questions, and provide the opportunity for research, concerning the secretion and maturation of conotoxins along the venom duct, a process which is currently not fully understood. This research uses the two mass spectrometric techniques of isotope Coded Affinity Tagging (ICAT) and label-free quantification to study each of five portions of the venom duct of Conus textile snails from New Caledonia. Fifteen conotoxins, several with different post-translational modifications (PTMs) were identified and quantified. One hundred and forty three non-identified conotoxins were also quantified. Distinctive patterns emerged, with the largest group of conotoxins increasing, then peaking in the central-proximal part, before decreasing; whilst the second largest group peaked in the distal region, generally displaying nothing in the first parts. Conotoxins from different superfamilies were commonly found to have similar distributions. A new conotoxin, PCCSKLHDNSCCGL*, was sequenced. A comparison is made with other studies to see how the process varies in cone snails from different regions.

    Conopeptide gene expression and gene evolution in Turridae and Conus

    Olivera BM, Watkins M, Bandyopadhyay P, Imperial JS, de la Cotera EP, Aguilar MB, Vera EL, Concepcion GP, Lluisma A. (2012) Adaptive radiation of venomous marine snail lineages and the accelerated evolution of venom peptide genes. Ann N Y Acad Sci. 1267: 61-70.

    Biology Department, University of Utah, Salt Lake City, Utah, USA. olivera@biology.utah.edu

    Abstract: An impressive biodiversity (>10,000 species) of marine snails (suborder Toxoglossa or superfamily Conoidea) have complex venoms, each containing approximately 100 biologically active, disulfide-rich peptides. In the genus Conus, the most intensively investigated toxoglossan lineage (>500 species), a small set of venom gene superfamilies undergo rapid sequence hyperdiversification within their mature toxin regions. Each major lineage of Toxoglossa has its own distinct set of venom gene superfamilies. Two recently identified venom gene superfamilies are expressed in the large Turridae clade, but not in Conus. Thus, as major venomous molluscan clades expand, a small set of lineage-specific venom gene superfamilies undergo accelerated evolution. The juxtaposition of extremely conserved signal sequences with hypervariable mature peptide regions is unprecedented and raises the possibility that in these gene superfamilies, the signal sequences are conserved as a result of an essential role they play in enabling rapid sequence evolution of the region of the gene that encodes the active toxin.

    2 September 2012

    High molecular mass peptides in Conus consors

    Leonardi A, Biass D, Kordiš D, Stöcklin R, Favreau P, Križaj I. (2012) Conus consors snail venom proteomics proposes functions, pathways and novel families involved in its venomic system. J Proteome Res. 2012 Aug 29. [Epub ahead of print] PubMed PMID: 22928724.


    Abstract:For some decades, cone snail venoms have been providing peptides, generally termed conopeptides, that exhibit a large diversity of pharmacological properties. However, little attention has been devoted to the high molecular mass (HMM) proteins in venoms of mollusks. In order to shed more light on cone snail venom HMM components, the proteins of dissected and injected venom of a fish-hunting cone snail, Conus consors, were extensively assessed. HMM venom proteins were separated by two-dimensional polyacrylamide gel electrophoresis and analyzed by mass spectrometry (MS). The MS data were interpreted using UniProt database, EST libraries from C. consors venom duct and salivary gland, and their genomic information. Numerous protein families were discovered in the lumen of the venom duct and assigned a biological function, thus pointing to their potential role in venom production and maturation. Interestingly, the study also revealed original proteins defining new families of unknown function. Only two groups of HMM proteins passing the venom selection process - echotoxins and hyaluronidases - were clearly present in the injected venom. They are suggested to contribute to the envenomation process. This newly devised integrated HMM proteomic analysis is a big step towards identification of the protein arsenal used in a cone snail venom apparatus for venom production, maturation and function.

    Ligands that discriminate between alpha-conotoxin receptor subtypes

    Hone AJ, Scadden M, Gajewiak J, Christensen S, Lindstrom JM, McIntosh JM. (2012) alpha-Conotoxin PeIA[S9H,V10A,E14N] Potently and Selectively Blocks alpha6beta2beta3 versus alpha6beta4 Nicotinic Acetylcholine Receptors. Mol Pharmacol. 82:972-82.

    University of Utah

    Abstract:Nicotinic acetylcholine receptors (nAChRs) containing ?6 and ?2 subunits modulate dopamine release in the basal ganglia and are therapeutically relevant targets for treatment of neurological and psychiatric disorders including Parkinson's disease and nicotine dependence. However, the expression profile of ?2 and ?4 subunits overlap in a variety of tissues including locus coeruleus, retina, hippocampus, dorsal root ganglia, and adrenal chromaffin cells. Ligands that bind ?6?2 nAChRs also potently bind the closely related ?6?4 subtype. To distinguish between these two subtypes, we synthesized novel analogs of a recently described ?-conotoxin, PeIA. PeIA is a peptide antagonist that blocks several nAChR subtypes, including ?6/?3?2?3 and ?6/?3?4 nAChRs, with low nanomolar potency. We systematically mutated PeIA and evaluated the resulting analogs for enhanced potency and/or selectivity for ?6/?3?2?3 nAChRs expressed in Xenopus oocytes (?6/?3 is a subunit chimera that contains the N-terminal ligand binding domain of the ?6 subunit). Based on these results, second generation analogs were then synthesized. The final analog, PeIA[S9H,V10A,E14N], potently blocked acetylcholine-gated currents mediated by ?6/?3?2?3 and ?6/?3?4 nAChRs with IC(50) values of 223 pM and 65 nM, respectively, yielding a >290-fold separation between the two subtypes. Kinetic studies of ligand binding to ?6/?3?2?3 nAChRs yielded a k(off) of 0.096 ± 0.001 min(-1) and a k(on) of 0.23 ± 0.019 min(-1) M(-9). The synthesis of PeIA[S9H,V10A,E14N] demonstrates that ligands can be developed to discriminate between ?6/?3?2?3 and ?6/?3?4 nAChRs.

    Multienzyme complexes aid folding of conotoxins. Studies in Conus imperialis and Conus geographus

    Safavi-Hemami H, Gorasia DG, Steiner AM, Williamson NA, Karas JA, Gajewiak J, Olivera BM, Bulaj G, Purcell AW (2012). Modulation of conotoxin structure and function is achieved through a multienzyme complex in the venom glands of cone snails. J Biol Chem. 2012 Aug 13. [Epub ahead of print] PubMed PMID: 22891240.

    University of Melbourne, Australia;

    Abstract:The oxidative folding of large polypeptides has been investigated in detail, however, comparatively little is known about the enzyme-assisted folding of small, disulfide-containing peptide substrates. To investigate the concerted effect of multiple enzymes on the folding of small disulfide-rich peptides, we sequenced and expressed protein disulfide isomerase (PDI), peptidyl-prolyl isomerase (PPI) and immunoglobulin binding protein (BiP) from Conus venom glands. Conus PDI was shown to catalyze the oxidation and reduction of disulfide bonds in two conotoxins alpha-GI and alpha-ImI. Oxidative folding rates were further increased in the presence of Conus PPI with the maximum effect observed in the presence of both enzymes. In contrast, Conus BiP was only observed to assist folding in the presence of microsomes, suggesting additional cofactors were involved. The identification of a complexs between BiP, PDI and nascent conotoxins further suggests that the folding and assembly of conotoxins is highly regulated multienzyme-assisted process. Unexpectedly, all three enzymes contributed to the folding the ribbon isomer of alpha-ImI. Here, we identify this alternative disulfide linked species in the venom of Conus imperialis providing the first evidence for the existence of a non-native peptide isomer in the venom of cone snails. Thus, ER-resident enzymes act in concert to accelerate the oxidative folding of conotoxins and modulate their conformation and function by reconfiguring disulphide connectivities. This study has evaluated the role of a number of ER-resident enzymes in the folding of conotoxins providing novel insights into the enzyme-guided assembly of these small, disulfide-rich peptides.

    Pain killers from Spiders and Cone shells

    de Souza AH, Castro CJ Jr, Rigo FK, de Oliveira SM, Gomez RS, Diniz DM, Borges MH, Cordeiro MN, Silva MA, Ferreira J, Gomez MV. (2012) An Evaluation of the Antinociceptive Effects of Ph?1?, a Neurotoxin from the Spider Phoneutria nigriventer, and ?-Conotoxin MVIIA, a Cone Snail Conus magus Toxin, in Rat Model of Inflammatory and Neuropathic Pain. Cell Mol Neurobiol. 2012 Aug 7. [Epub ahead of print] PubMed PMID: 22869352.

    Programa de Pós-Graduaçăo em Medicina Molecular, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.

    Abstract:Voltage-sensitive calcium channels (VSCCs) underlie cell excitability and are involved in the mechanisms that generate and maintain neuropathic and inflammatory pain. We evaluated in rats the effects of two VSCC blockers, ?-conotoxin MVIIA and Ph?1?, in models of inflammatory and neuropathic pain induced with complete Freund's adjuvant (CFA) and chronic constrictive injury (CCI), respectively. We also evaluated the effects of the toxins on capsaicin-induced Ca(2+) influx in dorsal root ganglion (DRG) neurons obtained from rats exposed to both models of pain. A single intrathecal injection of Ph?1? reversibly inhibits CFA and CCI-induced mechanical hyperalgesia longer than a single injection of ?-conotoxin MVIIA. Ph?1? and MVIIA also inhibited capsaicin-induced Ca(2+) influx in DRG neurons. The inhibitory effect of Ph?1? on capsaicin-induced calcium transients in DRG neurons was greater in the CFA model of pain, while the inhibitory effect of ?-conotoxin MVIIA was greater in the CCI model. The management of chronic inflammatory and neuropathic pain is still a major challenge for clinicians. Ph?1?, a reversible inhibitor of VSCCs with a preference for N-type Ca(2+) channels, has potential as a novel therapeutic agent for inflammatory and neuropathic pain. Clinical studies are necessary to establish the role of Ph?1? in the treatment of chronic pain.

    REVIEW - Cone shell therapeutics

    Vetter I, Lewis RJ. (2012) Therapeutic Potential of Cone Snail Venom Peptides(conopeptides. Curr Top Med Chem. 2012 Jul 17. [Epub ahead of print] PubMed PMID: 22827523.

    Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, 4072, Brisbane, Australia.

    Abstract:Cone snails have evolved many 1000s of small, structurally stable venom peptides (conopeptides) for prey capture and defense. Whilst <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 (?-conopeptide analogue Xen2174) and one marketed (?-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.

    REVIEW - Disulfide rich cyclic peptides

    Cemazar M, Kwon S, Mahatmanto T, Ravipati AS, Craik DJ (2012). Discovery and Applications of Disulfide-rich Cyclic Peptides. Curr Top Med Chem. 2012 Jul 17. [Epub ahead of print] PubMed PMID: 22827522.

    Institute for Molecular Bioscience, Division of Chemistry & Structural Biology, The University of Queensland, Brisbane, Queensland, 4072, Australia. d.craik@imb.uq.edu.au.

    Abstract:Cyclic peptides typically have much higher stability and improved biopharmaceutical properties over their linear counterparts. Our work focuses on the discovery of naturally occurring disulfide-rich cyclic peptides and their applications in drug design. These peptides provide a design basis for re-engineering natural acyclic peptides to improve their biopharmaceutical properties by chemically linking their termini. Here we describe examples of the discovery of the cyclotide family of peptides, their chemical re-engineering to introduce desired pharmaceutical activities, studies of their biopharmaceutical properties and applications of cyclization technologies to naturally occurring toxins, including conotoxins and scorpion toxins. In the case of the conotoxin Vc1.1, we produced an orally active peptide with potential for the treatment of neuropathic pain by cyclising the native peptide.In the case of the scorpion toxin chlorotoxin, a cyclised derivative had improved biopharmaceutical properties as a tumour imaging agent over the naturally occurring linear chlorotoxin. Ongoing chemical and structural studies of these classes of disulfide-rich peptides promise to increase their value for use in dissecting biological processes in plants and mammals while also providing leads to new classes of biopharmaceuticals.

    A 5-cysteine containing conopeptide from Conus generalis

    Gao B, Zhangsun D, Wu Y, Lin B, Zhu X, Luo S. (2012) Expression, renaturation and biological activity of recombinant conotoxin GeXIVAWT. Appl Microbiol Biotechnol. 2012 Jul 24. [Epub ahead of print] PubMed PMID: 22825834.

    Key Laboratory of Tropical Biological Resources, Ministry of Education, Hainan University, Haikou, Hainan, 570228, China.

    Abstract:Conotoxins are a diverse array of small peptides mostly with multiple disulfide bridges. These peptides become an increasing significant source of neuro-pharmacological probes and drugs as a result of the high selectivity for ion channels and receptors. Conotoxin GeXIVAWT (CTX-GeXIVAWT) is a 28-amino acid peptide containing five cysteines isolated from the venom of Conus generalis. Here, we present a simple and fast strategy of producing disulfide-rich conotoxins via recombinant expression. The codes of novel conotoxin gene GeXIVAWT were optimized and generated two pairs of primers by chemical synthesis for construction of expression vector. Recombinant expression vector pET22b(+)-GeXIVAWT fused with pelB leader and His-tag was successfully expressed as an insoluble body in Escherichia coli BL21(DE3) cells. Recombinant conotoxin GeXIVAWT (rCTX-GeXIVAWT) was obtained by dissolving the insoluble bodies and purifying with a Ni-NTA affinity column, which was further purified using reverse-phase high-performance liquid chromatography and identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The rCTX-GeXIVAWT renatured in vitro could inhibited the growth of Sf9 cell with biological activity assay. This expression system may prove valuable for future structure-function studies of conotoxins.

    REVIEW - Therapeutic potential of conopeptides

    Essack M, Bajic VB, Archer JA.(2012) Conotoxins that Confer Therapeutic Possibilities. Mar Drugs. 2012 Jun;10(6):1244-65. Epub 2012 Jun 4. PubMed PMID: 22822370; PubMed Central PMCID: PMC3397437.

    Computational Bioscience Research Center (CBRC), King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Jeddah, Kingdom of Saudi Arabia; Email: magbubah.essack@kaust.edu.sa (M.E.); vladimir.bajic@kaust.edu.sa (V.B.B.).

    Abstract:Cone snails produce a distinctive repertoire of venom peptides that are used both as a defense mechanism and also to facilitate the immobilization and digestion of prey. These peptides target a wide variety of voltage- and ligand-gated ion channels, which make them an invaluable resource for studying the properties of these ion channels in normal and diseased states, as well as being a collection of compounds of potential pharmacological use in their own right. Examples include the United States Food and Drug Administration (FDA) approved pharmaceutical drug, Ziconotide (Prialt(®); Elan Pharmaceuticals, Inc.) that is the synthetic equivalent of the naturally occurring ?-conotoxin MVIIA, whilst several other conotoxins are currently being used as standard research tools and screened as potential therapeutic drugs in pre-clinical or clinical trials. These developments highlight the importance of driving conotoxin-related research. A PubMed query from 1 January 2007 to 31 August 2011 combined with hand-curation of the retrieved articles allowed for the collation of 98 recently identified conotoxins with therapeutic potential which are selectively discussed in this review. Protein sequence similarity analysis tentatively assigned uncharacterized conotoxins to predicted functional classes. Furthermore, conotoxin therapeutic potential for neurodegenerative disorders (NDD) was also inferred.

    Interaction of 4/7 alpha-conotoxins with the nicotinic receptor

    Beissner M, Dutertre S, Schemm R, Danker T, Sporning A, Grubmuller H, Nicke A.(2012) Efficient Binding of 4/7 ?-conotoxins to Nicotinic ?4?2 Receptors is Prevented by R185 and P195 in the ?4 Subunit. Mol Pharmacol. 2012 Jul 16. [Epub ahead of print] PubMed PMID: 22802270.

    1 Max Planck Institute for Brain Research; &?-

    Abstract:Conotoxins are subtype-selective nAChR antagonists. While potent [alpha]3?2 nAChR-selective ?-contoxins have been identified, currently characterized ?-conotoxins show no or only weak affinity for ?4?2 nAChRs, which are besides ?7 receptors the most abundant nAChRs in the mammalian brain. To identify the determinants responsible for this difference, we substituted selected amino acid residues in the ligand binding domain of the ?4 subunit by the corresponding residues in the ?3 subunit. Two-electrode voltage-clamp analysis of these mutants revealed increased affinity of ?-conotoxins MII, TxIA, and [A10L]TxIA at the ?4(R185I)?2 receptor. Conversely, ?-conotoxin potency was reduced at the reverse ?3(I186R)?2 mutant. Replacement of ?4R185 by alanine, glutamate, and lysine demonstrated that a positive charge in this position prevents ?-conotoxin binding. Combination of the R185I mutation with a P195Q mutation outside the binding site but in loop C completely transferred high ?-conotoxin potency to the ?4?2 receptor. Molecular dynamics simulations of homology models with docked ?-conotoxin indicate that these residues control access to the ?-conotoxin binding site.

    A Tyr alpha-conotoxin MII suitable for labelling

    [Effect of alpha-conotoxin MII and its N-terminal derivatives on Ca2+ and Na+ signals induced by nicotine in neuroblastoma cell line SH-SY5Y]. Bioorg Khim. 2012 Mar-Apr;38(2):214-22. Russian. PubMed PMID: 22792725.

    [Article in Russian] [No authors listed]

    Abstract:Nicotinic acetylcholine receptors (nAChRs) are implicated in the regulation ofintracellular Ca2+-dependent processes in cells both in normal and pathological states, alpha-Conotoxins isolated from Conus snails venom are a valuable tool for the study of pharmacological properties and functional role of nAChRs. In the present study the alpha-conotoxin MII analogue with the additional tyrosine attached to the N terminus (Y0-MII) was prepared. Also we synthesized analogs with the N-terminal glycine residue labeled with the Bolton- Hunter reagent (BH-MII) or fluorestsein isothiocyanate (FITC-MII). Fluorescence microscopy studies of the neuroblastoma SH-SY5Y cells loaded with Ca2+ indicator Fura-2 or with Ca2+ and Na+ indicators Fluo-4 and SBFI were performed to examine effect of MII modification on its ability to inhibit nicotin-induced increases in intracellular free Ca2+ and Na+ concentrations ([Ca2+] and [Na+]i respectively). Monitoring of individual cell [Ca2+]i and [Na+]i signals revealed different kinetics of [Ca2+]i and [Na+]i rise and decay in responses to brief nicotine (Nic) applications (10-30 microM, 3-5 min), which indicates to different mechanisms of Ca2+ and Na+ homeostasis control in SH-SY5Y cells. MII inhibited in concentration-dependent manner the both [Ca2+]i and [Na+]i increase induced by Nic. Additional tyrosine in the Y0-MII or, especially, more sizeable label in FITC-MII significantly reduced the inhibitory effect of MII. Whereas the efficiency of the Ca2+ response inhibition by BH-MII was found to be close to the efficiency of its inhibition by natural alpha-conotoxin MII, radioiodinated derivatives BH-MII can be used in radioligand assay.

    12 August 2012

    Studies on Vc1.1 from Conus victoriae

    Production of a cyclic bioengineered conopeptide: Fig. 3 from Bingham et al (2012)

    Research on mechanisms of action of conotoxin Vc1.1

    In addition to the interest by Dr David Adams, RMIT University, Melbourne (who proposed GABAB receptor interaction) and by Dr David Craik, IMB, Brisbane and colleagues (who synthesised a cyclized orally active form of Vc1.1, reviewed here), two other investigators Dr Ray Norton and Dr Andrea Robinson from Monash University, Clayton, Victoria, have recently indicated continued interest in conotoxin Vc1.1 with student projects progressing through 2012.

    The July 2012 Newsletter of the Malacological Society of Australasia (No. 144, July 2012) announced among the highly commended research projects submitted, one by Samuel Robinson, Monash University “Novel peptides from the venom of Conus victoriae”.

    A search on Google brought to light the presentations at the 6th Annual Postgraduate Research Symposium (September 2011), Monash University, at this site, where you can read, on page 32, a summary of Sam Robinson’s presentation for the project titled “Non-reducible analogues of alpha conotoxin Vc1.1”. Sam’s supervisor is Dr Ray Norton (Pharmacy and Pharmaceutical Sciences).

    “The aim of this study is to investigate the effect of replacing individual disulfides with dicarba bridges on both the structure and activity of this peptide using NMR spectroscopy and electrophysiology, respectively. Peptides with dicarba bonds in place of each disulfide have been synthesized. The structural consequences of dicarba bridges in alpha-conotoxin Vc1.1 will be discussed. Inclusion of dicarba bridges is a viable option for improving the stability of alpha-conotoxin Vc1.1 and enhancing its therapeutic potential”

    Projects listed for Honours 2012 within Pharmacy and Pharmaceutical Sciences at Monash include one with the title “Peptide interactions with Nicotinic Acetylcholine and GABAB Receptors: Structural Basis for the Potent Analgesic Activity of alpha-conotoxins”. This is a multidisciplinary project, spanning structural biology, molecular modelling and molecular design, with the opportunity to gain experience in peptide synthesis. The supervisors are listed as Dr Ray Norton, Dr Elizabeth Yuriev and Dr Andrea Robinson. See page 16 at this site.

    As summarized nicely in Lewis, RJ et al (2012) Pharmacological Reviews 64 (2) 259-298, “it is now possible to engineer potentially more stable N-C-cyclized forms as exemplified by using alpha-conotoxin Vc1.1, which retained analgesic activity even after oral dosing (Clark et al 2010).”

    This is all encouraging and indicates that interest in the conopeptides from Conus victoria remains high.

    16 July 2012

    Evolutionary role of disulfide bridges in conotoxins

    Gowd KH, Blais KD, Elmslie KS, Steiner AM, Olivera BM, Bulaj G. (2012) Dissecting a role of evolutionary-conserved but noncritical disulfide bridges in cysteine-rich peptides using ?-conotoxin GVIA and its selenocysteine analogs. Biopolymers. 2012;98(3):212-23. doi: 10.1002/bip.22047.

    Department of Biology, University of Utah, Salt Lake City, UT 84112; Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.

    Abstract:Conotoxins comprise a large group of peptidic neurotoxins that use diverse disulfide-rich scaffolds. Each scaffold is determined by an evolutionarily conserved pattern of cysteine residues. Although many structure-activity relationship studies confirm the functional and structural importance of disulfide crosslinks, there is growing evidence that not all disulfide bridges are critical in maintaining activities of conotoxins. To answer the fundamental biological question of what the role of noncritical disulfide bridges is, we investigated function and folding of disulfide-depleted analogs of ?-conotoxin GVIA (GVIA) that belongs to an inhibitory cystine knot motif family and blocks N-type calcium channels. Removal of a noncritical Cys1-Cys16 disulfide bridge in GVIA or its selenopeptide analog had, as predicted, rather minimal effects on the inhibitory activity on calcium channels, as well as on in vivo activity following intracranial administration. However, the disulfide-depleted GVIA exhibited significantly lower folding yields for forming the remaining two native disulfide bridges. The disulfide-depleted selenoconotoxin GVIA analog also folded with significantly lower yields, suggesting that the functionally noncritical disulfide pair plays an important cooperative role in forming the native disulfide scaffold. Taken together, our results suggest that distinct disulfide bridges may be evolutionarily preserved by the oxidative folding or/and stabilization of the bioactive conformation of a disulfide-rich scaffold. © 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 212-223, 2012. Copyright © 2012 Wiley Periodicals, Inc. PMCID: PMC3395435 [Available on 2013/1/1] PMID: 22782563 [PubMed - in process]

    Diversity and evolution of conotoxins from South China Sea

    Liu Z, Li H, Liu N, Wu C, Jiang J, Yue J, Jing Y, Dai Q (2012) Diversity and evolution of conotoxins in Conus virgo, Conus eburneus, Conus imperialis and Conus marmoreus from the South China Sea. Toxicon. 2012 Jul 7. [Epub ahead of print]

    Beijing Institute of Biotechnology, Beijing 100071, China.

    Abstract:The venom peptides of cone snails are encoded by a large gene family, and can selectively bind to voltage-gated ion channels (Na(+), K(+) and Ca(2+) channels) and to membrane receptors (nAChR, 5-HT3R, NMDAR). To identify novel conotoxin genes and analyze the evolution of typical conotoxin superfamily genes from different Conus species, we have constructed cDNA libraries derived from the venom ducts of C. virgo, C. eburneus, C. imperialis and C. marmoreus, which were collected from the South China Sea. 1312 transcripts from four Conus venom duct cDNA libraries were analyzed and 38.7-49.6% of the transcripts encoded conotoxin sequences. In addition to known conotoxins, 34 novel conotoxins have been identified and can be classified into eleven superfamilies, some of which showed unique patterns of cysteines or different signal peptide sequences. The evolutionary trees of T- and A-superfamily conotoxins were analyzed. Likelihood approaches revealed that T-superfamily conotoxins from the four Conus species undergo positive selection, mostly located in the mature toxin region. These findings contribute to a better understanding of the diversity and evolution of conotoxins from the South China Sea, and some novel conotoxins are valuable for further functional investigations. Copyright © 2012. Published by Elsevier Ltd. PMID: 22781954 [PubMed - as supplied by publisher]

    Novel peptides from Conus pictus and Conus natalis

    Peigneur S, Van Der Haegen A, Möller C, Waelkens E, Diego-García E, Marí F, Naudé R, Tytgat J. (2012) Unraveling the peptidome of the South African cone snails Conus pictus and Conus natalis. Peptides. 2012 Jul 6. [Epub ahead of print]

    Laboratory of Toxicology, University of Leuven (K.U.Leuven), O&N 2, PO Box 922, Herestraat 49, 3000 Leuven, Belgium.

    Abstract:Venoms from cone snails (genus Conus) can be seen as an untapped cocktail of biologically active compounds, being increasingly recognized as an emerging source of peptide-based therapeutics. Cone snails are considered to be specialized predators that have evolved the most sophisticated peptide chemistry and neuropharmacology system for their own biological purposes by producing venoms which contains a structural and functional diversity of neurotoxins. These neurotoxins or conotoxins are often small cysteine-rich peptides which have shown to be highly selective ligands for a wide range of ion channels and receptors. Local habitat conditions have constituted barriers preventing the spreading of Conus species occurring along the coast of South Africa. Due to their scarceness, these species remain, therefore, extremely poorly studied. In this work, the venoms of two South African cone snails, Conus pictus, a vermivorous snail and Conus natalis, a molluscivorous snail, have been characterized in depth. In total, 26 novel peptides were identified. Comparing the venoms of both snails, interesting differences were observed regarding venom composition and molecular characteristics of these components. Copyright © 2012. Published by Elsevier Inc. PMID: 22776330 [PubMed - as supplied by publisher]

    Sensitivity of rat vs. human alpha9alpha10 receptors

    Azam L, McIntosh JM. (2012) Molecular basis for the differential sensitivity of rat and human alpha9alpha10 nAChRs to alpha-conotoxin RgIA. J Neurochem. 2012 Jul 9. doi: 10.1111/j.1471-4159.2012.07867.x. [Epub ahead of print]

    Department of Biology and Department of Psychiatry, University of Utah, Salt Lake City, UT 84112.

    Abstract:The alpha9alpha10 nicotinic acetylcholine receptor (nAChR) may be a potential target in pathophysiology of the auditory system, chronic pain and breast and lung cancers. Alpha-conotoxins, from the predatory marine snail Conus, are potent nicotinic antagonists, some of which are selective for the alpha9alpha10 nAChR. Here we report a two-order of magnitude species difference in the potency of alpha-conotoxin RgIA for the rat vs. human alpha9alpha10 nAChR. We investigated the molecular mechanism of this difference. Heterologous expression of the rat alpha9 with the human alpha10 subunit in Xenopus oocytes resulted in a receptor that was blocked by RgIA with potency similar to that of the rat alpha9alpha10 nAChR. Conversely, expression of the human alpha9 with that of the rat alpha10 subunit resulted in a receptor that was blocked by RgIA with potency approaching that of the human alpha9alpha10 receptor. Systematic substitution of residues found in the human alpha9 subunit into the homologous position in the rat alpha9 subunit revealed that a single point mutation, Thr56 to Ile56, primarily accounts for this species difference. Remarkably, although the alpha9 nAChR subunit has previously been reported to provide the principal (+) binding face for binding of RgIA, Thr56 is located in the (-) complementary binding face.

    Design of peptides based on mu-conotoxin

    Stevens M, Peigneur S, Dyubankova N, Lescrinier E, Herdewijn P, Tytgat J (2012) Design of bioactive peptides from naturally occurring mu-conotoxin structures. J Biol Chem. 2012 Jul 6. [Epub ahead of print]

    Lab of Toxicology, KU Leuven, Belgium;

    Abstract:To date, cone snail toxins ( "conotoxins") are of great interest in the pursuit of novel subtype-selective modulators of voltage-gated sodium channels (Na(v)s). Na(v)s participate in a wide range of electrophysiological processes. Consequently, their malfunctioning has been associated with numerous diseases. The development of subtype-selective modulators of Na(v)s remains highly important in the treatment of such disorders. In current research, a series of novel, synthetic and bioactive compounds were designed based on two naturally occurring ?-conotoxins that target Na(v)s. The initial designed peptide contains solely 13 amino acids and was therefore named "Mini peptide". It was derived from the mu-conotoxins KIIIA and BuIIIC. Based on this Mini peptide, ten analogues were subsequently developed, comprising 12 to 16 AAs with two disulfide bridges. Following appropriate folding and mass verification, blocking effects on Na(v)s were investigated. The most promising compound established an IC(50) of 34.1 nM ± 0.01 (R2-Midi on Na(v)1.2). An NMR structure of one of our most promising compounds was determined. Surprisingly, this structure does not reveal an alpha-helix. We prove that it is possible to design small peptides based on known pharmacophores of mu-conotoxins, without losing their potency and selectivity. This data can provide crucial material for further development of conotoxin-based therapeutics.

    The work of Baldomero Olivera

    From DNA enzymes to cone snail venom: the work of Baldomero M. Olivera. PMCID: PMC3391119 J Biol Chem. 2012 Jun 29;287(27):23020-3. Full Text .pdf available here.

    [The two papers being recognized here as JBC Classics] are:

    Processivity of DNA Exonucleases (Thomas, K. R., and Olivera, B. M. (1978) J. Biol. Chem. 253, 424–429) and

    Neuronal Calcium Channel Inhibitors. Synthesis of omega-Conotoxin GVIA and Effects on 45Ca Uptake by Synaptosomes (Rivier, J., Galyean, R., Gray, W. R., Azimi-Zonooz, A., McIntosh, J. M., Cruz, L. J., and Olivera, B. M. (1987) J. Biol. Chem. 262, 1194–1198).

    Alpha6 receptor selectivity

    Kim HW, McIntosh JM.(2012) alpha6 nAChR subunit residues that confer alpha-conotoxin BuIA selectivity. FASEB J. 2012 Jul 2. [Epub ahead of print]

    *Department of Marine Biology, Pukyong National University, Busan, South Korea; and.

    Abstract:Nicotinic acetylcholine receptors (nAChRs) containing alpha6 and/or alpha4 subunits modulate the release of dopamine. However, few compounds can effectively discriminate between ligand-binding sites that contain alpha6 vs. alpha4 nAChR subunits. Using a chimeric (alpha6/alpha4) subunit, we showed that alpha-conotoxin BuIA binds the extracellular rat alpha6alpha2 vs. alpha4alpha2 interface with >60,000-fold selectivity. Chimeras containing residues from the alpha6 subunit were inserted into the homologous position of the alpha4 subunit to identify critical sequence segments. The region between residues 184 and 207 in the alpha6 subunit accounted for the potency difference. Chimeras within this region followed by point mutations were constructed for further definition. alpha6 Lys185, Thr187, and Ile188 form a triad of key residues that influence BuIA binding; when these 3 alpha6 residues were inserted into the alpha4 subunit, there was an >2000-fold increase in toxin potency. We used a crystal structure of BuIA bound to the acetylcholine-binding protein together with the structure of the Torepedo marmorata nAChR to build a homology model of BuIA bound to the interface between alpha6 and alpha2 subunits. The results indicate that the triad of alpha6 residues lies outside the C loop and is distantly located from BuIA (>10 Ĺ). This suggests that alterations in potency are not caused by the direct interaction between the triad and BuIA. Instead, alterations in C-loop 3-dimensional structure and/or flexibility may account for differential potency. Thr198 and Tyr205 also contributed to BuIA potency. In addition, Thr198 caused BuIA potency differences between the closely related alpha6 and alpha3 subunits. Together, the findings provide insight into differences between the alpha6 and other alpha subunits that may be exploited by alpha-conotoxins to achieve binding selectivity.

    Envenomation strategy of Conus geographus

    Hu H, Bandyopadhyay PK, Olivera BM, Yandell M. (2012) Elucidation of the molecular envenomation strategy of the cone snail Conus geographus through transcriptome sequencing of its venom duct. BMC Genomics. 2012 Jun 28;13(1):284. [Epub ahead of print]

    Abstract: BACKGROUND: The fish-hunting cone snail, Conus geographus, is the deadliest snail on earth. In the absenceof medical intervention, 70% of human stinging cases are fatal. Although, its venom isknown to consist of a cocktail of small peptides targeting different ion-channels andreceptors, the bulk of its venom constituents, their sites of manufacture, relative abundancesand how they function collectively in envenomation has remained unknown. RESULTS: We have used transcriptome sequencing to systemically elucidate the contents the C.geographus venom duct, dividing it into four segments in order to investigate each segment'smRNA contents. Three different types of Calcium channel (each targeted by unrelated,entirely distinct venom peptides) and at least two different nicotinic receptors appear to betargeted by the venom. Moreover, the most highly expressed venom component is notparalytic, but causes sensory disorientation and is expressed in a different segment of thevenom duct from venoms believed to cause sensory disruption. We have also identifiedseveral new toxins of interest for pharmaceutical and neuroscience research. CONCLUSIONS: Conus geographus is believed to prey on fish hiding in reef crevices at night. Our datasuggest that disorientation of prey is central to its envenomation strategy. Furthermore,venom expression profiles also suggest a sophisticated layering of venom-expression patternswithin the venom duct, with disorientating and paralytic venoms expressed in different regions. Thus, our transcriptome analysis provides a new physiological framework forunderstanding the molecular envenomation strategy of this deadly snail. PMID: 22742208 [PubMed - as supplied by publisher]

    30 June 2012

    Effect of N- and C-terminal extensions of mu-conotoxins

    Schroeder CI, Adams D, Thomas L, Alewood PF, Lewis RJ. (2012) N- and C-terminal extensions of mu-conotoxins increase potency and selectivity for neuronal sodium channels. Biopolymers. 2012;98(2):161-5. doi: 10.1002/bip.22032. Epub 2012 Feb 10.

    Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.

    Abstract: mu-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 mu-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 mu-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 mu-conotoxins at voltage-gated sodium channels, revealing that binding interactions outside the cysteine-stablilized loops can contribute to mu-conotoxin affinity and sodium channel selectivity.

    Conus ventricosus: insecticidal activity

    Spiezia MC, Chiarabelli C, Polticelli F. (2012) Recombinant expression and insecticidal properties of a Conus ventricosus conotoxin-GST fusion protein. Toxicon. 2012 Jun 21. [Epub ahead of print]

    Department of Biology, University of Roma Tre, Viale Guglielmo Marconi 446, 00146 Rome, Italy.

    Abstract: A novel conotoxin, conotoxin Vn2, was recently isolated from the venom of Conus ventricosus, a worm-hunting cone snail species living in the Mediterranean Sea. Analysis of conotoxin Vn2 amino acid sequence suggested that it is a member of the O1 superfamily of conotoxins. Conotoxin Vn2 displays quite a high degree of sequence similarity with bioactive peptides targeting calcium channels and in particular with the ? conotoxin PnVIB, extracted from the venom of the molluscivorous cone snail Conus pennaceus. In this work we describe the development of a heterologous expression system to obtain a glutathione-S-transferase (GST) fusion product of conotoxin Vn2 in a pure form and in a sufficient amount to characterize its bioactivity. The fusion product has been expressed in recombinant form in Escherichia coli cells, purified, and its neurotoxic activity has been assayed on the larvae of the moth Galleria mellonella, a simple experimental model to test the toxicity of compounds in insects. Moreover the conotoxin Vn2 Asp2His mutant has been produced to analyse the role of this aspartic acid residue in the toxin bioactivity, as an acidic amino acid is conserved in this position in all the O1 superfamily Conus ventricosus conotoxins. Results obtained indicate that indeed conotoxin Vn2 has strong insecticidal properties at a dose of only 100 pmol/g of body weight. Surprisingly, mutation of Asp2 to His leads to enhanced toxicity in the larvae model system opening up interesting possibilities for the use of conotoxin Vn2 variants in environmental friendly crop protection applications.

    Calcium channel blockers for neuropathic pain

    Vink S, Alewood PF. (2012) Targeting voltage-gated calcium channels: developments in peptide and small molecule inhibitors for the treatment of neuropathic pain. Br J Pharmacol. 2012 Jun 22. doi: 10.1111/j.1476 5381.2012.02082.x. [Epub ahead of print]

    Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia.

    Abstract: Chronic pain affects approximately 20% of people worldwide and places a large economic and social burden on society. Despite the availability of a range of analgesics this condition is inadequately treated, with complete alleviation of symptoms rarely occurring. In the last thirty years, the voltage-gated calcium channels (VGCCs) have been recognised as potential targets for analgesic development. Although the majority of the research has been focused on Ca(v) 2.2 in particular, other VGCC subtypes such as Ca(v) 3.2 have recently come to the forefront of analgesic research. Venom peptides from marine cone snails have proven to be a valuable tool in neuroscience playing a major role in the identification and characterisation of VGCC subtypes and producing the first conotoxin-based drug on the market, the omega-conotoxin, ziconotide. This peptide potently and selectively inhibits Ca(v) 2.2, resulting in analgesia in chronic pain states. However, this drug is only available via intrathecal administration and adverse effects and a narrow therapeutic window has limited its use in the clinic. Other Ca(v) 2.2 inhibitors are currently in development and offer the promise of an improved route of administration and safety profile. This review assesses the potential of targeting VGCCs for analgesic development with a main focus on conotoxins that block Ca(v) 2.2 and the developments made to transform them into therapeutics.

    Sequencing of conotoxins

    Bhatia S, Kil YJ, Ueberheide B, Chait BT, Tayo L, Cruz L, Lu B, Yates JR, Bern M. (2012) Constrained De Novo Sequencing of Conotoxins. J Proteome Res. 2012 Jun 18. [Epub ahead of print]


    Abstract: De novo peptide sequencing by mass spectrometry (MS) can determine the amino acid sequence of an unknown peptide without reference to a protein database. MS-based de novo sequencing assumes special importance in focused studies of families of biologically active peptides and proteins, such as hormones, toxins, and antibodies, for which amino acid sequences may be difficult to obtain through genomic methods. These protein families often exhibit sequence homology or characteristic amino acid content, yet current de novo sequencing approaches do not take advantage of this prior knowledge and hence search an unnecessarily large space of possible sequences. Here, we describe an algorithm for de novo sequencing that incorporates sequence constraints into the core graph algorithm, and thereby reduces the search space by many orders of magnitude. We demonstrate our algorithm in a study of cysteine-rich toxins from two cone snail species (Conus textile and Conus stercusmuscarum), and report 13 de novo and about 60 total toxins.

    Conantokin G for pain treatment

    Bossowska A, Majewski M. (2012) Conantokin G-induced changes in the chemical coding of dorsal root ganglion neurons supplying the porcine urinary bladder. Pol J Vet Sci. 2012;15(1):101-9.

    Department of Human Physiology, Faculty of Medical Sciences, University of Warmia and Mazury, Warszawska 30, 10-082 Olsztyn, Poland. Agnieszka.Bossowska@uwm.edu.pl

    Abstract: Conantokin G (CTG), isolated from the venom of the marine cone snail Conus geographus, is an antagonist of N-methyl-d-aspartate receptors (NMDARs), the activation of which, especially those located on the central afferent terminals and dorsal horn neurons, leads to hypersensitivity and pain. Thus, CTG blocking of NMDARs, has an antinociceptive effect, particularly in the case of neurogenic pain treatment. As many urinary bladder disorders are caused by hyperactivity of sensory bladder innervation, it seems useful to estimate the influence of CTG on the plasticity of sensory neurons supplying the organ. Retrograde tracer Fast Blue (FB) was injected into the urinary bladder wall of six juvenile female pigs. Three weeks later, intramural bladder injections of CTG (120 microg per animal) were carried out in all animals. After a week, dorsal root ganglia of interest were harvested from all animals and neurochemical characterization of FB+ neurons was performed using a routine double-immunofluorescence labeling technique on 10-microm-thick cryostat sections. CTG injections led to a significant decrease in the number of FB+ neurons containing substance P (SP), pituitary adenylate cyclase activating polypeptide (PACAP), somatostatin (SOM), calbindin (CB) and nitric oxide synthase (NOS) when compared with healthy animals (20% vs. 45%, 13% vs. 26%, 1.3% vs. 3%, 1.2 vs. 4% and 0.9% vs. 6% respectively) and to an increase in the number of cells immunolabelled for galanin (GAL, 39% vs. 6.5%). These data demonstrated that CTG changed the chemical coding of bladder sensory neurons, thus indicating that CTG could eventually be used in the therapy of selected neurogenic bladder illnesses.

    Injected venom from Conus consors

    Violette A, Biass D, Dutertre S, Koua D, Piquemal D, Pierrat F, Stöcklin R, Favreau P. (2012) Large-scale discovery of conopeptides and conoproteins in the injectable venom of a fish-hunting cone snail using a combined proteomic and transcriptomic approach. J Proteomics. 2012 Jun 13. [Epub ahead of print]

    Atheris Laboratories, Case postale 314, CH-1233 Bernex-Geneva, Switzerland.

    Abstract: Predatory marine snails of the genus Conus use venom containing a complex mixture of bioactive peptides to subdue their prey. Here we report on a comprehensive analysis of the protein content of injectable venom from Conus consors, an indo-pacific fish-hunting cone snail. By matching MS/MS data against an extensive set of venom gland transcriptomic mRNA sequences, we identified 105 components out of ~400 molecular masses detected in the venom. Among them, we described new conotoxins belonging to the A, M- and O1-superfamilies as well as a novel superfamily of disulphide free conopeptides. A high proportion of the deduced sequences (36%) corresponded to propeptide regions of the A- and M-superfamilies, raising the question of their putative role in injectable venom. Enzymatic digestion of higher molecular mass components allowed the identification of new conkunitzins (~7kDa) and two proteins in the 25 and 50kDa molecular mass ranges respectively characterised as actinoporin-like and hyaluronidase-like protein. These results provide the most exhaustive and accurate proteomic overview of an injectable cone snail venom to date, and delineate the major protein families present in the delivered venom. This study demonstrates the feasibility of this analytical approach and paves the way for transcriptomics-assisted strategies in drug discovery.


    Koua D, Brauer A, Laht S, Kaplinski L, Favreau P, Remm M, Lisacek F, Stöcklin R. (2012) ConoDictor: a tool for prediction of conopeptide superfamilies. Nucleic Acids Res. 2012 Jul 1;40(W1):W238-W241. Epub 2012 May 31.

    Atheris Laboratories, Case postale 314, CH-1233 Bernex-Geneva, Switzerland, Proteome Informatics Group, Swiss Institute of Bioinformatics, CH-1211 Geneva, Switzerland and Bioinformatics Workgroup, Estonian Biocentre, EE-51010 Tartu, Estonia.

    Abstract: ConoDictor is a tool that enables fast and accurate classification of conopeptides into superfamilies based on their amino acid sequence. ConoDictor combines predictions from two complementary approaches-profile hidden Markov models and generalized profiles. Results appear in a browser as tables that can be downloaded in various formats. This application is particularly valuable in view of the exponentially increasing number of conopeptides that are being identified. ConoDictor was written in Perl using the common gateway interface module with a php submission page. Sequence matching is performed with hmmsearch from HMMER 3 and ps_scan.pl from the pftools 2.3 package. ConoDictor is freely accessible at http://conco.ebc.ee.

    Peptide purification using bacterial flagellum.

    Singer HM, Erhardt M, Steiner AM, Zhang MM, Yoshikami D, Bulaj G, Olivera BM, Hughes KT.(2012) Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System. MBio. 2012 May 29;3(3). pii: e00115-12. doi:10.1128/mBio.00115-12. Print 2012.

    Dčpartement de Médecine, Université de Fribourg, Fribourg, Switzerland.

    ABSTRACT: The structure, assembly, and function of the bacterial flagellum involves about 60 different proteins, many of which are selectively secreted via a specific type III secretion system (T3SS) (J. Frye et al., J. Bacteriol. 188:2233-2243, 2006). The T3SS is reported to secrete proteins at rates of up to 10,000 amino acid residues per second. In this work, we showed that the flagellar T3SS of Salmonella enterica serovar Typhimurium could be manipulated to export recombinant nonflagellar proteins through the flagellum and into the surrounding medium. We translationally fused various neuroactive peptides and proteins from snails, spiders, snakes, sea anemone, and bacteria to the flagellar secretion substrate FlgM. We found that all tested peptides of various sizes were secreted via the bacterial flagellar T3SS. We subsequently purified the recombinant mu-conotoxin SIIIA (rSIIIA) from Conus striatus by affinity chromatography and confirmed that T3SS-derived rSIIIA inhibited mammalian voltage-gated sodium channel Na(V)1.2 comparably to chemically synthesized SIIIA. IMPORTANCE: Manipulation of the flagellar secretion system bypasses the problems of inclusion body formation and cellular degradation that occur during conventional recombinant protein expression. This work serves as a proof of principle for the use of engineered bacterial cells for rapid purification of recombinant neuroactive peptides and proteins by exploiting secretion via the well-characterized flagellator type III secretion system.

    Origin of After-Discharges in persistent pain

    Yamamoto S, Tanabe M, Ono H. (2012) N- and L-Type Voltage-Dependent Ca2+ Channels Contribute to the Generation of After-Discharges in the Spinal Ventral Root After Cessation of Noxious Mechanical Stimulation. J Pharmacol Sci. 2012 May 17;119(1):82-90. Epub 2012 Apr 28.

    Laboratory of CNS Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Japan.

    Abstract: Voltage-dependent Ca2+ channels (VDCCs) play a crucial role in the spinal pain transduction. We previously reported that nociceptive mechanical stimuli to the rat hindpaw evoked two types of ventral root discharges that increased during stimulation (during-discharges) and after cessation of stimulation (after-discharges). To explore the involvement of VDCCs in these ventral root discharges, several VDCC blockers were applied directly to the surface of the spinal cord. Spinalized rats were laminectomized. The fifth lumbar ventral root was sectioned and used for multi-unit efferent discharges recording. An agar pool was constructed on the first lumbar vertebra for drug application. Ethosuximide (a T-type VDCC blocker) had no effect on ventral root discharges. ?-Conotoxin GVIA (an N-type VDCC blocker) preferentially suppressed after-discharges. ?-Agatoxin IVA (a P/Q-type VDCC blocker), diltiazem, and verapamil (L-type VDCC blockers) nonselectively depressed both during- and after-discharges. The more selective L-type VDCC blocker nicardipine depressed only after-discharges and the depression was exhibited when nicardipine was microinjected into the dorsal horn, but not into the ventral horn. These findings suggested that N- and L-type VDCCs in the dorsal horn were involved in the generation of after-discharges and these blockers might be useful for treatment of persistent pain that involves the spinal pathway.

    Molecular docking with alpha-conotoxin GIC

    Lee C, Lee SH, Kim DH, Han KH (2012). Molecular docking study on the alpha3beta2 neuronal nicotinic acetylcholine receptor complexed with alpha-Conotoxin GIC. BMB Rep. 2012 May;45(5):275-80. [BMB reports 2012; 45(5): 275-280].

    Biomedical Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Korea khhan600@kribb.re.kr.

    Abstract: Nicotinic acetylcholine receptors (nAChRs) are a diverse family of homo- or heteropentameric ligand-gated ion channels. Understanding the physiological role of each nAChR subtype and the key residues responsible for normal and pathological states is important. alpha-Conotoxin neuropeptides are highly selective probes capable of discriminating different subtypes of nAChRs. In this study, we performed homology modeling to generate the neuronal alpha3, beta2 and beta4 subunits using the x-ray structure of the alpha1 subunit as a template. The structures of the extracellular domains containing ligand binding sites in the alpha3beta2 and alpha3beta4 nAChR subtypes were constructed using MD simulations and ligand docking processes in their free and ligand-bound states using alpha-conotoxin GIC, which exhibited the highest alpha3beta2 vs. alpha3beta4 discrimination ratio. The results provide a reasonable structural basis for such a discriminatory ability, supporting the idea that the present strategy can be used for future investigations on nAChR-ligand complexes.

    Both GABAB subunits required for analgesic action of alpha-Vc1.1 and RgIa

    Cuny H, de Faoite A, Huynh TG, Yasuda T, Berecki G, Adams DJ (2012). Gamma-aminobutyric acid type B (GABAB) receptor expression is needed for inhibition of N-type (Cav2.2) calcium channels by analgesic alpha-conotoxins. J Biol Chem. 2012 May 21. [Epub ahead of print]

    RMIT University, Australia;

    Abstract: alpha-Conotoxins Vc1.1 and RgIA are small peptides isolated from the venom of marine cone snails. They have effective anti-nociceptive actions in rat models of neuropathic pain. Pharmacological studies in rodent dorsal root ganglion (DRG) show their analgesic effect is mediated by inhibition of N-type (Ca(v)2.2) calcium channels via a pathway involving ?-aminobutyric acid type B (GABA(B)) receptor. However, there is no direct demonstration that functional GABA(B) receptors are needed for inhibition of Ca(v)2.2 channel by analgesic alpha-conotoxins. This study examined the effect of the GABA(B) agonist baclofen and alpha-conotoxins Vc1.1 and RgIA on calcium channel currents following transient knockdown of the GABA(B) receptor using RNA interference. Isolated rat DRG neurons were transfected with small interfering RNAs (siRNAs) targeting GABA(B) subunits R1 and R2. Efficient knockdown of GABA(B) receptor expression at mRNA and protein levels was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and immunocytochemical analysis, respectively. Whole-cell patch clamp recordings conducted 2-4 days after transfection showed that inhibition of N-type calcium channels in response to baclofen, Vc1.1 and RgIA was significantly reduced in GABA(B) receptor knockdown DRG neurons. In contrast, neurons transfected with a scrambled, non-targeting siRNA were indistinguishable from untransfected neurons. In the HEK 293 cell heterologous expression system, Vc1.1 and RgIA inhibition of Ca(v)2.2 channels needed functional expression of both human GABA(B) receptor subunits. Together, these results confirm that GABA(B) receptors must be activated for the modulation of N-type (Ca(v)2.2) calcium channels by analgesic alpha-conotoxins Vc1.1 and RgIA.

    Chemotherapy-evoked neuropathic pain

    Wala EP, Crooks PA, McIntosh JM, Holtman JR Jr.(2012) Novel Small Molecule alpha9alpha10 Nicotinic Receptor Antagonist Prevents and Reverses Chemotherapy-Evoked Neuropathic Pain in Rats. Anesth Analg. 2012 May 18. [Epub ahead of print]

    From the *Department of Anesthesiology, College of Medicine, and.

    Background: Peripheral neuropathy is a common dose-limiting side effect of chemotherapy. There are no clinically proven analgesics for the treatment of this condition. Drugs from different classes have been tested with mixed results. Identification of novel molecular targets for analgesic(s) is important. Antagonism of the ?9?10 nicotinic acetylcholine receptor (nAChR) subtype (absent in brain) is thought to underlie analgesic efficacy of peptide alpha-conotoxins. We found novel nonpeptide small molecule analogs from a family of tetrakis-, tris-, and bis-azaaromatic quaternary ammonium salts (high potency with selectivity as antagonists at the alpha9alpha10 nAChRs) to produce dose-related analgesia in rat models of nerve injury-evoked neuropathy and persistent inflammatory pain. No tests were done in a model of neuropathy induced by drug administration (ie, chemotherapy). Methods: In this study, a lead bis-analog, ZZ1-61c, was characterized in a rat model of vincristine-evoked neuropathy. Male Sprague-Dawley rats were repeatedly dosed with the vinca-alkaloid, vincristine (100 ?g/kg/day IP, days 1 to 5 and 8 to 12). ZZ1-61c (100 ?g/kg/day IP) was given either along with or after completion of vincristine (commencing by day 15 when neuropathy was maximum). Responsiveness was assessed with von Frey hairs and the paw-pressure test. The effects of ZZ1-61c on motor function (rotarod) and muscle strength (grip test) were characterized in naďve rats. Results: The development of neuropathy was demonstrated with repeated dosing of vincristine (pain hypersensitivity in response to mechanical stimulation). ZZ1-61c showed both preventive and restorative effects on this condition: (1) vincristine-evoked sensitivity to pressure was reduced by coadministration of ZZ1-61c; (2) established neuropathy was diminished by ZZ1-61c after cessation of chemotherapy. ZZ1-61c did not cause motor dysfunction (rotarod) or muscular weakness (the grip test).Conclusions: This study suggests that ZZ1-61c, a novel compound with a unique mechanism of antagonistic action at the alpha9alpha10 nAChR, may be a potential drug candidate for prevention and attenuation of neuropathic pain resulting from chemotherapy. Such a strategy may provide effective treatment that circumvents toxicity of centrally acting agonists at nAChR.

    A novel muO-conotoxin from Conus magnificus

    Vetter I, Dekan Z, Knapp O, Adams DJ, Alewood PF, Lewis RJ (2012). Isolation, characterization and total regioselective synthesis of the novel muO-conotoxin MfVIA from Conus magnificus that targets voltage-gated sodium channels. Biochem Pharmacol. 2012 May 16. [Epub ahead of print]

    Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Australia.

    Abstract: The muO-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 muO-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 muO-conotoxin MrVIB. To overcome the synthetic challenges posed by muO-conotoxins due to their hydrophobic nature and difficult folding, we developed a novel regioselective approach for the synthesis of muO-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 (IC(50) 95.9±74.3nM) and Na(v)1.4 (IC(50) 81±16nM), with significantly lower affinity for other Na(v) subtypes (IC(50) 431-6203nM; Na(v)1.5>1.6?1.7?1.3?1.1?1.2). This improved approach to muO-conotoxin synthesis will facilitate the optimization of muO-conotoxins as novel analgesic molecules to improve pain management.

    Conantokins derived from Asprella

    Gowd KH, Han TS, Twede V, Gajewiak J, Smith MD, Watkins M, Platt RJ, Toledo G, White HS, Olivera BM, Bulaj G. (2012). Conantokins Derived from the Asprella Clade Impart conRl-B, an N-Methyl d-Aspartate Receptor Antagonist with a Unique Selectivity Profile for NR2B Subunits. Biochemistry. 2012 May 30. [Epub ahead of print]

    Department of Biology, ‡Department of Pharmacology/Toxicology, §Department of Pathology, and Department of Medicinal Chemistry, University of Utah , Salt Lake City, Utah 84112, United States.

    Abstract: Using molecular phylogeny has accelerated the discovery of peptidic ligands targeted to ion channels and receptors. One clade of venomous cone snails, Asprella, appears to be significantly enriched in conantokins, antagonists of N-methyl d-aspartate receptors (NMDARs). Here, we describe the characterization of two novel conantokins from Conus rolani, including conantokin conRl-B that has shown an unprecedented selectivity for blocking NMDARs that contain NR2B subunits. ConRl-B shares only some sequence similarity with the most studied NR2B selective conantokin, conG. The divergence between conRl-B and conG in the second inter-Gla loop was used to design analogues for structure-activity studies; the presence of Pro10 was found to be key to the high potency of conRl-B for NR2B, whereas the e-amino group of Lys8 contributed to discrimination in blocking NR2B- and NR2A-containing NMDARs. In contrast to previous findings for Tyr5 substitutions in other conantokins, conRl-B[L5Y] showed potencies on the four NR2 NMDA receptor subtypes that were similar to those of the native conRl-B. When delivered into the brain, conRl-B was active in suppressing seizures in the model of epilepsy in mice, consistent with NR2B-containing NMDA receptors being potential targets for antiepileptic drugs. Circular dichroism experiments confirmed that the helical conformation of conRl-B is stabilized by divalent metal ions. Given the clinical applications of NMDA antagonists, conRl-B provides a potentially important pharmacological tool for understanding the differential roles of NMDA receptor subtypes in the nervous system. This work shows the effectiveness of coupling molecular phylogeny, chemical synthesis, and pharmacology for discovering new bioactive natural products.

    Pharmacophore grafting

    Dekan Z, Wang CI, Andrews RK, Lewis RJ, Alewood PF (2012). Conotoxin engineering: dual pharmacophoric noradrenaline transport inhibitor/integrin binding peptide with improved stability. Org Biomol Chem. 2012 May 11. [Epub ahead of print]

    Institute for Molecular Bioscience, The University of Queensland, St Lucia 4072, Queensland, Australia. p.alewood@imb.uq.edu.au.

    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 Chi-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.

    Unbinding of conotoxin ImI

    Yu R, Kaas Q, Craik DJ (2012). Delineation of the Unbinding Pathway of alpha-Conotoxin ImI from the alpha7 Nicotinic Acetylcholine Receptor. J Phys Chem B. 2012 May 31;116(21):6097-105. Epub 2012 May 18.

    Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland , Brisbane, Queensland 4072, Australia.

    Abstract: alpha-Conotoxins potently and specifically inhibit isoforms of nicotinic acetylcholine receptors (nAChRs) and are used as molecular probes and as drugs or drug leads. Interactions occurring during binding and unbinding events are linked to binding kinetics, and knowledge of these interactions could help in the development of alpha-conotoxins as drugs. Here, the unbinding process for the prototypical alpha-conotoxin ImI/alpha7-nAChR system was investigated theoretically, and three exit routes were identified using random accelerated molecular dynamics simulations. The route involving the smallest conformation perturbation was further divided into three subpathways, which were studied using steered molecular dynamics simulations. Of the three subpathways, two had better experimental support and lower potential of mean force, indicating that they might be sampled more frequently. Additionally, these subpathways were supported by previous experimental studies. Several pairwise interactions, including a cation-Pi interaction and charge and hydrogen bond interactions, were identified as potentially playing important roles in the unbinding event.

    Venomics applied to conopeptide discovery

    Prashanth JR, Lewis RJ, Dutertre S. (2012) Towards an integrated venomics approach for accelerated conopeptide discovery. Toxicon. 2012 Apr 28. [Epub ahead of print]

    The Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia.

    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.

    mu-conopeptide CnIIIC

    Markgraf R, Leipold E, Schirmeyer J, Paolini-Bertrand M, Hartley O, Heinemann SH.(2012) Mechanism and molecular basis for the sodium channel subtype specificity of µ-conopeptide CnIIIC.Br J Pharmacol. 2012 Apr 27. doi: 10.1111/j.1476-5381.2012.02004.x. [Epub ahead of print]

    Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University of Jena & Jena University Hospital, Hans-Knoell-Str. 2, D-07745 Jena, Germany, Department of Structural Biology and Bioinformatics, University of Geneva, 1 Rue Michel Servet, CH-1211, Geneva, Switzerland.

    Abstract: BACKGROUND AND PURPOSE: Voltage-gated sodium channels (Na(V) channels) are key players in the generation and propagation of action potentials, and selective blockade of these channels is a promising strategy for clinically useful suppression of electrical activity. The conotoxin µ-CnIIIC from the cone snail Conus consors exhibits myorelaxing activity in rodents through specific blockade of skeletal muscle (Na(V) 1.4) Na(V) channels (Favreau et al., 2012). EXPERIMENTAL APPROACH: Here we investigate the activity of µ-CnIIIC towards human Na(V) channels and characterize its inhibitory mechanism as well as the molecular basis for its channel specificity. KEY RESULTS: Like for rat paralogs, human Na(V) 1.4 and Na(V) 1.2 were potently blocked by µ-CnIIIC, the sensitivity of Na(V) 1.7 was intermediate, and Na(V) 1.5 and Na(V) 1.8 were insensitive. Half-channel chimeras revealed that determinants for the insensitivity of Na(V) 1.8 must reside in both the first and second halves of the channel, while those for Na(V) 1.5 are restricted to domains I and II. Furthermore, domain I pore loop affects the total block and therefore harbors the major determinants for the subtype specificity. Domain II pore loop only affects the kinetics of toxin binding and dissociation. Blockade by µ-CnIIIC of Na(V) 1.4 is virtually irreversible but leaves a residual current of about 5%, reflecting a "leaky" block: while Na(+) ions can still pass through µ-CnIIIC-occupied Na(V) 1.4 to some extent, TTX is excluded from its binding site, but can be trapped inside the pore by µ-CnIIIC. CONCLUSIONS AND IMPLICATIONS: Of clinical significance, µ-CnIIIC is a potent and persistent blocker of human skeletal muscle Na(V) 1.4 that does not affect activity of cardiac Na(V) 1.5.


    24 April 2012

    Toxins as weapons

    Anderson PD. Bioterrorism: toxins as weapons (2012). J Pharm Pract. 25(2):121-9. PubMed PMID: 22523138

    Forensic Pharmacologist, Private Practice, Adjunct Associate Professor of Pharmacy Practice, University of Rhode Island, Randolph, MA, USA.

    Abstract: The potential for biological weapons to be used in terrorism is a real possibility. Biological weapons include infectious agents and toxins. Toxins are poisons produced by living organisms. Toxins relevant to bioterrorism include ricin, botulinum, Clostridium perfrigens epsilson toxin, conotoxins, shigatoxins, saxitoxins, tetrodotoxins, mycotoxins, and nicotine. Toxins have properties of biological and chemical weapons. Unlike pathogens, toxins do not produce an infection. Ricin causes multiorgan toxicity by blocking protein synthesis. Botulinum blocks acetylcholine in the peripheral nervous system leading to muscle paralysis. Epsilon toxin damages cell membranes. Conotoxins block potassium and sodium channels in neurons. Shigatoxins inhibit protein synthesis and induce apoptosis. Saxitoxin and tetrodotoxin inhibit sodium channels in neurons. Mycotoxins include aflatoxins and trichothecenes. Aflatoxins are carcinogens. Trichothecenes inhibit protein and nucleic acid synthesis. Nicotine produces numerous nicotinic effects in the nervous system.

    Dicarba bridge (staple) stabilization of conantokins

    Platt RJ, Han TS, Green BR, Smith MD, Skalicky J, Gruszczynski P, White HS, Olivera B, Bulaj G, Gajewiak J (2012). Stapling Mimics Noncovalent Interactions of gamma-Carboxyglutamates in Conantokins, Peptidic Antagonists of N-Methyl-D-Aspartic Acid (NMDA) Receptors. J Biol Chem. Apr 19. [Epub ahead of print]

    University of Utah, United States;

    Abstract: Conantokins are short peptides derived from the venoms of marine cone snails that act as antagonists of the N-methyl-D-aspartate (NMDA) receptor family of excitatory glutamate receptors. These peptides contain gamma-carboxyglutamic acid residues (Gla) typically spaced at i,i+4 and/or i,i+7 intervals, which by chelating divalent cations, induce and stabilize helical conformation of the peptide. Introduction of a dicarba bridge (or a staple) can covalently stabilize peptide helicity and improve its pharmacological properties. To test the hypothesis that stapling can effectively replace Gla residues in stabilizing the helical conformation of conantokins, we designed, synthesized and characterized several stapled analogs of conantokin G (conG), with varying connectivities in terms of staple length and location along the face of the alpha-helix. NMR studies confirmed that the ring closing metathesis reaction yielded a single product with the Z configuration of the olefinic bond. Based on circular dichroism and molecular modeling, the stapled analogs exhibited significantly enhanced helicity compared to the native peptide in a metal-free environment. Stapling i,i+4 was benign with respect to effects on in vitro and in vivo pharmacological properties. One analog, namely conG[11-15,S(i,i+4)S(8)], blocked NR2B-containing NMDA receptors with IC(50) = 0.7 ?M and provided significant protection in the 6 Hz psychomotor model of pharmaco-resistant epilepsy in mice. Remarkably, unlike native conG, conG[11-15,S(i,i+4)S(8)] produced no behavioral motor toxicity. Our results extend the applications of peptide stapling to helical peptides with extracellular targets and provide a means for engineering conantokins with improved pharmacological properties.

    A novel alpha-conotoxin MII sensitive nAChR modulates GABA release

    McClure-Begley T, Wageman C, Grady S, Marks M, McIntosh J, Collins A, Whiteaker P. (2012) A novel alpha-conotoxin MII sensitive nicotinic acetylcholine receptor modulates [(3) H]-GABA release in the superficial layers of the mouse superior colliculus. J Neurochem. 2012 Apr 16. doi: 10.1111/j.1471-4159.2012.07759.x.[Epub ahead of print] PubMed PMID: 22506481.

    Institute for Behavioral Genetics, 447 UCB, University of Colorado, Boulder, CO 80309. Department of Integrative Physiology, 447 UCB, University of Colorado, Boulder, CO 80309. Departments of Psychiatry and Biology, University of Utah, Salt Lake City, UT 84112. Division of Neurobiology, Barrow Neurological Institute, St. Joseph's Hospital, Phoenix, AZ 85013.

    Abstract: Mouse superficial superior colliculus (SuSC) contains dense GABAergic innervation and diverse nicotinic acetylcholine receptor subtypes. Pharmacological and genetic approaches were used to investigate the subunit compositions of nicotinic acetylcholine receptors (nAChR) expressed on mouse SuSC GABAergic terminals. [(125) I]-Epibatidine competition binding studies revealed that the alpha3beta2* and alpha6beta2* nicotinic subtype-selective peptide alpha-conotoxinMII blocked binding to 40 +/- 5% of SuSC nAChRs. Acetylcholine-evoked [(3) H]-GABA release from SuSC crude synaptosomal preparations is calcium dependent, blocked by the voltage-sensitive calcium channel blocker, cadmium, and the nAChR antagonist mecamylamine, but is unaffected by muscarinic, glutamatergic, P2X and 5-HT3 receptor antagonists. Approximately 50% of nAChR-mediated SuSC [(3) H]-GABA release is inhibited by alpha-conotoxinMII. However, the highly-alpha6beta2*-subtype-selective alpha-conotoxin PIA did not affect [(3) H]-GABA release. Nicotinic subunit-null mutant mouse experiments revealed that ACh-stimulated SuSC [(3) H]-GABA release is entirely beta2 subunit-dependent. alpha4 subunit deletion decreased total function by >90%, and eliminated alpha-conotoxin MII-resistant release. ACh-stimulated SuSC [(3) H]-GABA release was unaffected by beta3, alpha5 or alpha6 nicotinic subunit deletions. Together, these data suggest that a significant proportion of mouse SuSC nicotinic agonist-evoked GABA-release is mediated by a novel, alpha-conotoxin MII-sensitive alpha3alpha4beta2 nAChR. The remaining alpha-conotoxin MII-resistant, nAChR agonist-evoked SuSC GABA release appears to be mediated via alpha4beta2* subtype nAChRs. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

    Milked venom from Conus purpurascens - a source of novel conopeptides

    Chun JB, Baker MR, Kim DH, Leroy M, Toribo P, Bingham JP. (2012) Cone snail milked venom dynamics - A quantitative study of Conus purpurascens. Toxicon. Apr 5. [Epub ahead of print] PubMed PMID: 22497788.

    Department of Molecular Biosciences and Bioengineering, College of Tropical Agriculture and Human Resources, University of Hawaii, Honolulu, HI 96822, USA.

    Abstract: Milked venom from cone snails represent a novel biological resource with a proven track record for drug discovery. To strengthen this correlation, we undertook a chromatographic and mass spectrometric study of individual milked venoms from Conus purpurascens. Milked venoms demonstrate extensive peptide differentiation amongst individual specimens and during captivity. Individual snails were found to lack a consistent set of described conopeptides, but instead demonstrated the ability to change venom expression, composition and post-translational modification incorporation; all variations contribute to an increase in chemical diversity and prey targeting strategies. Quantitative amino acid analysis revealed that milked venom peptides are expressed at ranges up to 3.51-121.01 uM within single milked venom samples. This provides for a 6.37-20,965 fold-excess of toxin to induce apparent IC(50) for individual conopeptides identified in this study. Comparative molecular mass analysis of duct venom, milked venom and radula tooth extracts from single C. purpurascens specimens demonstrated a level of peptide continuity. Numerous highly abundant and unique conopeptides remain to be characterized. This study strengthens the notion that approaches in conopeptide drug lead discovery programs will potentially benefit from a greater understanding of the toxinological nature of the milked venoms of Conus. Copyright © 2012 Elsevier Ltd. All rights reserved. PMID: 22497788 [PubMed - as supplied by publisher]

    Folding structures of kappa-conotoxin RIIJ

    Yan S, Wu G. (2012) Detailed Folding Structures of Kappa-conotoxin RIIIJ and Its Mutageneses Obtained from 2-Dimensional HP Model. Protein Pept Lett. 19:567-572. PubMed PMID: 22486650.

    State Key Laboratory of Nonfood Biomass Enzyme Technology, National Engineering Research Center for Non-food Biorefinery, Guangxi Key Laboratory of Biorefinery, Guangxi Academy of Sciences, 98 Daling Road, Nanning, Guangxi, 530007, China. hongguanglishibahao@yahoo.com.

    Abstract: Kappa-conotoxin RIIIJ is a conopeptide to inhibit voltage-gated potassium channels, however, its detailed folding structures have yet to be studied. With the advance in computing power, it is possible to use the HP model to analyze all its possible folding structures. In this study, the amino acid sequences of kappa-conotoxin RIIIJ and its four mutageneses were converted into ten HP sequences according to the normalized hydrophobicity index. All 282 429 536 481 possible folding structures in each HP sequence were found using the 2-dimensional HP model, and the detailed folding structures at native state were studied. The results showed that kappa-conotoxin RIIIJ had 180 and 90 folding structures at their native state with minimal energy of -9 and -10 at pH 2 and pH 7; its mutagenesis (6-8) TPP - > SLN increased the numbers of the folding structures to 456 and 564 at pH 2 and pH 7; whereas its mutageneses (6-11) TPPKKH - > SLNLRL, (9- 11) KKH - > LRL, and (10-11) KH - > RL decreased the numbers of the folding structures to 60, 30 and 90 at both pH levels, respectively. Thereafter, the normalized hydrophobicity index was employed to distinguish those native states, and attempts were made to explain the effect of mutageneses on potassium channels in terms of the number of folding structures and numerical native states. PMID: 22486650 [PubMed - in process]

    Modelling electrostatic interactions in solvated alpha-conotoxins.

    Jiang N, Ma J. (2012) Multi-layer coarse-graining polarization model for treating electrostatic interactions of solvated ?-conotoxin peptides. J Chem Phys. 2012 Apr 7;136(13):134105. PubMed PMID: 22482538.

    School of Chemistry and Chemical Engineering, Institute of Theoretical and Computational Chemistry, Key Laboratory of Mesoscopic Chemistry of MOE, Nanjing University, Nanjing 210093, People's Republic of China.

    Abstract: A multi-layer coarse-graining (CG) model is presented for treating the electrostatic interactions of solvated alpha-conotoxin peptides. According to the sensitivity to the electrostatic environment, a hybrid set of electrostatic parameters, such as secondary-structure- and residue-based dipoles, and atom-centered partial charges, are adopted. For the polarization "inert" secondary-structures and residues, the fragment dipole moments are distributed within narrow ranges with the magnitude close to zero. The coarse-graining fragment dipoles are parameterized from a large training set (10 000 configurations) to reproduce the electrostatic features of molecular fragments. In contrast, the electrostatically "sensitive" atoms exhibit large fluctuations of charges with the varied environments. The environment-dependent variable charges are updated in each energetic calculation. The electrostatic interaction of the whole chemical system is hence partitioned into several sub-terms coming from the fragment dipole-dipole, (fragment) dipole-(atom) charge, and atom charge-charge interactions. A large number of test calculations on the relative energies of cyclo-peptide conformers have demonstrated that the multi-layer CG electrostatic model presents better performance than the non-polarized force fields, in comparison with the density-functional theory and the fully polarized force field model. The selection of CG fragment centers, mass or geometric center, has little influence on the fragment-based dipole-dipole interactions. The multi-layer partition of electrostatic polarization is expected to be applied to many biologically interesting and complicated phenomena. PMID: 22482538 [PubMed - in process]

    2 April 2012

    Genome mining reveals novel peptides in social ants

    Gruber CW, Muttenthaler M. (2012) Discovery of defense- and neuropeptides in social ants by genome-mining. PLoS One. 2012;7(3):e32559. Epub 2012 Mar 20. PubMed PMID: 22448224; PubMed Central PMCID: PMC3308954.

    Medical University of Vienna, Center for Physiology and Pharmacology, Vienna, Austria.

    Abstract: Natural peptides of great number and diversity occur in all organisms, but analyzing their peptidome is often difficult. With natural product drug discovery in mind, we devised a genome-mining approach to identify defense- and neuropeptides in the genomes of social ants from Atta cephalotes (leaf-cutter ant), Camponotus floridanus (carpenter ant) and Harpegnathos saltator (basal genus). Numerous peptide-encoding genes of defense peptides, in particular defensins, and neuropeptides or regulatory peptide hormones, such as allatostatins and tachykinins, were identified and analyzed. Most interestingly we annotated genes that encode oxytocin/vasopressin-related peptides (inotocins) and their putative receptors. This is the first piece of evidence for the existence of this nonapeptide hormone system in ants (Formicidae) and supports recent findings in Tribolium castaneum (red flour beetle) and Nasonia vitripennis (parasitoid wasp), and therefore its confinement to some basal holometabolous insects. By contrast, the absence of the inotocin hormone system in Apis mellifera (honeybee), another closely-related member of the eusocial Hymenoptera clade, establishes the basis for future studies on the molecular evolution and physiological function of oxytocin/vasopressin-related peptides (vasotocin nonapeptide family) and their receptors in social insects. Particularly the identification of ant inotocin and defensin peptide sequences will provide a basis for future pharmacological characterization in the quest for potent and selective lead compounds of therapeutic value.

    That this can yield therapeutic drug leads was shown with conotoxin Vc1.1, which was identified via DNA sequencing, where surprisingly only the non-modified sequence and not the native, expressed and post-translational modified sequences was active in chronic pain models, .

    See also: Gruber CW, Muttenthaler M, Freissmuth M. (2010) Ligand-based peptide design and combinatorial peptide libraries to target G protein-coupled receptors. Curr Pharm Des. 2010;16(28):3071-88. Review. PubMed PMID: 20687879.

    Conkunitzin-S1 (Conk-S1): selectively blocks K(v) 1.7 and increases glucose-stimulated insulin secretion

    Finol-Urdaneta RK, Remedi MS, Raasch W, Becker S, Clark RB, Strüver N, Pavlov E, Nichols CG, French RJ, Terlau H.(2012) Block of K(v) 1.7 potassium currents increases glucose-stimulated insulin secretion. EMBO Mol Med. 2012 Mar 21. doi: 10.1002/emmm.201200218. [Epub ahead of print] PubMed PMID: 22438204.

    Faculty of Medicine, Department of Physiology and Pharmacology, and HBI, University of Calgary, Calgary, AB, Canada; Max-Planck-Institute for Experimental Medicine, Göttingen, Germany.

    Abstract: Glucose-stimulated insulin secretion (GSIS) relies on repetitive, electrical spiking activity of the beta cell membrane. Cyclic activation of voltage-gated potassium channels (K(v) ) generates an outward, 'delayed rectifier' potassium current, which drives the repolarizing phase of each spike and modulates insulin release. Although several K(v) channels are expressed in pancreatic islets, their individual contributions to GSIS remain incompletely understood. We take advantage of a naturally occurring cone-snail peptide toxin, Conkunitzin-S1 (Conk-S1), which selectively blocks K(v) 1.7 channels to provide an intrinsically limited, finely graded control of total beta cell delayed rectifier current and hence of GSIS. Conk-S1 increases GSIS in isolated rat islets, likely by reducing K(v) 1.7-mediated delayed rectifier currents in beta cells, which yields increases in action potential firing and cytoplasmic free calcium. In rats, Conk-S1 increases glucose-dependent insulin secretion without decreasing basal glucose. Thus, we conclude that K(v) 1.7 contributes to the membrane-repolarizing current of beta cells during GSIS and that block of this specific component of beta cell K(v) current offers a potential strategy for enhancing GSIS with minimal risk of hypoglycaemia during metabolic disorders such as Type 2 diabetes.

    Antiallodynic effect of gabapentin

    Morimoto SI, Ito M, Oda S, Sugiyama A, Kuroda M, Adachi-Akahane S.(2012) Spinal Mechanism Underlying the Antiallodynic Effect of Gabapentin Studied in the Mouse Spinal Nerve Ligation Model. J Pharmacol Sci. 2012 Mar 14. [Epub ahead of print] PubMed PMID: 22447299.

    Department of Pharmacology, School of Medicine, Faculty of Medicine, Toho University, Japan.

    Abstract: We studied the antiallodynic effect of gabapentin (GBP) in the mouse model of neuropathic pain, aiming at clarifying the underlying mechanism. The L5 spinal nerve ligation induced tactile allodynia, an increase of CD11b expression, and an increase in the protein expression level of the voltage-dependent Ca(2+) channel alpha(2)/delta-1 subunit in the spinal dorsal horn on the injured side. The chronic intrathecal administration of GBP (100 µg/body per day) as well as omega-conotoxin MVIIA, an N-type Ca(2+)-channel blocker, completely suppressed allodynia, but did not attenuate the CD11b expression. The antiallodynic effect of GBP lasted for several days after the termination of the drug, while that of omega-conotoxin MVIIA disappeared immediately after the termination. GBP suppressed the elevation of the protein level of the alpha(2)/delta-1 subunit in the spinal dorsal horn, although it did not affect its mRNA level in the L5 DRG. These results suggest that GBP inhibits the development of allodynia by suppressing the up-regulation of N-type Ca(2+) channels, through normalization of the protein level of the alpha(2)/delta-1 subunit at the primary afferent nerve terminal via the inhibition of its anterograde transport. In addition, we propose that the nerve injury enhances the expression level of alpha(2)/delta-1 in the downstream of the activation of microglia.

    Peptide rigidity does not correlate with desorption/ionization efficiency in MALDI-MS

    Shigeri Y, Inazumi S, Hagihara Y, Yasuda A, Kawasaki H, Arakawa R, Nakata M(2012). Desorption/Ionization efficiency of peptides containing disulfide bonds in matrix-assisted laser desorption/ionization mass spectrometry. Anal Sci. 2012;28(3):295. PubMed PMID: 22451371.

    Health Research Institute, National Institute of Advanced Industrial Science and Technology.

    Abstract: In order to elucidate the role of desorption/ionization efficiency of peptides in MALDI-MS, we focused on peptides with disulfide bonds, which form a rigid tertiary structure. We synthesized seven sets of peptides with one disulfide bond (oxytocin, somatostatin, [Arg(8)]-vasopressin, [Arg(8)]-vasotocin, cortistatin, melanin-concentrating hormone, urotensin II-related peptide) and five sets of peptides with two disulfide bonds (tertiapin, alpha-conotoxin GI, alpha-conotoxin ImI, alpha-conotoxin MI and alpha-conotoxin SI). Each peptide set consisted of three peptides: the oxidized form (S-S type), the reduced form (SH type), and an internal standard peptide in which all cysteine residues were substituted with alanine residues. In the case of urotensin II-related peptide, tertiapin, alpha-conotoxin ImI and alpha-conotoxin MI, the reduced form showed higher desorption/ionization efficiency than the oxidized form. In contrast, the other peptides revealed higher desorption/ionization efficiency in the oxidized form relative to the reduced form. These results imply that a rigid structure of peptides formed by disulfide bonds does not correlate with desorption/ionization efficiency in MALDI-MS.

    Nociceptor-specific ligand targeting: MrVIB has a binding preference for Na(v) 1.8

    Knapp O, Nevin ST, Yasuda T, Lawrence N, Lewis RJ, Adams DJ.(2012) Biophysical properties of Na(v) 1.8/Na(v) 1.2 chimeras and inhibition by µO-conotoxin MrVIB. Br J Pharmacol. 2012 Mar 27. doi: 10.1111/j.1476-5381.2012.01955.x. [Epub ahead of print] PubMed PMID: 22452751.

    Health Innovations Research Institute, RMIT University, Melbourne, Victoria 3083, Australia Institute for Molecular Bioscience Queensland Brain Institute, University of Queensland, Brisbane, Queensland 4072, Australia.

    Abstract: Background and purpose: Voltage-gated sodium channels are expressed primarily in excitable cells and play a pivotal role in the initiation and propagation of action potentials. Nine subtypes of the pore-forming ?-subunit have been identified, each with a distinct tissue distribution, biophysical properties and sensitivity to tetrodotoxin (TTX). Na(v) 1.8, a TTX-resistant (TTX-R) subtype, is selectively expressed in sensory neurons and plays a pathophysiological role in neuropathic pain. In comparison with TTX-sensitive (TTX-S) Na(v) alpha-subtypes in neurons, Na(v) 1.8 is most strongly inhibited by the muO-conotoxin MrVIB from Conus marmoreus. To determine which domain confers Na(v) 1.8 alpha-subunit its biophysical properties and MrVIB binding, we constructed various chimeric channels incorporating sequence from Na(v) 1.8 and the TTX-S Na(v) 1.2, using a domain exchange strategy. Experimental approach: Wild-type and chimeric Na(v) channels were expressed in Xenopus oocytes and depolarization-activated Na(+) currents were recorded using the two-electrode voltage clamp technique. Key results: MrVIB (1 uM) reduced Na(v) 1.2 current amplitude to 69 ± 12 % whereas Na(v) 1.8 current was reduced to 31 ± 3%, confirming that MrVIB has a binding preference for Na(v) 1.8. A similar reduction in Na(+) current amplitude was observed when MrVIB was applied to chimeras containing the region extending from S6 segment of domain I through the S5-S6 linker of domain II of Na(v) 1.8. In contrast, MrVIB had only a small effect on Na(+) current for chimeras containing the corresponding region of Na(v) 1.2. Conclusions and implications: Taken together, these results suggest that domain II of Na(v) 1.8 is an important determinant of MrVIB affinity, highlighting a region of the ?-subunit that may allow further nociceptor-specific ligand targeting.

    Reagentless Oxidative Folding of Disulfide-Rich Peptides

    Steiner AM, Woycechowsky KJ, Olivera BM, Bulaj G. (2012) Reagentless Oxidative Folding of Disulfide-Rich Peptides Catalyzed by an Intramolecular Diselenide. Angew Chem Int Ed Engl. 2012 Mar 27. doi: 10.1002/anie.201200062. [Epub ahead of print] PubMed PMID: 22454362.

    Department of Medicinal Chemistry, University of Utah, 421 Wakara Way, Suite 360, Salt Lake City, UT 84108 (USA).

    Abstract: Building bridges: In cysteine-rich peptides, diselenides can be used as a proxy for disulfide bridges as the energetic preference for Se=Se bonds over mixed Se=S bonds simplifies folding. An intramolecular diselenide bond efficiently catalyzes the oxidative folding of selenopeptide analogues of conotoxins, and serves as a reagentless method to accelerate formation of various native disulfide bridging patterns.

    22 March 2012

    Analgesic conotoxins - Review

    Adams DJ, Callaghan B, Berecki G. (2012) Analgesic Conotoxins: Block and G Protein-Coupled Receptor Modulation of N-Type (Ca(V) 2.2) Calcium Channels. Br J Pharmacol. 2011 Nov 18. doi: 10.1111/j.1476-5381.2011.01781.x. [Epub ahead of print]

    Health Innovations Research Institute, RMIT University, Melbourne, Victoria 3083 Australia.

    Abstract: Conotoxins (conopeptides) are small disulfide bonded peptides from the venom of marine cone snails. These peptides target a wide variety of membrane receptors, ion channels and transporters, and have enormous potential for a range of pharmaceutical applications. Structurally related omega-conotoxins bind directly to and selectively inhibit neuronal (N)- type voltage-gated calcium channels (VGCCs) of nociceptive primary afferent neurones. Among these, omega-conotoxin MVIIA (Prialt) is approved by the FDA as an alternative intrathecal analgesic for the management of chronic intractable pain, particularly in patients refractory to opioids. A series of newly discovered omega-conotoxins from Conus catus, including CVID-F, are potent and selective antagonists of N-type VGCCs. In spinal cord slices, these peptides reversibly inhibit excitatory synaptic transmission between primary afferents and dorsal horn superficial lamina neurones and in the rat partial sciatic nerve ligation model of neuropathic pain, significantly reduce allodynic behaviour. Another family of conotoxins, the alpha-conotoxins, are competitive antagonists of mammalian nicotinic acetylcholine receptors (nAChRs). alpha-Conotoxins Vc1.1 and RgIA possess two disulfide bonds and are currently in development as a treatment for neuropathic pain. It was initially proposed that the primary target of these peptides is the alpha9alpha10 neuronal nAChR. Surprisingly, however, alpha-conotoxins Vc1.1, RgIA and PeIA more potently inhibit N-type VGCC currents via a GABA(B) G protein-coupled receptor mechanism in rat sensory neurones. This inhibition is largely voltage-independent and involves complex intracellular signalling. Understanding the molecular mechanisms of conotoxin action will lead to new ways to regulate VGCC block and modulation in normal and diseased states of the nervous system.

    18 March 2012

    chi-conopeptides as analgesics - Review

    Lewis RJ. (2012) Discovery and development of the chi-conopeptide class of analgesic peptides. Toxicon.59: 524-528.

    Institute for Molecular Bioscience, The University of Queensland, Australia.

    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 chi-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 chi-MrIA revealed this class of peptides had a unusual cysteine-stabilised scaffold that presented a gamma-turn in an optimised conformation for high affinity interactions with NET. chi-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 for chi-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.

    Violette A, Leonardi A, Piquemal D, Terrat Y, Biass D, Dutertre S, Noguier F, Ducancel F, Stöcklin R, Križaj I, Favreau P. (2012) Recruitment of glycosyl hydrolase proteins in a cone snail venomous arsenal: further insights into biomolecular features of conus venoms. Mar Drugs. 10: 258-280.

    Atheris Laboratories, Case postale 314, CH-1233 Bernex-Geneva, Switzerland; Email: daniel.biass@atheris.ch (D.B.); reto.stocklin@atheris.ch (R.S.); philippe.favreau@atheris.ch (P.F.).

    Abstract: Cone snail venoms are considered an untapped reservoir of extremely diverse peptides, named conopeptides, displaying a wide array of pharmacological activities. We report here for the first time, the presence of high molecular weight compounds that participate in the envenomation cocktail used by these marine snails. Using a combination of proteomic and transcriptomic approaches, we identified glycosyl hydrolase proteins, of the hyaluronidase type (Hyal), from the dissected and injectable venoms ("injectable venom" stands for the venom variety obtained by milking of the snails. This is in contrast to the "dissected venom", which was obtained from dissected snails by extraction of the venom glands) of a fish-hunting cone snail, Conus consors (Pionoconus clade). The major Hyal isoform, Conohyal-Cn1, is expressed as a mixture of numerous glycosylated proteins in the 50 kDa molecular mass range, as observed in 2D gel and mass spectrometry analyses. Further proteomic analysis and venom duct mRNA sequencing allowed full sequence determination. Additionally, unambiguous segment location of at least three glycosylation sites could be determined, with glycans corresponding to multiple hexose (Hex) and N-acetylhexosamine (HexNAc) moieties.
    With respect to other known Hyals, Conohyal-Cn1 clearly belongs to the hydrolase-type of Hyals, with strictly conserved consensus catalytic donor and positioning residues. Potent biological activity of the native Conohyals could be confirmed in degrading hyaluronic acid. A similar Hyal sequence was also found in the venom duct transcriptome of C. adamsonii (Textilia clade), implying a possible widespread recruitment of this enzyme family in fish-hunting cone snail venoms. These results provide the first detailed Hyal sequence characterized from a cone snail venom, and to a larger extent in the Mollusca phylum, thus extending our knowledge on this protein family and its evolutionary selection in marine snail venoms.

    Vetter I, Mozar CA, Durek T, Wingerd JS, Alewood PF, Christie MJ, Lewis RJ (2012). Characterisation of Na(v) types endogenously expressed in human SH-SY5Y neuroblastoma cells. Biochem Pharmacol. 2012 Mar 3. [Epub ahead of print] PubMed PMID: 22410003.

    Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia.

    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) ? and ? 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 N (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. ?-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.

    Pohanka M. (2012) Alpha7 nicotinic acetylcholine receptor is a target in pharmacology and toxicology. Int J Mol Sci. 13: 2219-2238.

    Faculty of Military Health Sciences, University of Defence, Trebesska 1575, 50001 Hradec Kralove, Czech Republic; E-Mail: miroslav.pohanka@gmail.com ; Tel.: +420-973253091

    Abstract: Alpha7 nicotinic acetylcholine receptor (?7 nAChR) is an important part of the cholinergic nerve system in the brain. Moreover, it is associated with a cholinergic anti-inflammatory pathway in the termination of the parasympathetic nervous system. Antagonists of ?7 nAChR are a wide group represented by conotoxin and bungarotoxin. Even Alzheimer's disease drug memantine acting as an antagonist in its side pathway belongs in this group. Agonists of ?7 nAChR are suitable for treatment of multiple cognitive dysfunctions such as Alzheimer's disease or schizophrenia. Inflammation or even sepsis can be ameliorated by the agonistic acting compounds. Preparations RG3487, SEN34625/WYE-103914, SEN12333, ABT-107, Clozapine, GTS-21, CNI-1493, and AR-R17779 are representative examples of the novel compounds with affinity toward the ?7 nAChR. Pharmacological, toxicological, and medicinal significance of ?7 nAChR are discussed throughout this paper.

    Lewis RJ, Dutertre S, Vetter I, Christie MJ. (2012) Conus Venom Peptide Pharmacology. Pharmacol Rev. 64: 259-298.

    Institute for Molecular Bioscience, the University of Queensland, Q4072, Australia. r.lewis@imb.uq.edu.au.

    Abstract: Conopeptides are a diverse 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 diverse 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 diversity, pharmacology, structure-activity relationships, and therapeutic potential of cone snail venom peptide families acting at voltage-gated ion channels (omega-, mu-, muO-, delta-, tor, iota-, and kappa-conotoxins), ligand-gated ion channels (alpha-conotoxins, sigma-conotoxin, ikot-ikot, and conantokins), G-protein-coupled receptors (rho-conopeptides, conopressins, and contulakins), and neurotransmitter transporters (chi-conopeptides), with expanded discussion on the clinical potential of sodium and calcium channel inhibitors and alpha-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.

    Tietze AA, Tietze D, Ohlenschläger O, Leipold E, Ullrich F, Kühl T, Mischo A, Buntkowsky G, Görlach M, Heinemann SH, Imhof D (2012). Structurally Diverse ?-Conotoxin PIIIA Isomers Block Sodium Channel Na(V) 1.4. Angew Chem Int Ed Engl. 2012 Mar12. doi: 10.1002/anie.201107011. [Epub ahead of print] PubMed PMID: 22407516.

    Pharmaceutical Chemistry I, Institute of Pharmacy, University of Bonn, Brühler Strasse 7, 53119 Bonn (Germany).

    Abstract: The one and only fold? Three chemically synthesized ?-conotoxin PIIIA isomers, which contain different disulfide connectivity, block the skeletal muscle voltage-gated sodium channel Na(V) 1.4 with similar, yet distinguishable potency. Hence, bioactivity of this ?-conotoxin is not strictly coupled to its native fold. Future development of conotoxin-derived analgesics may benefit from such a widened structural repertoire.

    Lin ZY, Chen LM, Zhang J, Pan XD, Zhu YG, Ye QY, Huang HP, Chen XC.(2012) Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels in cultured rat hippocampal neurons. Acta Pharmacol Sin. 2012 Mar 12. doi: 10.1038/aps.2011.181. [Epub ahead of print] PubMed PMID: 22407229.

    [1] Fujian Institute of Geriatri cs, the Affiliated Union Hospital of Fujian Medical University, Fuzhou 350001, China [2] These authors contributed equally to this paper.

    Abstract: Aim:To investigate the effect of ginsenoside Rb1 on voltage-gated calcium currents in cultured rat hippocampal neurons and the modulatory mechanism.Methods:Cultured hippocampal neurons were prepared from Sprague Dawley rat embryos. Whole-cell configuration of the patch-clamp technique was used to record the voltage-gated calcium currents (VGCCs) from the hippocampal neurons,and the effect of Rb1 was examined.Results:Rb1 (2-100 ?mol/L) inhibited VGCCs in a concentration-dependent manner, and the current was mostly recovered upon wash-out. The specific L-type Ca(2+) channel inhibitor nifedipine (10 ?mol/L) occluded Rb1-induced inhibition on VGCCs. Neither the selective N-type Ca(2+) channel blocker ?-conotoxin-GVIA (1 ?mol/L), nor the selective P/Q-type Ca(2+) channel blocker ?-agatoxin IVA (30 nmol/L) diminished Rb1-sensitive VGCCs. Rb1 induced a leftward shift of the steady-state inactivation curve of I(Ca) to a negative potential without affecting its activation kinetics or reversal potential in the I-V curve. The inhibitory effect of Rb1 was neither abolished by the adenylyl cyclase activator forskolin (10 ?mol/L), nor by the PKA inhibitor H-89 (10 ?mol/L).Conclusion:Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels, without affecting the N-type or P/Q-type Ca(2+) channels in hippocampal neurons. cAMP-PKA signaling pathway is notinvolved in this effect.

    Ye M, Khoo KK, Xu S, Zhou M, Boonyalai N, Perugini MA, Shao X, Chi C, Galea CA, Wang C, Norton RS (2012). A helical conotoxin from Conus imperialis has a novel cysteine framework and defiines a new superfamily. J Biol Chem. 2012 Mar 7. [Epub ahead of print] PubMed PMID: 22399292.

    Institute of Protein Research, Tongji University, China;

    Abstract: Cone snail venoms are a rich source of peptides, many of which are potent and selective modulators of ion channels and receptors. Here we report the isolation and characterization of two novel conotoxins from the venom of Conus imperialis. These two toxins contain a novel cysteine framework, C-C-C-CC-C, which has not been found in other conotoxins described to date. We name it framework XXIII and designate the two toxins im23a and im23b, respectively; cDNAs of these toxins exhibit a novel signal peptide sequence, which defines a new K-superfamily The disulfide connectivity of im23a has been mapped by chemical mapping of partially reduced intermediates and by NMR structure calculations, both of which establish a I-II, III-IV, V-VI pattern of disulfide bridges. This pattern was also confirmed by synthesis of im23a with orthogonal protection of individual cysteine residues. The solution structure of im23a reveals that im23a adopts a novel helical hairpin fold. A cluster of acidic residues on the surface of the molecule is able to bind calcium. The biological activity of the native and recombinant peptides was tested by injection into mice intracranially and intravenously to assess the effects on the central and peripheral nervous systems, respectively. Intracranial injection of im23a or im23b into mice induced excitatory symptoms; however, the biological target of these new toxins has yet to be identified.

    27 February 2012

    Conotoxins as anti-cancer agents - Review

    Lahiry A, Dave K. (2012) Conotoxins: Review and Docking Studies to determine potentials of Conotoxin as an Anticancer Drug Molecule. Curr Top Med Chem. Feb 14. [Epub ahead of print] PubMed PMID: 22352912.

    G.H. Patel P.G.Dept of Computer Science and Technology. Sardar Patel University, Vallabh Vidyanagar; Gujarat; India; kirtandave11@gmail.com.

    Abstract: It is known that potassium channels are important for cell proliferation. HERG, a potassium channel protein, is a transmembrane protein, which increases in concentration on the cell surface of cancer cells. Apart from cancer cells, this protein is found only in the brain & heart tissue, in very low number. The proliferation of cells in cancer is dependent on activation of this protein, and it has been noted that blocking of this protein with drug molecule, helps inhibit the proliferation of the cells further. The current work aims to study the binding potentials of kappa-PVIIA, conotoxin isolated from Conus purpurascens venom with HERG K+ channel of tumor cells, where HERG mutation has been noted. The toxin under consideration i.e. kappa-conotoxins-PVIIA (kappa-PVIIA) is a 27 residue peptide. The docking studies suggest that the conotoxin binds stably to the HERG protein. The study shows that the peptide interacts with the charged extracellular unit of the HERG protein, i.e. the extracellular portion of the S5 domain named S5-P extracellular linker. Study of binding of toxins of similar origin, with normal potassium channels has been studied in silico. Further, wet laboratory work needs to be conducted for development of a drug molecule from this toxin, to treat some number of cancers.

    Gene duplication facilitates rapid evolution of conotoxin A-superfamily genes

    Chang D, Duda TF Jr. (2012) Extensive And Continuous Duplication Facilitates Rapid Evolution And Diversification Of Gene Families. Mol Biol Evol. Feb 15. [Epub ahead of print] PubMed PMID: 22337864.

    Department of Ecology and Evolutionary Biology and Museum of Zoology, University of Michigan, Ann Arbor, Michigan, United States of America, 48109.

    Abstract: The origin of novel gene functions through gene duplication, mutation and natural selection represents one of the mechanisms by which organisms diversify and one of the possible paths leading to adaptation. Nonetheless, the extent, role and consequences of duplications in the origins of ecological adaptations, especially in the context of species interactions, remain unclear. To explore the evolution of a gene family that is likely linked to species associations, we investigated the evolutionary history of the A-superfamily of conotoxin genes of predatory marine cone snails (Conus species). Members of this gene family are expressed in the venoms of Conus species and are presumably involved in predator-prey associations because of their utility in prey capture. We recovered sequences of this gene family from genomic DNA of four closely related species of Conus and reconstructed the evolutionary history of these genes. Our study is the first to directly recover conotoxin genes from Conus genomes to investigate the evolution of conotoxin gene families. Our results revealed a phenomenon of rapid and continuous gene turnover that is coupled with heightened rates of evolution. This continuous duplication pattern has not been observed previously and the rate of gene turnover is at least two times higher than estimates from other multi-gene families. Conotoxin genes are among the most rapidly evolving protein-coding genes in metazoans, a phenomenon that may be facilitated by extensive gene duplications and have driven changes in conotoxin functions through neofunctionalization. Together these mechanisms led to dramatically divergent arrangements of A-superfamily conotoxin genes among closely related species of Conus. Our findings suggest that extensive and continuous gene duplication facilitates rapid evolution and drastic divergence in venom compositions among species, processes that may be associated with evolutionary responses to predator-prey interactions.

    Novel venom peptides from Conus pulicarius

    Lluisma AO, Milash BA, Moore B, Olivera BM, Bandyopadhyay PK. (2012) Novel venom peptides from the cone snail Conus pulicarius discovered through next-generation sequencing of its venom duct transcriptome. Mar Genomics. Mar;5:43-51. Epub 2012 Jan 9. PubMed PMID: 22325721.

    Department of Biology, University of Utah, Salt Lake City, UT, USA; Marine Science Institute, University of the Philippines, Quezon City, Philippines.

    Abstract: The venom peptides (i.e., conotoxins or conopeptides) that species in the genus Conus collectively produce are remarkably diverse, estimated to be around 50,000 to 140,000, but the pace of discovery and characterization of these peptides have been rather slow. To date, only a minor fraction have been identified and studied. However, the advent of next-generation DNA sequencing technologies has opened up opportunities for expediting the exploration of this diversity. The whole transcriptome of a venom duct from the vermivorous marine snail C. pulicarius was sequenced using the 454 sequencing platform. Analysis of the data set resulted in the identification of over eighty unique putative conopeptide sequences, the highest number discovered so far from a Conus venom duct transcriptome. More importantly, majority of the sequences were potentially novel, many with unexpected structural features, hinting at the vastness of the diversity of Conus venom peptides that remains to be explored. The sequences represented at least 14 major superfamilies/types (disulfide- and non-disulfide-rich), indicating the structural and functional diversity of conotoxins in the venom of C. pulicarius. In addition, the contryphans were surprisingly more diverse than what is currently known. Comparative analysis of the O-superfamily sequences also revealed insights into the complexity of the processes that drive the evolution and diversification of conotoxins.

    mu-Conotoxin PIIIA blocks bacterial Na(V)Ab sodium channel

    Chen R, Chung SH. (2012) Binding Modes of ?-Conotoxin to the Bacterial Sodium Channel (Na(V)Ab). Biophys J. Feb 8;102(3):483-8. Epub 2012 Feb 7. PubMed PMID: 22325270; PubMed Central PMCID: PMC3274799.

    Computational Biophysics Group, Research School of Biology, Australian National University, Canberra, Australia.

    Abstract: Polypeptide toxins isolated from the venom of cone snails, known as mu-conotoxins, block voltage-gated sodium channels by physically occluding the ion-conducting pathway. Using molecular dynamics, we show that one subtype of mu-conotoxins, PIIIA, effectively blocks the bacterial voltage-gated sodium channel Na(V)Ab, whose crystal structure has recently been elucidated. The spherically shaped toxin, carrying a net charge of +6 e with six basic residues protruding from its surface, is attracted by the negatively charged residues on the vestibular wall and the selectivity filter of the channel. The side chain of each of these six arginine and lysine residues can wedge into the selectivity filter, whereas the side chains of other basic residues form electrostatic complexes with two acidic residues on the channel. We construct the profile of potential of mean force for the unbinding of PIIIA from the channel, and predict that PIIIA blocks the bacterial sodium channel with subnanomolar affinity.

    Cupricyclins: novel metallopeptides based on conotoxin scaffold

    Barba M, Sobolev AP, Zobnina V, Bonaccorsi di Patti MC, Cervoni L, Spiezia MC, Schininŕ ME, Pietraforte D, Mannina L, Musci G, Polticelli F. (2012) Cupricyclins, novel redox-active metallopeptides based on conotoxins scaffold. PLoS One. 2012;7(2):e30739. Epub Feb 3. PubMed PMID: 22319584; PubMed Central PMCID: PMC3272027.

    Department of Biology, University Roma Tre, Rome, Italy.

    Abstract: Highly stable natural scaffolds which tolerate multiple amino acid substitutions represent the ideal starting point for the application of rational redesign strategies to develop new catalysts of potential biomedical and biotechnological interest. The knottins family of disulphide-constrained peptides display the desired characteristics, being highly stable and characterized by hypervariability of the inter-cysteine loops. The potential of knottins as scaffolds for the design of novel copper-based biocatalysts has been tested by engineering a metal binding site on two different variants of an ?-conotoxin, a neurotoxic peptide belonging to the knottins family. The binding site has been designed by computational modelling and the redesigned peptides have been synthesized and characterized by optical, fluorescence, electron spin resonance and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupricyclin-1 and -2, bind one Cu(2+) ion per molecule with nanomolar affinity. Cupricyclins display redox activity and catalyze the dismutation of superoxide anions with an activity comparable to that of non-peptidic superoxide dismutase mimics. We thus propose knottins as a novel scaffold for the design of catalytically-active mini metalloproteins.

    12 February 2012

    Nicotinic receptor types involved in neuropathic pain reversal

    Napier IA, Klimis H, Rycroft BK, Jin AH, Alewood PF, Motin L, Adams DJ, Christie MJ. (2012) Intrathecal alpha-conotoxins Vc1.1, AuIB and MII acting on distinct nicotinic receptor subtypes reverse signs of neuropathic pain. Neuropharmacology. 2012 Jan 27. [Epub ahead of print] PubMed PMID: 22306793.

    Discipline of Pharmacology, The University of Sydney, NSW 2006, Australia.

    Abstract: The large diversity of peptides from venomous creatures with high affinity for molecules involved in the development and maintenance of neuropathic pain has led to a surge in venom-derived analgesic research. Some members of the alpha-conotoxin family from Conus snails which specifically target subtypes of nicotinic acetylcholine receptors (nAChR) have been shown to be effective at reducing mechanical allodynia in neuropathic pain models. We sought to determine if three such peptides, Vc1.1, AuIB and MII were effective following intrathecal administration in a rat neuropathic pain model because they exhibit different affinities for the major putative pain relieving targets of alpha-conotoxins. Intrathecal administration of alpha-conotoxins, Vc1.1, AuIB and MII into neuropathic rats reduced mechanical allodynia for up to 6 h without significant side effects. In vitro patch-clamp electrophysiology of primary afferent synaptic transmission revealed the mode of action of these toxins was not via a GABA(B)-dependant mechanism, and is more likely related to their action at nAChRs containing combinations of alpha3, alpha7 or other subunits. Intrathecal nAChR subunit-selective conotoxins are therefore promising tools for the effective treatment of neuropathic pain. Copyright © 2012. Published by Elsevier Ltd. PMID: 22306793 [PubMed - as supplied by publisher]

    The Cone Collector Vol. 19

    Conus textile from the Gold Coast, Australia [Image courtesy of Thierry Vulliet]

    The Cone Collector: Volume 19, Visaya Net - February 6, 2012, Antonio Monteiro Background: The first issue for 2012 (actually this is the twenty-first issue, since publication began with a trial issues number 0 and along the way included a special issue 14A). Contributors to this issue include: Remy Devorsine, Edward J. Petuch, Philippe Quiquandon, José Rosado, Jon F. Singleton, Manuel J. Tenorio, David Touitou, Thierry Vulliet and Paul Wilson.

    Includes announcement about the 2nd InternationalCone Meeting that will take place at the end of September 2012 at La Rochelle, France.

    Edited by António Monteiro, with layout by André Poremski.

    Click here to download The Cone Collector #19 (PDF - 4.2 MB).
    [Earlier issues, #0 - #14, are available for download at http://www.conchology.be/?t=42]

    See interviews of notable cone shell collectors at the Seashell Collector site:

    Balsara R, Li N, Weber-Adrian D, Huang L, Castellino FJ. (2012) Opposing action of conantokin-G on synaptically and extrasynaptically-activated NMDA receptors. Neuropharmacology. 2012 Jan 27. [Epub ahead of print] PubMed PMID: 22306487.

    W.M. Keck Center for Transgene Research and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.

    Abstract: Synaptic and extrasynaptic activation of the N-methyl-d-aspartate receptor (NMDAR) has distinct consequences on cell signaling and neuronal survival. Since conantokin (con)-G antagonism is NR2B-selective, which is the key subunit involved in extrasynaptic activation of the receptor, its ability to specifically elicit distinct signaling outcomes in neurons with synaptically or extrasynaptically-activated NMDARs was evaluated. Inhibition of Ca(2+) influx through extrasynaptic NMDAR ion channels was neuroprotective, as it effectively enhanced levels of activated extracellular signal-regulated kinase 1/2 (ERK1/2), activated cAMP response element binding protein (CREB), enhanced mitochondrial viability, and attenuated the actin disorganization observed by extrasynaptic activation of NMDARs. Conversely, the pro-signaling pathways stimulated by synaptically-induced Ca(2+) influx were abolished by con-G. Furthermore, subunit non-selective con-T was unable to successfully redress the impairments in neurons caused by extrasynaptically-activated NMDARs, thus indicating that NR2B-specific antagonists are beneficial for neuron survival. Neurons ablated for the NR2B subunit showed weak synaptic Ca(2+) influx, reduced sensitivity to MK-801 blockage, and diminished extrasynaptic current compared to WT and NR2A(-/-) neurons. This indicates that the NR2B subunit is an integral component of both synaptic and extrasynaptic NMDAR channels. Altogether, these data suggest that con-G specifically targets the NR2B subunit in the synaptic and extrasynaptic locations, resulting in the opposing action of con-G on differentially activated pools of NMDARs.

    Toll L, Zaveri NT, Polgar WE, Jiang F, Khroyan TV, Zhou W, Xie XS, Stauber GB, Costello MR, Leslie FM. (2012) AT-1001: A High Affinity and Selective ?3?4 Nicotinic Acetylcholine Receptor Antagonist Blocks Nicotine Self-Administration in Rats. Neuropsychopharmacology. 2012 Jan 25. doi: 10.1038/npp.2011.322. [Epub ahead of print] PubMed PMID: 22278092.

    SRI International, Menlo Park, CA, USA.

    Abstract: Genomic and pharmacologic data have suggested the involvement of the ?3?4 subtype of nicotinic acetylcholine receptors (nAChRs) in drug seeking to nicotine and other drugs of abuse. In order to better examine this receptor subtype, we have identified and characterized the first high affinity and selective ?3?4 nAChR antagonist, AT-1001, both in vitro and in vivo. This is the first reported compound with a Ki below 10?nM at ?3?4 nAChR and >90-fold selectivity over the other major subtypes, the ?4?2 and ?7 nAChR. AT-1001 competes with epibatidine, allowing for [(3)H]epibatidine binding to be used for structure-activity studies, however, both receptor binding and ligand-induced Ca(2+) flux are not strictly competitive because increasing ligand concentration produces an apparent decrease in receptor number and maximal Ca(2+) fluorescence. AT-1001 also potently and reversibly blocks epibatidine-induced inward currents in HEK cells transfected with ?3?4 nAChR. Importantly, AT-1001 potently and dose-dependently blocks nicotine self-administration in rats, without affecting food responding. When tested in a nucleus accumbens (NAcs) synaptosomal preparation, AT-1001 inhibits nicotine-induced [(3)H]dopamine release poorly and at significantly higher concentrations compared with mecamylamine and conotoxin MII. These results suggest that its inhibition of nicotine self-administration in rats is not directly due to a decrease in dopamine release from the NAc, and most likely involves an indirect pathway requiring ?3?4 nAChR. In conclusion, our studies provide further evidence for the involvement of ?3?4 nAChR in nicotine self-administration. These findings suggest the utility of this receptor as a target for smoking cessation medications, and highlight the potential of AT-1001 and congeners as clinically useful compounds.

    Laht S, Koua D, Kaplinski L, Lisacek F, Stöcklin R, Remm M. (2012) Identification and classification of conopeptides using profile Hidden Markov Models. Biochim Biophys Acta. 2011 Dec 30;1824(3):488-492. [Epub ahead of print] PubMed PMID: 22244925.

    Estonian Biocentre, Riia 23, 51010, Tartu, Estonia.

    Abstract: Conopeptides are small toxins produced by predatory marine snails of the genus Conus. They are studied with increasing intensity due to their potential in neurosciences and pharmacology. The number of existing conopeptides is estimated to be 1 million, but only about 1000 have been described to date. Thanks to new high-throughput sequencing technologies the number of known conopeptides is likely to increase exponentially in the near future. There is therefore a need for a fast and accurate computational method for identification and classification of the novel conopeptides in large data sets. 62 profile Hidden Markov Models (pHMMs) were built for prediction and classification of all described conopeptide superfamilies and families, based on the different parts of the corresponding protein sequences. These models showed very high specificity in detection of new peptides. 56 out of 62 models do not give a single false positive in a test with the entire UniProtKB/Swiss-Prot protein sequence database. Our study demonstrates the usefulness of mature peptide models for automatic classification with accuracy of 96% for the mature peptide models and 100% for the pro- and signal peptide models. Our conopeptide profile HMMs can be used for finding and annotation of new conopeptides from large datasets generated by transcriptome or genome sequencing. To our knowledge this is the first time this kind of computational method has been applied to predict all known conopeptide superfamilies and some conopeptide families.

    Favreau P, Benoit E, Hocking HG, Carlier L, D'hoedt D, Leipold E, Markgraf R, Schlumberger S, Córdova MA, Gaertner H, Paolini-Bertrand M, Hartley O, Tytgat J, Heinemann SH, Bertrand D, Boelens R, Stöcklin R, Molgó J. (2012) Pharmacological characterization of a novel mu-conopeptide, CnIIIC, indicates potent and preferential inhibition of sodium channel subtypes (Na(V) 1.2/1.4) and reveals unusual activity on neuronal nicotinic acetylcholine receptors. Br J Pharmacol. 2012 Jan 9. doi: 10.1111/j.1476-5381.2012.01837.x. [Epub ahead of print] PubMed PMID: 22229737.

    Atheris Laboratories, Case Postale 314, CH-1233 Bernex-Geneva, Switzerland, the CNRS, Institut de Neurobiologie Alfred Fessard - FRC2118, Laboratoire de Neurobiologie et Développement - UPR3294, 91198 Gif sur Yvette cedex, France, the Bijvoet Center for Biomolecular Research, Padualaan 8, NL-3584, Utrecht, Netherlands, the Department of Neuroscience, 1 rue Michel Servet, CH-1211, Geneva, Switzerland, the Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University of Jena and Jena University Hospital, Hans-Knöll-St. 2, D-07745 Jena, Germany, the Laboratory of Marine Toxins, Program of Physiology and Biophysics, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago 6530499, Chile, the Laboratorium voor Toxicologie, Campus Gasthuisberg, Onderwijs en Navorsing 2, Herestraat 49, Bus 922, Leuven B-3000, Belgium, and the Department of Structural Biology and Bioinformatics, 1 rue Michel Servet, CH-1211, Geneva, Switzerland.

    Abstract: BACKGROUND AND PURPOSE? The µ-conopeptide family is defined by its ability to block voltage-gated sodium channels (VGSCs), a property that can be used for the development of myorelaxants and analgesics. We performed a pharmacological characterisation of a new µ-conopeptide (µ-CnIIIC) on multiple preparations and molecular targets to gauge its potential as a myorelaxant. EXPERIMENTAL APPROACH? The µ-CnIIIC was sequenced, synthesized, and characterized by its ability to block directly-elicited twitch tension in mouse skeletal muscle and action potentials in mouse sciatic and pike olfactory nerves. µ-CnIIIC was also studied on HEK-293 cells expressing various rodent VGSCs. Pharmacological investigations were extended to voltage-gated potassium channels and nAChRs to assess cross-interactions. Nuclear magnetic resonance (NMR) experiments were carried out for structural data. KEY RESULTS? Synthetic µ-CnIIIC potently decreased twitch tension in mouse hemidiaphragms (IC(50) = 150 nM), and displayed a higher blocking effect in mouse extensor digitorum longus muscles (IC = 46 nM), as compared to µ-SIIIA, µ-SmIIIA and µ-PIIIA. µ-CnIIIC blocked Na(V) 1.4 (IC(50) = 1.3 nM) and Na(V) 1.2 in a long-lasting manner. Cardiac Na(V) 1.5 and DRG-specific Na(V) 1.8 were not blocked at 1 ?M. An activity was unveiled on the alpha3beta2 nAChR subtype (IC(50) = 450 nM) and, to a lesser extent, on the ?7 and alpha4beta2 subtypes. Structure determination of µ-CnIIIC revealed some similarities to ?-conotoxins acting on nAChRs. CONCLUSION AND IMPLICATIONS? µ-CnIIIC potently blocks VGSCs in skeletal muscle and nerve, and hence is applicable to myorelaxation. Its new atypical pharmacological profile suggests some common structural features between VGSCs and nAChR channels.

    Carstens BB, Clark RJ, Daly NL, Harvey PJ, Kaas Q, Craik DJ. (2011) Engineering of conotoxins for the treatment of pain. Curr Pharm Des. 2011 Dec;17(38):4242-53. PubMed PMID: 22204425.

    The University of Queensland, Institute for Molecular Bioscience, Brisbane QLD 4072, Australia.

    Abstract: The peptides present in the venoms of marine snails are used by the snails to capture prey, but they have also attracted the interest of drug designers because of their potent activity against therapeutically important targets. These peptides are typically disulfiderich and target a wide range of ion channels, transporters and receptors with exquisite selectivity. In this article, we discuss structural and biological studies on several classes of conotoxins that have potential as drug leads for the treatment of pain. The chemical re-engineering of conotoxins via cyclization has been particularly valuable in improving their biopharmaceutical properties. An excellent example is the ?-conotoxin Vc1.1, for which several cyclized analogs have been made. One of them was shown to be orally active in a rat pain model and this analog is currently undergoing pre-clinical development for the treatment of neuropathic pain. Several other ?-conotoxins, including ImI, AuIB and MII, have proved amenable to cyclization and in all cases improvements in stability are obtained upon cyclization, suggesting that cyclization is a generally applicable approach to conotoxin stabilization. A variety of other chemical re-engineering approaches have also been used. Minor re-engineering of ?-conotoxin MrIa to convert its N-terminal residue to pyroglutamic acid proved particularly successful and the modified derivative, Xen2174, is currently in clinical trials for neuropathic pain.

    Muttenthaler M, Akondi KB, Alewood PF. (2011) Structure-activity studies on alpha-conotoxins. Curr Pharm Des. 2011 Dec;17(38):4226-41. PubMed PMID: 22204424.

    Institute for Molecular Bioscience, The University of Queensland, St Lucia Queensland 4072, Australia.

    Abstract: Conotoxins are small bioactive highly structured peptides from the venom of marine cone snails (genus Conus). Over the past 50 million years these molluscs have developed a complex venom cocktail for each species that is comprised of 100-2000 distinct cysteine- rich peptides for prey capture and defence. This review focuses on an important and well-studied class of conotoxins, the ?- conotoxins. These ?-conotoxins are potent and selective antagonists of various subtypes of the nicotinic acetylcholine receptors (nAChRs). Key structure-activity relationship studies are presented to illustrate the common motifs, structural features and pharmacophores that define this interesting peptide class. Additionally, their synthesis, chemical modifications, the development of more selective and stable analogues and their therapeutic potential are discussed.



    30 October 2011

    Embryonic expression of conotoxins

    Safavi-Hemami H, Siero WA, Kuang Z, Williamson NA, Karas JA, Page LR, MacMillan D, Callaghan B, Kompella SN, Adams DJ, Norton RS, Purcell AW (2011)). Embryonic toxin expression in the cone snail Conus victoriae: primed to kill or divergent function? . J Biol Chem. 286(25):22546-22557.

    Department of Biochemistry and Molecular Biology, University of Melbourne, 3010 Victoria, Australia.

    Abstract: Predatory marine cone snails (genus Conus) utilize complex venoms mainly composed of small peptide toxins that target voltage- and ligand-gated ion channels in their prey. Although the venoms of a number of cone snail species have been intensively profiled and functionally characterized, nothing is known about the initiation of venom expression at an early developmental stage. Here, we report on the expression of venom mRNA in embryos of Conus victoriae and the identification of novel ?- and O-conotoxin sequences. Embryonic toxin mRNA expression is initiated well before differentiation of the venom gland, the organ of venom biosynthesis. Structural and functional studies revealed that the embryonic ?-conotoxins exhibit the same basic three-dimensional structure as the most abundant adult toxin but significantly differ in their neurological targets. Based on these findings, we postulate that the venom repertoire of cone snails undergoes ontogenetic changes most likely reflecting differences in the biotic interactions of these animals with their prey, predators, or competitors. To our knowledge, this is the first study to show toxin mRNA transcripts in embryos, a finding that extends our understanding of the early onset of venom expression in animals and may suggest alternative functions of peptide toxins during development.

    Conotoxin isoforms in Conus victoriae and Conus novaehollandiae

    Safavi-Hemami H, Siero WA, Gorasia DG, Young ND, Macmillan D, Williamson NA, Purcell AW. (2011)). Specialisation of the venom gland proteome in predatory cone snails reveals functional diversification of the conotoxin biosynthetic pathway. J Proteome Res. 10(9):3904-3919.

    Department of Biochemistry and Molecular Biology, University of Melbourne, Victoria, Australia.

    Abstract: Conotoxins, venom peptides from marine cone snails, diversify rapidly as speciation occurs. It has been suggested that each species can synthesize between 1000 and 1900 different toxins with little to no interspecies overlap. Conotoxins exhibit an unprecedented degree of post-translational modifications, the most common one being the formation of disulfide bonds. Despite the great diversity of structurally complex peptides, little is known about the glandular proteins responsible for their biosynthesis and maturation. Here, proteomic interrogations on the Conus venom gland led to the identification of novel glandular proteins of potential importance for toxin synthesis and secretion. A total of 161 and 157 proteins and protein isoforms were identified in the venom glands of Conus novaehollandiae and Conus victoriae, respectively. Interspecies differences in the venom gland proteomes were apparent. A large proportion of the proteins identified function in protein/peptide translation, folding, and protection events. Most intriguingly, however, we demonstrate the presence of a multitude of isoforms of protein disulfide isomerase (PDI), the enzyme catalyzing the formation and isomerization of the native disulfide bond. Investigating whether different PDI isoforms interact with distinct toxin families will greatly advance our knowledge on the generation of cone snail toxins and disulfide-rich peptides in general.



    Additional items from 1 January 2011 to 29 October 2011 to be added here.


    Additional items from 24 October to 31 December 2010 to be added here.

    Cone Snails: Versatile Hunters
    Dr. Jason Biggs of the University of Guam Marine Laboratory discusses the anatomy of cone snails and introduces us to a variety of cone snail species with different tactics to hunt and capture their prey. To view: CLICK HERE

    23 October 2010

    Predicting Conotoxin Superfamilies

    Fan YX, Song J, She HB (2010)). PredCSF: An Integrated Feature-Based Approach for Predicting Conotoxin Superfamily. Protein Pept Lett. 2010 Oct 18. [Epub ahead of print] PubMed PMID: 20955172.

    Institute of Image Processing & Pattern Recognition, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai, 200240, China. hbshen@sjtu.edu.cn

    Abstract: Conotoxins are small disulfide-rich peptides that are invaluable channel-targeted peptides and target neuronal receptors. They show prospects for being potent pharmaceuticals in the treatment of Alzheimer's disease, Parkinson's disease, and epilepsy. Accurate and fast prediction of conotoxin superfamily is very helpful towards the understanding of its biological and pharmacological functions especially in the post-genomic era. In the present study, we have developed a novel approach called PredCSF for predicting the conotoxin superfamily from the amino acid sequence directly based on fusing different kinds of sequential features by using modified one-versus-rest SVMs. The input features to the PredCSF classifiers are composed of physicochemical properties, evolutionary information, predicted second structure and amino acid composition, where the most important features are further screened by random forest feature selection to improve the prediction performance. The prediction results show that PredCSF can obtain an overall accuracy of 90.65% based on a benchmark dataset constructed from the most recent database, which consists of 4 main conotoxin superfamilies and 1 class of non-conotoxin class. Systematic experiments also show that combing different features is extremely helpful for enhancing the prediction power when dealing with complex biological problems. PredCSF is expected to be a powerful tool for in silico identification of novel conotonxins and is freely available for academic use at http://www.csbio.sjtu.edu.cn/bioinf/PredCSF. PMID: 20955172 [PubMed - as supplied by publisher]

    Benchmark Datasets
    Online Supporing Infomation A: The accession numbers and mature peptide of the 261 toxin sequences and 60 negative datasets in the benchmark dataset are classified into 5 types (subsets):

  • (1) A-conotoxin superfamily 63
  • (2) M-conotoxin superfamily 48
  • (3) O-conotoxin superfamily 95
  • (4) T-conotoxin superfamily 55, and
  • (6) Negative dataset 60.
    None of proteins included here has more than 20% pairwise sequence identity to any other in a same subset. See the text of the paper for further explanation. To download the data in the Online Supporting Information A, click Supp-A

    Online Supporing Infomation B: The accession numbers and full sequence of the 261 toxin sequences and 60 negative dataset in the benchmark dataset. See the text of the paper for further explanation. To download the data in the Online Supporting Information B, click Supp-B


    Targeting conantokin-G receptors to NMDA receptors using gold nanoparticles

    Maus L, Dick O, Bading H, Spatz JP, Fiammengo R (2010). Conjugation of Peptides to the Passivation Shell of Gold Nanoparticles for Targeting of Cell-Surface Receptors. ACS Nano. 2010 Oct 12. [Epub ahead of print] PubMed PMID: 20939520.

    Department of New Materials and Biosystems, Max Planck Institute for Metals Research, Stuttgart, Germany, and Department of Biophysical Chemistry, University of Heidelberg, Germany, postal address: Heisenbergstrasse 3, 70569 Stuttgart, Germany.

    Abstract: We report the preparation of gold nanoparticles (AuNPs) functionalized with the peptide-toxin conantokin-G and their selective binding to N-methyl-d-aspartate (NMDA) receptors recombinantly expressed by transfected HEK 293 cells. The AuNPs are passivated with a mixed self-assembled monolayer of omega-carboxy- and omega-amino-polyethylene glycol (PEG) thiols. We compare two different passivation systems: the alkyl-PEG600 system is characterized by a C(11)-alkyl chain between the thiol group and the PEG segment, whereas the PEG3000 system lacks this alkyl-chain. We show that only the alkyl-PEG600 passivation system allows selective conjugation of cysteine-terminated peptides to the periphery of the passivation layer via a heterobifunctional linker strategy. In contrast, using the PEG3000 passivation system, peptides are immobilized both on the passivation layer and directly on the gold surface via concurrent place-exchange reaction. We therefore recommend the use of the alkyl-PEG600 system to precisely control the number of immobilized peptides on AuNPs. In fact, we show that the number of conjugated peptides per particle can be varied with good control simply by varying the composition of the self-assembled monolayer. Finally, we demonstrate that conjugation of the conantokin-G peptide to the solvent-exposed interface of the passivation layer results in maximal binding interaction between the peptide-functionalized AuNPs and the targeted NMDA receptors on the cell surface. Conantokin G-coupled AuNP may be used to spatially restrict NMDA-receptor-blockade on neuronal surfaces.
    PMID: 20939520 [PubMed - as supplied by publisher]

    12 October 2010

    South African cone shell toxins

    Kauferstein S, Porth C, Kendel Y, Wunder C, Nicke A, Kordis D, Favreau P, Koua D, Stöcklin R, Mebs D (2010). Venomic study on cone snails (Conus spp.) from South-Africa. Toxicon. 2010 Oct 6. [Epub ahead of print] PubMed PMID: 20933537.

    Institute of Legal Medicine, University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt, Germany.

    Abstract: From six Conus species (C. coronatus, C. lividus, C. mozambicus f. lautus, C. pictus, C. sazanka, C. tinianus) collected off the eastern coast of South-Africa the venoms were analyzed using MALDI-TOF mass spectrometry. Between 56 to 151 molecular masses most in a range of 1,000 to 2,500 Da, were identified. Among the six venoms, between 0 to 27% (C. coronatus versus C. sazanka) of the peptide masses were found to be similar. In a study on venoms from 6 Conus species collected in the Philippines, the percentage of identical masses was between none to 9% only. The venoms from the South-African Conus species antagonized the rat neuronal nicotinic acetylcholine receptors (nAChRs) alpha3beta2, alpha4beta2, and alpha7, except for C. coronatus venom which blocked the alpha4beta2 and alpba7 nAChRs only. HPLC-fractionation of C. tinianus venom led to the isolation of a peptide that is active on all three receptor subtypes. It consists of 16 amino acid residues cross-linked by two disulfide bridges as revealed by de novo sequencing using tandem mass spectrometry: GGCCSHPACQNNPDYC. Posttranslational modifications include C-terminal amidation and tyrosine sulfation. The new peptide is a member of the alpha-conotoxin family which are competitive antagonists of nAChRs. Phylogenetic analysis of the 16S RNA from numerous Conus species has clarified the evolutionary position of endemic South African Conus species and provided the first evidence for their close genetic relationships. PMID: 20933537 [PubMed - as supplied by publisher]

    Calcium channel toxin from Conus californicus

    Bernaldez J, López O, Licea A, Salceda E, Arellano RO, Vega R, Soto E (2010). Electrophysiological characterization of a novel small peptide from the venom of Conus californicus that targets voltage-gated neuronal Ca(2+) channels. Toxicon 2010 Oct 5. [Epub ahead of print] PubMed PMID: 20920515.

    Laboratorio de Inmunología Molecular y Biotoxinas, Departamento de Biotecnología Marina (C.I.C.E.S.E.), Ensenada, B.C., Mexico.

    Abstract: Conus californicus belongs to a genus of marine gastropods with more than 700 extant species. C. californicus has been shown to be distantly related to all Conus species, but showing unusual biological features. We report a novel peptide isolated from C. californicus with a significant inhibitory action over neuronal voltage-gated calcium channels. The new toxin is formed by 13-amino acid residues with two disulfide bonds, whose sequence (NCPAGCRSQGCCM) is strikingly different from regular omega-conotoxins. In the HPLC purification procedure, the venom fraction eluted in the first 10-15 min produced a significant decrease (54% ± 3%) of the Ca(2+) current in Xenopus laevis oocytes transfected with purified rat-brain mRNA. A specific peptide obtained from the elution at 13 min decreased the Ca(2+) current in the adult rat dorsal-root ganglion neurons in a primary culture by 34% ± 2%. The cysteine pattern of this peptide corresponds to the framework XVI described for the M-superfamily of conopeptides and is unprecedented among Conus peptides acting on Ca(2+) channels.

    5 October 2010

    How do alpha-conotoxins relieve allodynia?

    Klimis H, Adams DJ, Callaghan B, Nevin S, Alewood PF, Vaughan CW, Mozar CA, Christie MJ (2010). A novel mechanism of inhibition of high-voltage activated calcium channels by alpha-conotoxins contributes to relief of nerve injury-induced neuropathic pain. Pain 152, 259-266.

    Pain Management Research Institute, University of Sydney, Royal North Shore Hospital, St. Leonards NSW 2065, Australia; Brain and Mind Research Institute, University of Sydney, NSW 2006, Australia.

    Abstract: alpha-Conotoxins that are thought to act as antagonists of nicotinic acetylcholine receptors (nAChRs) containing alpha3-subunits are efficacious in several preclinical models of chronic pain. Potent interactions of Vc1.1 with other targets have suggested that the pain-relieving actions of alpha-conotoxins might be mediated by either alpha9alpha10 nAChRs or a novel GABA(B) receptor-mediated inhibition of N-type calcium channels. Here we establish that three alpha-conotoxins, Vc1.1, AuIB and MII have distinct selectivity profiles for these three potential targets. Their potencies after intramuscular administration were then determined for reversal of allodynia produced by partial nerve ligation in rats. Vc1.1, which potently inhibits alpha9alpha10 nAChRs and GABA(B)/Ca(2+) channels but weakly blocks alpha3beta2 and alpha3beta4 nAChRs, produced potent, long-lasting reversal of allodynia that were prevented by pre-treatment with the GABA(B) receptor antagonist, SCH50911. alpha-Conotoxin AuIB, a weak alpha3beta4 nAChR antagonist, inhibited GABA(B)/Ca(2+) channels but did not act on alpha9alpha10 nAChRs. AuIB also produced reversal of allodynia. These findings suggest that GABA(B) receptor-dependent inhibition of N-type Ca(2+) channels can mediate the sustained anti-allodynic actions of some alpha-conotoxins. However, MII, a potent alpha3beta2 nAChR antagonist but inactive on alpha9alpha10 and alpha3beta4 nAChRs and GABA(B)/Ca(2+) channels, was demonstrated to have short-acting anti-allodynic action. This suggests that alpha3beta2 nAChRs may also contribute to reversal of allodynia. Together, these findings suggest that inhibition of alpha9alpha10 nAChR is neither necessary nor sufficient for relief of allodynia and establish that alpha-conotoxins selective for GABA(B) receptor-dependent inhibition of N-type Ca(2+) channels relieve allodynia, and could therefore be developed to manage chronic pain.

    3 October 2010

    Proposed mechanism(s) for the antiallodynic conopeptide Vc1.1

    Zamponi GW (2010). Antiallodynic effects of a confused alpha-conotoxin: Vc1.1 relieves neuropathic pain via off target actions on GABA(B) receptors and N-type channels. COMMENTARY Pain Sep 27. [Epub ahead of print] PubMed PMID: 20880631. doi:10.1016/j.pain.2010.09.019

    Department of Physiology and Pharmacology, Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Dr. NW, Calgary, Canada T2N 4N1.

    Abstract: No Abstract. EXTRACT follows: "Venoms from predatory marine organisms such as fish-hunting cone snails are a rich source of bioactive compounds that bind to, and regulate a wide range of voltage- and ligand-gated ion channels. These snails use a cocktail of peptide toxins to rapidly stun and ultimately kill their prey. The individual toxins contained within the venom selectively target specific ion channel subfamilies [3]. For example, mu-conotoxins and omega-conotoxins, respectively, block voltage gated sodium and calcium channels, whereas the alpha-conotoxins are typically thought of as antagonists of nicotinic acetylcholine receptors [5]. Of course, the primary purpose of cone snail venom is to kill fish. However, because there is sufficient conservation in ion channel sequences among fish and mammals, many types of cone snail toxins also potently interact with human ion channel proteins [3]. On one hand, this poses a danger to humans coming into contact with various Conus species, but on the other hand, the selectivity and potency of individual peptide toxins can potentially be harnessed for therapeutic purposes. A prime example is the clinical use of omega-conotoxin MVIIA, a potent inhibitor of N-type calcium channels that can be delivered intrathecally to mediate analgesia in patients with severe pain [1]. A number of biotechnology companies and academic research laboratories are actively seeking other conotoxin molecules for use as therapeutics in humans [3]. This includes Vc1.1, an alpha-conotoxin from Conus victoriae that is currently in phase II clinical trials for neuropathic pain." [Conotoxin Vc1.1 also known as ACV1, completed Phase IIa clinical trials with no deleterious effects (BGL)]

    "Callaghan and colleagues recently reported a Vc1.1 mediated inhibition of N-type calcium channels in rat sensory neurons [2] These authors showed that this inhibition was dependent on a Vc1.1 action on GABAB receptors, rather than on its canonical target (i.e., the nicotinic receptor). While this is in itself unusual, what is even more perplexing is that when activated by Vc1.1, GABAB receptors appear to signal by a novel and still undefined pathway to the N-type channel protein. But irrespective of the precise mode of action, the fact that N-type channels are inhibited by Vc1.1 suggested the possibility that its therapeutic actions are also mediated via N-types, rather than an effect on nicotinic receptors."

    "In this issue of Pain, Kimlis and colleagues [4] examine the mechanism of Vc1.1 mediated analgesia in rats, along with that of two related alpha-conotoxins AuIB (Conus aulicus) and MII (Conus magus). The authors first characterized the effect of the three toxins on recombinant acetylcholine receptors expressed in Xenopus oocytes, and found that only Vc1.1 significantly inhibited a9a10 receptors The authors then demonstrated that Vc1.1 and AuIB, but not MII, triggered a GABAB receptor-mediated inhibition of native N-type currents in dorsal root ganglion neurons. The distinct target selectivities of the three toxins thus provided the authors with a unique tool kit to probe for the involvement of nicotinic and GABAB receptors in the afferent pain pathway. For this purpose, Kimlis and colleagues examined the effects of the three toxins on mechanical allodynia in a peripheral nerve ligation model. When injected intramuscularly, all three toxins produced an increase in mechanical paw withdrawal threshold with Vc1.1 being the most potent inhibitor, followed by AuIB and MII, and the effects of Vc1.1 were blocked by a GABAB receptor antagonist. Collectively, these data show that the antiallodynic actions of all three alpha-conotoxins are not mediated by a9a10 receptors and that a9a10 receptors are not significantly involved in the transmission of peripheral pain signals. Furthermore, the data provide compelling evidence that molecules such as Vc1.1 mediate their therapeutic actions via GABAB receptor induced inhibition of N-type calcium channels."

    "It is well established that inhibiting N-type calcium channels is a suitable objective for the development of analgesics, either directly, or indirectly via the activation of G protein coupled receptors [1]. The new findings with Vc1.1 fit with the latter notion, despite the fact that the Vc1.1 induced effect appears to involve a novel, and as yet not fully characterized mechanism of coupling between the receptor and the channel. The observation that MII also produced antiallodynic effects even though it did not act on either N-type channels or a9a10 receptors suggests that inhibition of other types of nicotinic receptors such as alpha3beta2 [5] may help to alleviate mechanical allodynia. If so, then it may well be possible that inhibition of both GABAB and nicotinic receptors could synergistically mediate analgesia. Future studies involving co-administration of compounds such as Vc1.1 and MII, or the identification of novel alpha-conotoxins with a combined action on both receptor types may be useful in testing such a possibility."


    [1] Altier C, Zamponi GW (2004). Targeting Ca2+ channels to treat pain: T-type versus Ntype. Trends Pharmacol Sci 25:465–467

    . [2] Callaghan B, Haythornthwaite A, Berecki G, Clark RJ, Craik DJ, Adams DJ (2008) Analgesic alpha-conotoxins Vc1.1 and Rg1A inhibit N-type calcium channels in rat sensory neurons via GABAB receptor activation. J Neurosci 28: 10943–10951.

    [3] Han TS, Teichert RW, Olivera BM, Bulaj G. (2008) Conus venoms – a rich source of peptide-based therapeutics. Curr Pharm Des 14: 2462–2479.

    [4] Klimis H, Adams DJ, Callghan B, Nevin S, Alewood PF, Vaughan CW, Mozar CA, Christie MJ (2010). A novel mechanism of inhibition of high-voltage activated calcium channels by alpha-conotoxins contributes to relief of nerve injury – induced neuropathic pain. Pain 152, 259e266 .

    [5] Olivera BM, Quik M, Vincler M, McIntosh JM (2008). Subtype-selective conopeptides targeted to nicotinic receptors: concerted discovery and biomedical applications. Channels (Austin) 2:143–152.

    BUT see critique in the review by McIntosh JM, Absalom N, Chebib M, Elgoyhen AB, Vincler M.(2009) Alpha9 nicotinic acetylcholine receptors and the treatment of pain. Biochem Pharmacol. 78: 693-702.

    Abstract: ......Pharmacological analysis indicates that RgIA and Vc1.1 are selective antagonists of alpha9alpha10 nAChRs. A recent study also reported that these alpha9alpha10 antagonists are also potent GABA-B agonists. In the current study, we were unable to detect RgIA or Vc1.1 binding to or action on cloned GABA-B receptors expressed in HEK cells or Xenopus oocytes. We review the background, findings and implications of use of compounds that act on alpha9* nAChRs.(1).

    Molluscan literature

    KOHN, 1991. Paintings of paralectotypes?: Conus of the Museum Ludovicae Ulricae. 12pp., 1fig., 2tabs., 2pls. Euro 2.25
    COOMANS & al., 1979. Alphabetical revision of the (sub) sp. in recent Conidae 1. Abbas to adansonii. 18pp., 25figs. Euro 2.70
    COOMANS & al., 1979. Alphabetical revision of the (sub) sp. in recent Conidae 2. adansoni to albuquerquei. 25pp., 50figs. Euro 3.75
    COOMANS & al., 1980. Alphabetical revision of the (sub) sp. in recent Conidae 3. albus to antillarum with the description of Conus algoensis agulhasi, n.ssp. 33pp., 43figs. Euro 4.95
    COOMANS, 1966. Boekbespreking [Marsh, 1964. Cone shells of the world]. 2pp. Euro 0.50

    Available from A.N. van der Bijl, Burgemeester van Bruggenstraat 41 1165 NV Halfweg, The Netherlands +31-20-4977772 (phone), anvdbijl@xs4all.nl (e-mail)

    24 September 2010

    Folding of cyclotides and conotoxins - REVIEW

    Craik DJ (2010). The Folding of Disulfide-Rich Proteins. Antioxid Redox Signal. 2010 Sep 20. [Epub ahead of print] PubMed PMID: 20849378.

    The Unversity of Queensland, Institute for Molecular Bioscience, Carmody Road, Brisbane, Queensland, Australia, 4069; d.craik@imb.uq.edu.au

    Abstract: The articles in this forum issue describe various aspects of the folding of disulfide-rich proteins. They include review articles using proteins such as bovine pancreatic trypsin inhibitor as models to highlight the range of folding pathways seen in disulfide-rich proteins, along with a detailed analysis of the methods used to study them. Following two comprehensive reviews on the methods and applications of protein folding, three original articles in this issue focus on two specific classes of disulfide-rich proteins that have applications in drug design and development, namely cyclotides and conotoxins. Cyclotides are head-to-tail cyclic and disulfide-rich proteins from plants and function as a defence against insect attack. Conotoxins are the disulfide-rich components of the venom of marine cone snails that is used to capture prey. These research articles report on factors that modulate protein folding pathways in these molecules and determine the outcomes of protein folding- i.e. yield and heterogeneity of products. Finally the issue concludes with a comprehensive review on a different type of disulfide bond, namely those that have a functional, rather than structural role in proteins, with a particular focus on allosteric disulfide bonds that modify protein function.

    The Conoidea superfamily: Teretoxins and conotoxins

    Puillandre N, Holford M (2010). The Terebridae and teretoxins: combining phylogeny and anatomy for concerted discovery of bioactive compounds. BMC Chem Biol. 2010 Sep 17;10(1):7. [Epub ahead of print] PubMed PMID: 20849634.

    Abstract: The Conoidea superfamily, comprised of cone snails, terebrids, and turrids, is an exceptionally promising group for the discovery of natural peptide toxins. The potential of conoidean toxins has been realized with the distribution of the first Conus (cone snail) drug, Prialt (ziconotide), an analgesic used to alleviate chronic pain in HIV and cancer patients. Cone snail toxins (conotoxins) are highly variable, a consequence of a high mutation rate associated to duplication events and positive selection. As Conus and terebrids diverged in the early Paleocene, the toxins from terebrids (teretoxins) may demonstrate highly divergent and unique functionalities. Recent analysis of the Terebridae, a largely distributed family with more than 300 described species, indicate they have evolutionary and pharmacological potential. Based on a three gene (COI, 12S and 16S) molecular phylogeny, including ~50 species from the West-Pacific, five main terebrid lineages were discriminated: Two of these lineages independently lost their venom apparatus, and one venomous lineage was previously unknown. Knowing the phylogenetic relationship of the Terebridae aids in effectively targeting divergent lineages with novel peptide toxins. Preliminary results indicate that teretoxins are similar in structure and composition to conotoxins, suggesting teretoxins are an attractive line of research to discover and develop new therapeutics that target ion channels and receptors. Using conotoxins as a guideline, and innovative natural products discovery strategies, such as the Concerted Discovery Strategy, the potential of the Terebridae and their toxins are explored as a pioneering pharmacological resource.

    A novel linker resin system for synthesis of 'difficult' conotoxins

    Alewood D, Hopping G, Brust A, Reid RC, Alewood PF (2010). Benzhydrylamine linker grafting: a strategy for the improved synthesis of C-terminal peptide amides. J Pept Sci. 2010 Oct;16(10):551-7. PubMed PMID: 20862722.

    Institute for Molecular Bioscience, The University of Queensland, St Lucia 4072, Australia.

    Abstract: The standard p-MBHA resin used during Boc-chemistry synthesis of peptides carrying C-terminal carboxamides is compromised by batch-to-batch variations in its performance. This can cause artificially 'difficult' couplings during peptide chain assembly, which may ultimately lead to failed syntheses given the inability to achieve acceptable coupling yields. To overcome these problems, we have developed a new approach by grafting a functionalized benzhydrylamine linker onto well-characterized and well-performing PAM resins. We combine optimized Boc-chemistry, high-performing PAM resins and new benzhydrylamine-based linkers to achieve improved syntheses of peptide amides. Here we present the synthesis of two new benzhydrylamine linkers and their attachment to selected PAM resins. This novel solid support was evaluated through the synthesis of selected 'difficult' conotoxins and monitoring the coupling efficiency using quantitative ninhydrin assay. The results show a superior performance of the novel linker solid support compared to the standard p-MBHA resins routinely used. In summary, we describe an alternative linker-resin system that allows improved access to C-terminal amide peptides employing Boc/Bzl chemistry. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.

    21 September 2010

    Determination of disulfide pairing in conotoxins by MS.

    Gupta K, Kumar M, Balaram P (2010). Disulfide Bond Assignments by Mass Spectrometry of Native Natural Peptides: Cysteine Pairing in Disulfide Bonded Conotoxins. Anal Chem. 2010 Sep 15. [Epub ahead of print] PubMed PMID: 20843009

    Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012, India, and National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore-560065, India.

    Abstract: The critical, and often most difficult, step in structure elucidation of diverse classes of natural peptides is the determination of correct disulfide pairing between multiple cysteine residues. Here, we present a direct mass spectrometric analytical methodology for the determination of disulfide pairing. Protonated peptides, having multiple disulfide bonds, fragmented under collision induced dissociation (CID) conditions and preferentially cleave along the peptide backbone, with occasional disulfide fragmentation either by C(?)-S bond cleavage through H(?) abstraction to yield dehydroalanine and cysteinepersulfide, or by S-S bond cleavage through H(?) abstraction to yield the thioaldehyde and cysteine. Further fragmentation of the initial set of product ions (MS(n)) yields third and fourth generation fragment ions, permitting a distinction between the various possible disulfide bonded structures. This approach is illustrated by establishing cysteine pairing patterns in five conotoxins containing two disulfide bonds. The methodology is extended to the Conus araneosus peptides Ar1446 and Ar1430, two 14 residue sequences containing 3 disulfide bonds. A distinction between 15 possible disulfide pairing schemes becomes possible using direct mass spectral fragmentation of the native peptides together with fragmentation of enzymatically nicked peptides.

    15 September 2010

    alpha-Conotoxin ImI disrupts swimming in Hirudo spp.

    Wagenaar DA, Gonzalez R, Ries DC, Jr WB, French KA (2010). Alpha-conotoxin ImI Disrupts Central Control of Swimming in the Medicinal Leech. Neurosci Lett. 2010 Sep 9. [Epub ahead of print] PubMed PMID: 20833225.

    California Institute of Technology, Broad Fellows Program and Division of Biology, 1200 E California Blvd 216-76, Pasadena, CA91125, United States.

    Abstract: Medicinal leeches (Hirudo spp.) swim using a metachronal, front-to-back undulation. The behavior is generated by central pattern generators (CPGs) distributed along the animal's midbody ganglia and is coordinated by both central and peripheral mechanisms. Here we report that a component of the venom of Conus imperialis, alpha-conotoxin ImI, known to block nicotinic acetyl-choline receptors in other species, disrupts swimming. Leeches injected with the toxin swam in circles with exaggerated dorsoventral bends and reduced forward velocity. Fictive swimming in isolated nerve cords was even more strongly disrupted, indicating that the toxin targets the CPGs and central coordination, while peripheral coordination partially rescues the behavior in intact animals.

    Calcium channel modulation in the auditory system

    Zorrilla de San Martín J, Pyott S, Ballestero J, Katz E (2010). Ca2+ and Ca2+-activated k+ channels that support and modulate transmitter release at the olivocochlear efferent-inner hair cell synapse. J Neurosci. 2010 Sep 8;30(36):12157-67. PubMed PMID: 20826678.

    Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, 1428 Buenos Aires, Argentina, Department of Biology and Marine Biology, University of North Carolina Wilmington, Wilmington, North Carolina 28403, and Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EGA Buenos Aires, Argentina.

    Abstract: In the mammalian auditory system, the synapse between efferent olivocochlear (OC) neurons and sensory cochlear hair cells is cholinergic, fast, and inhibitory. This efferent synapse is mediated by the nicotinic alpha9alpha10 receptor coupled to the activation of SK2 Ca(2+)-activated K(+) channels that hyperpolarize the cell. So far, the ion channels that support and/or modulate neurotransmitter release from the OC terminals remain unknown. To identify these channels, we used an isolated mouse cochlear preparation and monitored transmitter release from the efferent synaptic terminals in inner hair cells (IHCs) voltage clamped in the whole-cell recording configuration. Acetylcholine (ACh) release was evoked by electrically stimulating the efferent fibers that make axosomatic contacts with IHCs before the onset of hearing. Using the specific antagonists for P/Q- and N-type voltage-gated calcium channels (VGCCs), ?-agatoxin IVA and omega-conotoxin GVIA, respectively, we show that Ca(2+) entering through both types of VGCCs support the release process at this synapse. Interestingly, we found that Ca(2+) entering through the dihydropiridine-sensitive L-type VGCCs exerts a negative control on transmitter release. Moreover, using immunostaining techniques combined with electrophysiology and pharmacology, we show that BK Ca(2+)-activated K(+) channels are transiently expressed at the OC efferent terminals contacting IHCs and that their activity modulates the release process at this synapse. The effects of dihydropiridines combined with iberiotoxin, a specific BK channel antagonist, strongly suggest that L-type VGCCs negatively regulate the release of ACh by fueling BK channels that are known to curtail the duration of the terminal action potential in several types of neurons.

    Conotoxin-like proteins revealed among short viral proteins

    Naamati G, Askenazi M, Linial M (2010). A predictor for toxin-like proteins exposes cell modulator candidates within viral genomes. Bioinformatics. 2010 Sep 15;26(18):i482-i488. PubMed PMID: 20823311; PubMed Central PMCID: PMC2935411.

    School of Computer Science and Engineering, Department of Biological Chemistry, Sudarsky Center for Computational Biology, Hebrew University of Jerusalem, Israel and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USA.

    Abstract: MOTIVATION: Animal toxins operate by binding to receptors and ion channels. These proteins are short and vary in sequence, structure and function. Sporadic discoveries have also revealed endogenous toxin-like proteins in non-venomous organisms. Viral proteins are the largest group of quickly evolving proteomes. We tested the hypothesis that toxin-like proteins exist in viruses and that they act to modulate functions of their hosts. RESULTS: We updated and improved a classifier for compact proteins resembling short animal toxins that is based on a machine-learning method. We applied it in a large-scale setting to identify toxin-like proteins among short viral proteins. Among the approximately 26 000 representatives of such short proteins, 510 sequences were positively identified. We focused on the 19 highest scoring proteins. Among them, we identified conotoxin-like proteins, growth factors receptor-like proteins and anti-bacterial peptides. Our predictor was shown to enhance annotation inference for many 'uncharacterized' proteins. We conclude that our protocol can expose toxin-like proteins in unexplored niches including metagenomics data and enhance the systematic discovery of novel cell modulators for drug development. AVAILABILITY: ClanTox is available at http://www.clantox.cs.huji.ac.il CONTACT: michall@cc.huji.ac.il.

    Optimization of oxidative folding of conotoxins

    Steiner AM, Bulaj G (2010). Optimization of oxidative folding methods for cysteine-rich peptides: A study of conotoxins containing three disulfide bridges. J Pept Sci. 2010 Sep 2. [Epub ahead of print] PubMed PMID: 20814907.

    Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84108, USA.

    Abstract: The oxidative folding of small, cysteine-rich peptides to selectively achieve the native disulfide bond connectivities is critical for discovery and structure-function studies of many bioactive peptides. As the propensity to acquire the native conformation greatly depends on the peptide sequence, numerous empirical oxidation methods are employed. The context-dependent optimization of these methods has thus far precluded a generalized oxidative folding protocol, in particular for peptides containing more than two disulfides. Herein, we compare the efficacy of optimized solution-phase and polymer-supported oxidation methods using three disulfide-bridged conotoxins, namely micro-SIIIA, micro-KIIIA and omega-GVIA. The use of diselenide bridges as proxies for disulfide bridges is also evaluated. We propose the ClearOx-assisted oxidation of selenopeptides as a fairly generalized oxidative folding protocol. Copyright (c) 2010 European Peptide Society and John Wiley & Sons, Ltd. PMID: 20814907 [PubMed - as supplied by publisher]

    Chemical synthesis of conotoxins Mr1.1 and Lp1.4

    Peng C, Chen W, Sanders T, Chew G, Liu J, Hawrot E, Chi C (2010). Chemical synthesis and characterization of two {alpha}4/7-conotoxins. Acta Biochim Biophys Sin (Shanghai). 2010 Aug 27. [Epub ahead of print] PubMed PMID: 20801929.

    Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China.

    Abstract: alpha-Conotoxins are small disulfide-constrained peptides that act as potent and selective antagonists on specific subtypes of nicotinic acetylcholine receptors (nAChRs). We previously cloned two alpha-conotoxins, Mr1.1 from the molluscivorous Conus marmoreus and Lp1.4 from the vermivorous Conus leopardus. Both of them have the typical 4/7-type framework of the subfamily of alpha-conotoxins that act on neuronal nAChRs. In this work, we chemically synthesized these two toxins and characterized their functional properties. The synthetic Mr1.1 could primarily inhibit acetylcholine (ACh)-evoked currents reversibly in the oocyte-expressed rat alpha7 nAChR, whereas Lp1.4 was an unexpected specific blocker of the mouse fetal muscle alpha1beta1gammadelta receptor. Although their inhibition affinities were relatively low, their unique receptor recognition profiles make them valuable tools for toxin-receptor interaction studies. Mr1.1 could also suppress the inflammatory response to pain in vivo, suggesting that it should be further investigated with respect to its molecular role in analgesia and its mechanism or therapeutic target for the treatment of pain.

    9 September 2010

    Venom Peptides: Bachem and Atheris to collaborate on Melusine

    Press Release: Bachem (SIX: BANB) today announced that the company concluded an agreement with Atheris Laboratories to market their Melusine® libraries, consisting of natural products isolated from animal venoms. The agreement includes services to assist customers with the identification of the active ingredients and to synthesize individual compounds.

    These libraries, isolated from venomous animals, have been fractionated to provide well-defined mixtures. The main components are peptide toxins, a class of interest for the development of peptide based therapeutics and cosmetics. Atheris and Bachem intend to encourage the screening of these compounds to expand and support their applications as drugs.

    Melusine libraries are offered on microplates (96 or 384-wells plates) in multiple formats, from raw extracts to highly purified molecules.

    From the Melusine Shop you can obtain from their Royal Menu fractionated venoms for testing from the following Conus species: Conus ammiralis, Conus coelinae, Conus eburneus, Conus imperialis, Conus leopardis, Conus miles, Conus textile, Conus vexillum and Conus virgo

    For each species, the venom sample is submitted to a specific handling step, followed by a high resolution chromatographic separation into 80 fractions that are dispatched in ready-to-use microplates for HTS. For more information go to the Melusine web site at www.melusine.com

    31 August 2010

    Selectivity of {alpha}-Conotoxin BuIA[T5A;P6O]

    Azam L, Maskos U, Changeux JP, Dowell CD, Christensen S, De Biasi M, McIntosh JM (2010). {alpha}-Conotoxin BuIA[T5A;P6O]: a novel ligand that discriminates between {alpha}6{beta}4 and {alpha}6{beta}2 nicotinic acetylcholine receptors and blocks nicotine-stimulated norepinephrine release. FASEB J. 2010 Aug 25. [Epub ahead of print] PubMed PMID: 20739611.

    *Department of Biology andDepartment of Psychiatry, University of Utah, Salt Lake City, Utah, USA;Unité Neurobiologie Intégrative des Systčmes Cholinergiques, Institut Pasteur, Paris, France; and Department of Neuroscience and Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas, USA.

    Abstract: alpha6* (asterisk indicates the presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) are broadly implicated in catecholamine-dependent disorders that involve attention, motor movement, and nicotine self-administration. Different molecular forms of alpha6 nAChRs mediate catecholamine release, but receptor differentiation is greatly hampered by a paucity of subtype selective ligands. alpha-Conotoxins are nAChR-targeted peptides used by Conus species to incapacitate prey. We hypothesized that distinct conotoxin-binding kinetics could be exploited to develop a series of selective probes to enable study of native receptor subtypes. Proline6 of alpha-conotoxin BuIA was found to be critical for nAChR selectivity; substitution of proline6 with 4-hydroyxproline increased the IC50 by 2800-fold at alpha6/alpha3beta2beta3 but only by 6-fold at alpha6/alpha3beta4 nAChRs (to 1300 and 12 nM, respectively). We used conotoxin probes together with subunit-null mice to interrogate nAChR subtypes that modulate hippocampal norepinephrine release. Release was abolished in alpha6-null mutant mice. alpha-Conotoxin BuIA[T5A;P6O] partially blocked norepinephrine release in wild-type controls but failed to block release in beta4(-/-) mice. In contrast, BuIA[T5A;P6O] failed to block dopamine release in the wild-type striatum known to contain alpha6beta2* nAChRs. BuIA[T5A;P6O] is a novel ligand for distinguishing between closely related alpha6* nAChRs; alpha6beta4* nAChRs modulate norepinephrine release in hippocampus but not dopamine release in striatum.

    In vivo screening of toxins

    Mejia M, Heghinian MD, Busch A, Armishaw CJ, Marí F, Godenschwege TA (2010). A novel approach for in vivo screening of toxins using the Drosophila Giant Fiber circuit. Toxicon. 2010 Aug 17. [Epub ahead of print] PubMed PMID: 20723555.

    Department of Biological Sciences, Florida Atlantic University, 777 Glades Road, Boca Raton Fl 33431, USA.

    Abstract: Finding compounds that affect neuronal or muscular function is of great interest as potential therapeutic agents for a variety of neurological disorders. Alternative applications for these compounds include their use as molecular probes as well as insecticides. We have developed a bioassay that requires small amounts of compounds and allows for unbiased screening of biological activity in vivo. For this, we paired administering compounds in a non-invasive manner with simultaneous electrophysiological recordings from a well-characterized neuronal circuit, the Giant Fiber System of Drosophila melanogaster, which mediates the escape response of the fly. The circuit encompasses a variety of neurons with cholinergic, glutamatergic, and electrical synapses as well as neuromuscular junctions. Electrophysiological recordings from this system allow for the detection of compound-related effects against any molecular target on these components. Here, we provide evidence that this novel bioassay works with small molecules such as the cholinergic receptor blocker mecamylamine hydrochloride and the potassium channel blocker tetraethylammonium hydroxide, as well as with venom from Conus brunneus and isolated conopeptides. Conopeptides have been developed into powerful drugs, such as the painkillers Prialt and Xen2174. However, most conopeptides have yet to be characterized, revealing the need for a rapid and straightforward screening method. Our findings show that mecamylamine hydrochloride, as well as the alpha-conotoxin ImI, which is known to be an antagonist of the human alpha7 nicotinic acetylcholine receptor, efficiently disrupted the synaptic transmission of a Drosophila alpha7 nicotinic acetylcholine receptor-dependent pathway in our circuit but did not affect the function of neurons with other types of synapses. This demonstrates that our bioassay is a valid tool for screening for compounds relevant to human health.

    26 August 2010

    A new superfamily of E-conotoxins

    Liu Z, Yu Z, Liu N, Zhao C, Hu J, Dai Q (2010). cDNA cloning of conotoxins with framework XII from several Conus species. Acta Biochim Biophys Sin (Shanghai). 42: 656-661.

    Beijing Institute of Biotechnology, Beijing 100071, China.

    Abstract: In our efforts for cloning novel I(2)-superfamily conotoxins using the signal peptide sequence, we identified a novel conotoxin Lt12.4 from Conus litteratus. This gene has a framework XII (-C-C-C-C-CC-C-C-), which is distinct from the cysteine pattern I(2)-superfamily conotoxin (-C-C-CC-CC-C-C-). Subsequently, we found the signal peptide sequence of Lt12.4 by 5'-RACE. Using this new sequence, we identified another five novel conotoxins with this cysteine pattern from four Conus species (Conus eburneus, Conus imperialis, Conus marmoreus, and C. litteratus). These novel conotoxins have the same cysteine pattern as the reported Gla-TxX and Gla-MII, and may contain Gla residues. Furthermore, they have the highly conserved signal peptide and hypervariable mature peptide sequences, and widely exist in Conus species. Therefore, it could be defined as a new superfamily of E-conotoxins.

    Binding of mu-conotoxin KIIIA and TTX to sodium channels

    French RJ, Yoshikami D, Sheets MF, Olivera BM. (2010) The tetrodotoxin receptor of voltage-gated sodium channels--perspectives from interactions with mu-conotoxins. Mar Drugs. 8: 2153-2161

    Department of Physiology and Pharmacology, University of Calgary, and Hotchkiss Brain Institute, 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4N1, Canada. french@ucalgary.ca

    Abstract: Neurotoxin receptor site 1, in the outer vestibule of the conducting pore of voltage-gated sodium channels (VGSCs), was first functionally defined by its ability to bind the guanidinium-containing agents, tetrodotoxin (TTX) and saxitoxin (STX). Subsequent studies showed that peptide mu-conotoxins competed for binding at site 1. All of these natural inhibitors block single sodium channels in an all-or-none manner on binding. With the discovery of an increasing variety of mu-conotoxins, and the synthesis of numerous derivatives, observed interactions between the channel and these different ligands have become more complex. Certain mu-conotoxin derivatives block single-channel currents partially, rather than completely, thus enabling the demonstration of interactions between the bound toxin and the channel's voltage sensor. Most recently, the relatively small mu-conotoxin KIIIA (16 amino acids) and its variants have been shown to bind simultaneously with TTX and exhibit both synergistic and antagonistic interactions with TTX. These interactions raise new pharmacological possibilities and place new constraints on the possible structures of the bound complexes of VGSCs with these toxins.

    The alpha7 nAChR agonist, PNU-282987, protects against neuropathic pain

    Pacini A, Di Cesare Mannelli L, Bonaccini L, Ronzoni S, Bartolini A, Ghelardini C (2010). Protective effect of alpha7 nAChR: behavioural and morphological features on neuropathy. Pain. 150: 542-549.

    Dept of Anatomy Histology and Forensic Medicine, University of Florence, Viale Morgagni 85, 50139 Florence, Italy.

    Abstract: Traumatic, toxic or metabolic damage to the nervous system is the etiological foundation of neuropathic pain. Neuropathies are extremely difficult to treat and available drugs rarely joint an anti-hyperalgesic with a neurorestorative effect. From the literature, evidences support the alpha7 nicotinic receptor (nAChR) subtype as having a role in neuropathic pain as well as possessing neuroprotective properties. Aimed to inquire the anti-neuropathic effect of the alpha7 nAChR stimulation, we evaluated the pharmacological profile of the alpha7 nAChR agonist PNU-282987 on pain and on morphological alterations induced in the rat sciatic nerve by loose ligation (CCI). Acute administration of PNU-282987, 10 and 30 mg kg(-1) p.o. (15 days after ligation), was able to reduce hyperalgesia in a methyllicaconitine-reversed manner. This alpha7 nAChR agonist exerted no analgesic effects. Chronic PNU-282987 treatments, 30 mg kg(-1) once a day for 7 days and 10 mg kg(-1) for 28 days, were able to decrease pain perception. The histological studies highlighted that the ligation induces oedema and macrophagic infiltrate. Moreover, osmicated preparations revealed a decrease in axons' compactness and diameter, together with a significant loss of myelin sheath. Repeated treatment with PNU-282987 reduced the presence of oedema and macrophagic infiltrate and, on the coronal sections of the nerve, a significant higher myelin sheath, axonal diameter and number of fibers were observable. These results strongly suggest the pivotal role of alpha7 nAChR in the neuroprotection during neuropathy.

    18 August 2010

    Exploring Biodiversity: The Search for New Medicines

    The Howard Hughes Medical Institute has produced a DVD in which two leading biologists, Bonnie L. Bassler PhD and Baldomero Olivera PhD chart paths of medical discovery through a deeper understanding of biodiversity. These lectures are the outcome of the Howard Hughes Medical Institute 'Holiday Lectures on Science', for December 2009.

    Dr. Baldomero Olivera's presentation includes 4 animations:

  • Prialt blocks motor synapse in fish
  • Prialt blocks pain signaling in mice
  • Motor-cabal toxins block motor neuron synapses
  • Lightening-strike cabal acts like a Taser.

    The two lectures by Olivera are:

  • From Venoms to Drugs
  • Biodiversity at a Snail's Pace

    and numerous video clips demonstrating cone shell envenomation. For a printable list of the lecture summaries go to http://www.hhmi.org/biointeractive/hl/2009_summaries.html

    The two-disc set includes:

  • Four full-length presentations with detailed chapter stops.
  • Direct access to animations and video to illustrate a topic.
  • Special features on symbiotic bioluminescence
  • Philippine maritime culture, seashell taxonomy
  • Discussion on biodiversity and conservation featuring Dr. E.O. Wilson and Dr. Eric Chivian.

    For further information, go to http://www.hhmi.org/biointeractive/hl/.

    The Howard Hughes Medical Institute is a philanthropy that supports biomedical research and science education. As part of that mission, HHMI distributes educational materials free of charge to those that request them. These materials include printed matter as well as videos, CD-ROMs, and DVDs. The DVD version of the 2009 Holiday Lectures on Science can be ordered from http://www.hhmi.org/biointeractive/ordermaterials.html

    Seashell Phylogeny and Evolution Teacher Guide
    The seashell phylogeny and evolution activity is an interactive online activity that can be done in class or assigned as homework.

    Includes work sheets, information and videos on molluscs (in particular, cone shells). CLICK HERE

    13 August 2010

    Florida cones C. pealii and C. stearnsii

    As reported on CONCH-L mailing list, Marlo marlo@cfl.rr.com with help from Harry Lee, has taken a position on the “jaspideus question” as it applies to shells found in Florida and created a presentation on what is found in Florida and how to distinguish between the two species: C. pealii Green, 1830; and C. stearnsii Conrad, 1869, Stearns' Cone.

    10 August 2010

    Diselenium bonded analogues of conotoxins

    Raffa RB (2010). Diselenium, instead of disulfide, bonded analogs of conotoxins: novel synthesis and pharmacotherapeutic potential. Life Sci. 2010 Aug 4. [Epub ahead of print] PubMed PMID: 20691706.

    Abstract: The venoms of the cone snail (Conus) contain toxic peptides (conotoxins) that have remarkable selectivity for subtypes of a variety of mammalian voltage- and ligand-gated ion channels, G protein-coupled receptors, and neurotransmitter transporters. They thus have tremendous potential as pharmacologic tools. Less toxic analogs or mimetics could be highly-selective pharmacotherapeutic agents at their target sites. For this reason, conopeptides have been extensively studied and have progressed to clinical trials and even regulatory approval. However, the synthesis of the peptides remains difficult and stability and toxicity remain problems. A novel synthesis and testing of analogs incorporating diselenium bonds between selenocysteine residues in place of disulfide bonds between cysteine residues has recently been reported. The technique results in analogs that retain the folding of the native peptides, are more potent, and have the same or greater biological activity. Copyright © 2010. Published by Elsevier Inc.

    Two novel conotoxins from Conus pulicarius and Conus tessulatus

    Peng C, Ye M, Wang Y, Shao X, Yuan D, Liu J, Hawrot E, Wang C, Chi C (2010). A new subfamily of conotoxins belonging to the A-superfamily. Peptides. 2010 Aug 3.[Epub ahead of print] PubMed PMID: 20691232.

    Institute of Protein Research, Tongji University, Shanghai 200092, China; Ministry of Education Key Laboratory of Systems Biomedicine, Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, China.

    Abstract: Two novel conotoxins from vermivorous cone snails Conus pulicarius and Conus tessulatus, designated as Pu14.1 and ts14a, were identified by cDNA cloning and peptide purification, respectively. The signal sequence of Pu14.1 is identical to that of alpha-conotoxins, while its predicted mature peptide, pu14a, shares high sequence similarity with ts14a, with only one residue different in their first intercysteine loop, which contains ten residues and is rich in proline. Both pu14a and ts14a contain four separate cysteines in framework 14 (C-C-C-C). Peptide pu14a was chemically synthesized, air oxidized, and the connectivity of its two disulfide bonds was determined to be C1-C3, C2-C4, which is the same as found in alpha-conotoxins. The synthetic pu14a induced a sleeping symptom in mice and was toxic to freshwater goldfish upon intramuscular injection. Using the Xenopus oocyte heterologous expression system, 1muM of pu14a demonstrated to inhibit the rat neuronal alpha3beta2-containing as well as the mouse neuromuscular alpha1beta1gammadelta subtypes of nicotinic acetylcholine receptors, and then rapidly dissociated from the receptors. However, this toxin had no inhibitory effect on potassium channels in mouse superior cervical ganglion neurons. According to the identical signal sequence to alpha-conotoxins, the unique cysteine framework and molecular target of pu14a, we propose that pu14a and ts14a may represent a novel subfamily in the A-superfamily, designated as alpha1-conotoxins. Copyright © 2010. Published by Elsevier Inc.

    Selectivity of conantokins

    Sheng Z, Castellino FJ (2010). Specific determinants of conantokins that dictate their selectivity for the NR2B subunit of N-methyl-D-aspartate receptors. Neuroscience. 2010 Aug 2. [Epub ahead of print] PubMed PMID: 20688135.

    W.M. Keck Center for Transgene Research and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556.

    Abstract: Conantokins are naturally-occurring small peptide antagonists of ion flow through NMDA/glycine activated-N-methyl-D-aspartate receptor (NMDAR) ion channels. One member of the conantokin family, conantokin (con)-G, a 17-residue peptide, is selective for NMDARs containing the NR2B subunit, whereas the homologous peptides, con-T and con-R, show broader selectivity for NR2 subunits. In this study, con-G, con-R, and con-T variants were chemically synthesized and employed to investigate their subunit selectivities as inhibitors of agonist-evoked ion currents in HEK-293 cells expressing various combinations of NMDAR subunits that contain NR1a or NR1b combined with NR2A or NR2B. Using truncation and point mutants, as well as chimeric conantokins, we determined that the N-terminus of con-G contains all the determinants for NR2B selectivity. With this information, a large number of conantokin variants were synthesized and used to establish minimal sequence determinants for selectivity. Tyr at position 5 broadens the NR2 selectivity, and recovery of NR2B selectivity in Tyr5 peptides was achieved by incorporating Ala or Gly at position 8. NR2B selectivity in con-R can be conferred through deletion of the Ala at position 10, thereby shifting the gamma-carboxyglutamate (Gla) at position 11 to position 10, where a Gla naturally occurs in con-G and con-T. The nature of the amino acid at position 6 is also linked to subunit selectivity. Our studies suggest that the molecular determinants of conantokins that dictate NMDAR subunit selectivity are housed in specific residues of the N-termini of these peptides. Thus, it is possible to engineer desired NMDAR functional properties into conantokin-based peptides. Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

    Stability of conotoxin GI analogues

    Jiang N, Ma J (2010). Influence of Disulfide Connectivity, Electrostatics, and Hydrophobicity on the Conformational Variations of alpha-Conotoxin GI Single-Disulfide Analogues: Simulations with Polarizable Force Field. J Phys Chem B. 2010 Aug 5. [Epub ahead of print] PubMed PMID: 20687571.

    School of Chemistry and Chemical Engineering, Institute of Theoretical and Computational Chemistry, Key Laboratory of Mesoscopic Chemistry of MOE, Nanjing University, Nanjing, 210093, People's Republic of China.

    Abstract: The roles of the disulfide bridge, electrostatics, and hydrophobic/hydrophilic effects in the structural stability and conformational changes of six single-disulfide analogues of alpha-conotoxin GI(2-7;3-13) in aqueous solution are investigated by using molecular dynamics simulations with a fragment-based polarization model ( J. Phys. Chem. A 2008 , 112 , 9854. ). It is found that the relative stabilities are largely determined by the dipole-dipole interactions between secondary structure-based fragments, revealing the favorable effect of polar residues on conformational stabilities. The loop size closely correlates to not only the thermodynamic stability but also the local geometry of disulfide bridge. The disulfide loops with no more than five residues [GI(2-7), GI(3-7), and GI(7-13)] choose the left-handed disulfide conformation, while the larger loops [with nine and 10 residues in GI(3-13) and GI(2-13)] and a smaller disulfide loop [GI(2-3) without intercysteine residue] prefer the right-handed configuration. In the left-handed analogues, the dihedral angles concerning disulfide bonds decrease subtly along with the enlargement of disulfide loops. A converse dihedral angle and loop size relationship is found in the right-handed isomers. These results are rationalized by the strain energy of the disulfide bond as well as the electrostatic and van der Waals interactions between cysteine pairs. The single-disulfide analogues also exhibit much higher conformational diversity than the native GI. The important role of the size of hydrophobic core in the conformational evolution is also demonstrated in terms of the radius of gyration of the hydrophobic region. The radial distribution functions show the significant solvent-solute hydrogen bonding, implying that the interplay between the intermolecular and the intramolecular interactions control the dynamic process of GI single-disulfide analogues.

    Imaging alpha7 nicotinic receptors

    Hone AJ, Whiteaker P, Mohn JL, Jacob MH, McIntosh JM (2010). Alexa Fluor 546-ArIB[V11L;V16A] is a potent ligand for selectively labeling alpha7 nicotinic acetylcholine receptors. J Neurochem. 2010 Aug;114(4):994-1006. Epub 2010 May 18. PubMed PMID: 20492354.

    Interdepartmental Program in Neuroscience, University of Utah, Salt Lake City, Utah 84112, USA.

    Abstract: The alpha7* (*denotes the possible presence of additional subunits) nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the vertebrate nervous system and implicated in neuropsychiatric disorders that compromise thought and cognition. In this report, we demonstrate that the recently developed fluorescent ligand Cy3-ArIB[V11L;V16A] labels alpha7 nAChRs in cultured hippocampal neurons. However, photobleaching of this ligand during long image acquisition times prompted us to develop a new derivative. In photostability studies, this new ligand, Alexa Fluor 546-ArIB[V11L;V16A], was significantly more resistant to bleaching than the Cy3 derivative. The classic alpha7 ligand alpha-bungarotoxin binds to alpha1* and alpha9* nAChRs. In contrast, Alexa Fluor 546-ArIB[V11L;V16A] potently (IC(50) 1.8 nM) and selectively blocked alpha7 nAChRs but not alpha1* or alpha9* nAChRs expressed in Xenopus oocytes. Selectivity was further confirmed by competition binding studies of native nAChRs in rat brain membranes. The fluorescence properties of Alexa Fluor 546-ArIB[V11L;V16A] were assessed using human embryonic kidney-293 cells stably transfected with nAChRs; labeling was observed on cells expressing alpha7 but not cells expressing alpha3beta2, alpha3beta4, or alpha4beta2 nAChRs. Further imaging studies demonstrate that Alexa Fluor 546-ArIB[V11L;V16A] labels hippocampal neurons from wild-type mice but not from nAChR alpha7 subunit-null mice. Thus, Alexa Fluor 546-ArIB[V11L;V16A] represents a potent and selective ligand for imaging alpha7 nAChRs.

    4 August 2010

    Efficient regioselective folding of conopeptides

    Han TS, Zhang MM, Gowd KH, Walewska A, Yoshikami D, Olivera BM, Bulaj G (2010). Disulfide-Depleted Selenoconopeptides: a Minimalist Strategy to Oxidative Folding of Cysteine-Rich Peptides. ACS Med Chem Lett. 1: 140-144.

    Departments of Biology, University of Utah, Salt Lake City, Utah, 84112, USA.

    Abstract: Despite the therapeutic promise of disulfide-rich, peptidic natural products, their discovery and structure/function studies have been hampered by inefficient oxidative folding methods for their synthesis. Here we report that converting the three disulfide-bridged mu-conopeptide KIIIA into a disulfide-depleted selenoconopeptide (by removal of a noncritical disulfide bridge and substitution of a disulfide- with a diselenide-bridge) dramatically simplified its oxidative folding while preserving the peptide's ability to block voltage-gated sodium channels. The simplicity of synthesizing disulfide-depleted selenopeptide analogs containing a single disulfide bridge allowed rapid positional scanning at Lys7 of mu-KIIIA, resulting in the identification of K7L as a mutation that improved the peptide's selectivity in blocking a neuronal (Na(v)1.2) over a muscle (Na(v)1.4) subtype of sodium channel. The disulfide-depleted selenopeptide strategy offers regioselective folding compatible with high throughput chemical synthesis and on-resin oxidation methods, and thus shows great promise to accelerate the use of disulfide-rich peptides as research tools and drugs.

    Ziconotide combination therapy: Review

    Wallace MS, Rauck RL, Deer T (2010). Ziconotide Combination Intrathecal Therapy: Rationale and Evidence. Clin J Pain. 2010 Jul 15. [Epub ahead of print]

    University of California, San Diego, School of Medicine, La Jolla, CA Wake Forest University Health Sciences, Winston-Salem, NC; Center for Pain Relief, Charleston, WV.

    Abstract: BACKGROUND: Ziconotide is a nonopioid intrathecal analgesic used to manage moderate to severe chronic pain. Although ziconotide is approved in the United States for intrathecal monotherapy only, it is often used in combination with other intrathecal drugs in clinical practice. OBJECTIVES: The need exists for a critical assessment of the currently available published literature on ziconotide combination therapy. This review summarizes and evaluates the publications from preclinical and clinical peer-reviewed experiments that have investigated the safety and effectiveness of ziconotide in combination with a variety of other drugs. METHODS/RESULTS: Eleven relevant publications were identified through a systematic search of multiple databases. DISCUSSION: In preclinical studies, additive or synergistic antinociceptive effects were discovered when ziconotide was used in combination with morphine, clonidine, or baclofen; however, no additional antinociceptive effects were observed when bupivacaine was added to ziconotide therapy. Safety data from animal studies revealed that ziconotide did not exacerbate morphine-induced respiratory depression, or clonidine-induced hypotension or bradycardia; however, ziconotide did potentiate morphine-induced hypotension and inhibition of gastrointestinal tract motility. Results from 2 open-label trials indicated that combination ziconotide and morphine therapy produced greater analgesia than was produced by the use of either drug alone. Preliminary support for the use of ziconotide in combination with morphine, baclofen, or hydromorphone was provided by case studies. CONCLUSIONS: Although clinical and preclinical studies provide some support for the use of ziconotide in combination with morphine, hydromorphone, clonidine, or baclofen, strong evidence-based data are limited. Controlled, long-term clinical trials are warranted.

    26 July 2010

    Scientists engineer an orally active conotoxin from Conus victoriae

    Conus victoriae Conotoxin cVc1.1

    Article by Bethany Halford, "Pain Relief From Snail Spit" reported in Chemical & Engineering News 88 (30) 39-40, July 26, 2010

    Researchers led by David J. Craik of the Insittute for Molecular Bioscience at the University of Queensland discovered that by linking the N-terminus of alpha-conotoxin Vc1.1 - a compound derived from Conus victoriae to its C-terminus, they could make the 16-residue peptide orally active (Angew Chem Int Ed Engl. 122: 1-5 DOI:10.1002/anie.201000620). Craik's cyclized conotoxin "is a prime example of how peptide bioengineering can enhance bioavailability", comments Jon-Paul Bingham, a professor of molecular biosciences and bioengineering at the University of Hawaii, Manoa, noting that an orally active drug with the painkilling power of Prialt "would absolutely revolutionize how we manage chronic and terminal pain".

    For further information scroll down to 15 June entry.

    22 July 2010

    Western Atlantic Cones

    The Cone Collector: Special Issue, Volume 14A July 2010 contains a single important paper (166 pages) by John Tucker on Western Atlantic Cones.

    Background: John Tucker writes in his Introduction "In 1984, Danker Vink published part I of 'The Conidae of the Western Atlantic' in La Conchiglia. A total of 15 parts appeared between 1984 and 1990. It, thus, has been 20 years or longer since these parts appeared. Understanding of what Vink actually said appears to be fading among collectors. Because I think that the Vink papers are an important contribution to the systematics of Western Atlantic cone shells, I decided to review these papers. The goal is to review the 62 taxa that Vink covered in his 15 parts (Table 1), and to present images that are as close as possible to those used by Vink in his papers. I do suggest revisions, but I have tried to keep those in the context of the Vink species".

    Edited by António Monteiro, with layout by André Poremski.
    (See interview with António Monteiro on the Seashell Collector.com)

    Click here to download The Cone Collector #14A (PDF - 6 MB).
    [Earlier issues, #0 - #14, are available for download at http://www.conchology.be/?t=42]

    7 July 2010

    NPY-like conopeptides in the venom of Conus betulinus

    Wu X, Shao X, Guo ZY and Chi CW (2010) Identification of neuropeptide Y-like conopeptides from the venom of Conus betulinus Acta Biochim Biophys Sin. 42: 502-505

    Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China; State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China

    Correspondence E-mail: zhan-yun.guo@tongji.edu.cn (Z.-Y.G.). zwqi@sibs.ac.cn (C.-W.C.)

    Abstract: Neuropeptide Y (NPY) is a ubiquitous endocrine neuropeptide found in vertebrate and invertebrate. In our present work, two NPY-like exocrine conopeptides (designated as cono-NPYs) were first identified in the venom of cone snails. Both cono-NPYs showed sequence characteristics of invertebrate NPYs, suggesting that some exocrine venom peptides are probably evolved from the preexisting endocrine peptides during the evolution of cone snails.

    Development of mu-conotoxins as safe analgesics

    Zhang MM, Han TS, Olivera BM, Bulaj G, Yoshikami D (2010). Mu-conotoxin KIIIA derivatives with divergent affinities versus efficacies in blocking voltage-gated sodium channels. Biochemistry 49: 4804-4812.

    Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA.

    Abstract: The possibility of independently manipulating the affinity and efficacy of pore-blocking ligands of sodium channels is of interest for the development of new drugs for the treatment of pain. The analgesic mu-conotoxin KIIIA (KIIIA), a 16-residue peptide with three disulfide bridges, is a pore blocker of voltage-gated sodium channels, including neuronal subtype Na(V)1.2 (K(d) = 5 nM). At saturating concentrations, KIIIA incompletely blocks the sodium current of Na(V)1.2, leaving a 5% residual current (rI(Na)). Lys7 is an important residue: the K7A mutation decreases both the efficacy (i.e., increases rI(Na) to 23%) and the affinity of the peptide (K(d) = 115 nM). In this report, various replacements of residue 7 were examined to determine whether affinity and efficacy were inexorably linked. Because of their facile chemical synthesis, KIIIA analogues that had as a core structure the disulfide-depleted KIIIA[C1A,C2U,C9A,C15U] (where U is selenocysteine) or ddKIIIA were used. Analogues ddKIIIA and ddKIIIA[K7X], where X represents one of nine different amino acids, were tested on voltage-clamped Xenopus oocytes expressing rat Na(V)1.2 or Na(V)1.4. Their affinities ranged from 0.01 to 36 muM and rI(Na) values from 2 to 42%, and these two variables appeared to be uncorrelated. Instead, rI(Na) varied inversely with side chain size, and remarkably charge and hydrophobicity appeared to be inconsequential. The ability to manipulate a mu-conopeptide's affinity and efficacy, as well as its capacity to interfere with subsequent tetrodotoxin binding, greatly expands its scope as a reagent for probing sodium channel structure and function and may also lead to the development of mu-conotoxins as safe analgesics.

    mu-conotoxins KIIIA and KIIIA[K7A] act synergistically with TTX

    Zhang MM, Gruszczynski P, Walewska A, Bulaj G, Olivera BM, Yoshikami D (2010). Cooccupancy of the outer vestibule of voltage-gated sodium channels by mu-conotoxin KIIIA and saxitoxin or tetrodotoxin. J Neurophysiol. 104: 88-97.

    Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA.

    Abstract: The guanidinium alkaloids tetrodotoxin (TTX) and saxitoxin (STX) are classic ligands of voltage-gated sodium channels (VGSCs). Like TTX and STX, mu-conotoxin peptides are pore blockers but with greater VGSC subtype selectivity. mu-conotoxin KIIIA blocks the neuronal subtype Na(V)1.2 with nanomolar affinity and we recently discovered that KIIIA and its mutant with one fewer positive charge, KIIIA[K7A], could act synergistically with TTX in a ternary peptide x TTX x Na(V) complex. In the complex, the peptide appeared to trap TTX in its normal binding site such that TTX could not readily dissociate from the channel until the peptide had done so; in turn, the presence of TTX accelerated the rate at which peptide dissociated from the channel. In the present study we examined the inhibition of Na(V)1.2, exogenously expressed in Xenopus oocytes, by STX (a divalent cation) and its sulfated congener GTX2/3 (with a net +1 charge). Each could form a ternary complex with KIIIA and Na(V)1.2, as previously found with TTX (a monovalent cation), but only when STX or GTX2/3 was added before KIIIA. The KIIIA x alkaloid x Na(V) complex was considerably less stable with STX than with either GTX2/3 or TTX. In contrast, ternary KIIIA[K7A] x alkaloid x Na(V) complexes could be formed with either order of ligand addition and were about equally stable with STX, GTX2/3, or TTX. The most parsimonious interpretation of the overall results is that the alkaloid and peptide are closely apposed in the ternary complex. The demonstration that two interacting ligands ("syntoxins") occupy adjacent sites raises the possibility of evolving a much more sophisticated neuropharmacology of VGSCs.

    Conantokins enhance neuronal survival and plasticity

    Huang L, Balsara RD, Sheng Z, Castellino FJ.(2010) Conantokins inhibit NMDAR-dependent calcium influx in developing rat hippocampal neurons in primary culture with resulting effects on CREB phosphorylation. Mol Cell Neurosci. June 21. [Epub ahead of print] PubMed PMID: 20600930.

    W.M. Keck Center for Transgene Research and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556.

    Abstract: The effects of conantokin (con)-G, con-R[1-17], and con-T on ion flow through N-methyl-D-aspartate receptor (NMDAR) ion channels, were determined in cultured primary rat hippocampal neurons. The potency of con-G diminished, whereas inhibition by con-R[1-17] and con-T did not change, as the neurons matured. Con-G, con-R[1-17], and con-T effectively diminished NMDA-induced Ca(2+) influx into the cells. A similar age-dependent decrease in con-G-mediated inhibition of the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) was observed, compared to con-R[1-17] and con-T. The effects of the conantokins on NMDA-induced cAMP response element-binding protein (CREB) phosphorylation in immature (DIV 9) and mature (DIV 16) neurons showed that, at DIV 9, con-G, con-R[1-17], and con-T inhibited NMDA-mediated P-CREB levels, whereas in DIV 16 neurons the conantokins did not inhibit overall levels of NMDA-induced P-CREB. In contrast, P-CREB levels were enhanced through inhibition of the protein phosphatases, PP1 and PP2B (calcineurin). This ability of conantokins to sustain CREB phosphorylation can thus enhance neuronal survival and plasticity.

    4 July 2010

    Synthesis of Cyclic Peptides : Review

    Clark RJ, Craik DJ. (2010) Invited review : Native chemical ligation applied to the synthesis and bioengineering of circular peptides and proteins.Biopolymers. 94: 414-422

    Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.

    Abstract: Native chemical ligation methodology developed in the laboratory of Stephen Kent is a versatile approach to the linkage of peptide fragments using a native peptide bond. It is readily adaptable to the task of joining the N- and C-termini of peptides to produce cyclic molecules and we have used it for the cyclization of a range of disulfide-rich peptides. Specifically, it has been valuable for the synthesis of cyclotides, naturally occurring peptides characterized by a head-to-tail cyclized backbone and a knotted arrangement of three conserved disulfide bonds. Cyclotides have a diverse range of biological activities, including anti-HIV, antimicrobial, and insecticidal activities. They are ultrastable owing to their cyclic cystine knot motif, and native chemical ligation methodology has been invaluable in the synthesis of a range of native and modified cyclotides to explore their structure-activity relationships and applications in drug design. Similar studies have also been applied to a smaller cyclic peptide produced in sunflower seeds, sunflower trypsin inhibitor-1, which also shows promise as a template in drug design applications. We have also found native chemical ligation to be a valuable methodology for the cyclization of conotoxins, small disulfide-rich peptides from the venoms of marine cone snails. Conotoxins target a range of ions channels and receptors and are exciting leads in drug design applications. The synthetic cyclization of conotoxins with peptide linkers stabilizes them and improves their biopharmaceutical properties. In summary, this article illustrates the use of native chemical ligation technology in the cyclization of cyclotides, sunflower trypsin inhibitor-1, and conotoxins in our laboratory. (c) 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 414-422, 2010.

    26 June 2010

    Conantokin R1-A from Conus rolani

    Gowd KH, Watkins M, Twede VD, Bulaj GW, Olivera BM. (2010) Characterization of conantokin Rl-A: molecular phylogeny as structure/function study. J Pept Sci. 16(8):375-82.

    Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA.

    Abstract: A multidisciplinary strategy for discovery of new Conus venom peptides combines molecular genetics and phylogenetics with peptide chemistry and neuropharmacology. Here we describe application of this approach to the conantokin family of conopeptides targeting NMDA receptors. A new conantokin from Conus rolani, ConRl-A, was identified using molecular phylogeny and subsequently synthesized and functionally characterized. ConRl-A is a 24-residue peptide containing three gamma-carboxyglutamic acid residues with a number of unique sequence features compared to conantokins previously characterized. The HPLC elution of ConRl-A suggested that this peptide exists as two distinct, slowly exchanging conformers. ConRl-A is predominantly helical (estimated helicity of 50%), both in the presence and absence of Ca(++). The order of potency for blocking the four NMDA receptor subtypes by ConRl-A was NR2B > NR2D > NR2A > NR2C. This peptide has a greater discrimination between NR2B and NR2C than any other ligand reported so far. In summary, ConRl-A is a new member of the conantokin family that expands our understanding of structure/function of this group of peptidic ligands targeted to NMDA receptors. Thus, incorporating phylogeny in the discovery of novel ligands for the given family of ion channels or receptors is an efficient means of exploring the megadiverse group of peptides from the genus Conus. Copyright (c) 2010 European Peptide Society and John Wiley & Sons, Ltd.

    Six novel conopeptides from Conus parius

    Jimenez EC, Olivera BM. (2010) Divergent M- and O-superfamily peptides from venom of fish-hunting Conus parius. Peptides. 2010 Jun 4. [Epub ahead of print] PubMed PMID: 20570703.

    Department of Physical Sciences, College of Science, University of the Philippines Baguio, Baguio City 2600, Philippines; Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.

    Abstract: Six novel peptides from the piscivorous cone snail, Conus parius were purified by reverse-phase HPLC fractionation of crude venom. With the use of matrix-assisted laser desorption ionization mass spectrometry and standard Edman sequencing methods, the peptides were characterized. Two peptides were identified as members of the m-2 and m-4 branches of the M-superfamily and were designated as pr3a and pr3b, while four peptides were identified as members of the O-superfamily and were designated as pr6a, pr6b, pr6c and pr6d. Peptide pr3a differs from the majority of the M-superfamily peptides in the presence of two prolines, which are not modified to 4-trans-hydroxyproline. In peptide pr3b, five amino acids out of the 16 non-cysteine residues are identical with those of mu-GIIIA and mu-PIIIA, suggesting that pr3b may be a divergent mu-conotoxin. Peptide pr6a is notable because of its extreme hydrophobicity. Peptide pr6c has three prolines that are unhydroxylated. Peptides pr6b and pr6d differ from the previously characterized O-superfamily peptides in the presence of an extended N-terminus consisting of six amino acids. Peptides pr3a, pr3b, pr6a and pr6b were demonstrated to be biologically active when injected intraperitoneally in fish. The identification and characterization of these peptides in venom of a fish-hunting species establish the divergence of gene products and their patterns of post-translational modification within superfamilies in a single Conus species. Copyright © 2010 Elsevier Inc. All rights reserved.

    Large conopeptides from the venom of Conus purpuascens

    Möller C, Marí F. (2010) 9.3 kDa Components of the injected venom of Conus purpurascens define a New 5-disulfide conotoxin framework. Biopolymers. 2010 May 26. [Epub ahead of print] PubMed PMID: 20564010.

    Department of Chemistry & Biochemistry, Florida Atlantic University, Boca Raton, Fl 33431, USA.

    Abstract: The 83-residue conopeptide (p21a) and its corresponding non-hydroxylated analog were isolated from the injected venom of Conus purpurascens. The complete conopeptide sequences were derived from Edman degradation sequencing of fragments from tryptic, chymotryptic and cyanogen bromide digestions. p21a has a unique, ten-cystine/5-disulfide 7-loop framework with extended 10-residue N-terminus and a 5-residue C-terminus tails, respectively. p21a has a 48% sequence homology with a recently described 13-cystine conopeptide, con-ikot-ikot, isolated from the dissected venom of the fish-hunting snail Conus striatus. However, unlike con-ikot-ikot, p21a does not form a dimer of dimers. MALDI-TOF mass spectrometry suggests that p21a may form a non-covalent dimer. p21a is an unusually large conotoxin and insofar is the largest isolated from injected venom. p21a provides evidence that the Conus venom arsenal includes larger molecules that are directly injected into the prey. Therefore, cone snails can utilized toxins that are comparable in size to the ones commonly found in other venomous animals. (c) 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2010.

    16 June 2010

    Evolution of Conus californicus

    Biggs JS, Watkins M, Puillandre N, Ownby JP, Lopez-Vera E, Christensen S, Moreno KJ, Bernaldez J, Licea-Navarro A, Corneli PS, Olivera BM (2010) Evolution of Conus peptide toxins: analysis of Conus californicus Reeve, 1844. Mol Phylogenet Evol. 56: 1-12.

    University of Guam Marine Laboratory, UOG Station, Mangilao, GU 96923, USA. biggs.js@gmail.com

    Abstract: Conus species are characterized by their hyperdiverse toxins, encoded by a few gene superfamilies. Our phylogenies of the genus, based on mitochondrial genes, confirm previous results that C. californicus is highly divergent from all other species. Genetic and biochemical analysis of their venom peptides comprise the fifteen most abundant conopeptides and over 50 mature cDNA transcripts from the venom duct. Although C. californicus venom retains many of the general properties of other Conus species, they share only half of the toxin gene superfamilies found in other Conus species. Thus, in these two lineages, approximately half of the rapidly diversifying gene superfamilies originated after an early Tertiary split. Such results demonstrate that, unlike endogenously acting gene families, these genes are likely to be significantly more restricted in their phylogenetic distribution. In concordance with the evolutionary distance of C. californicus from other species, there are aspects of prey-capture behavior and prey preferences of this species that diverges significantly from all other Conus. Copyright 2010 Elsevier Inc. All rights reserved.

    29 June 2010

    US Patent on dicarba ImI

    Robinson A and Jamana E (2010) Conotoxin analogues and methods for synthesis of intramolecular dicarba bridge-containing peptides. US Patent #7,745,573

    Robinson, Andrea (St. Kilda, AU); Elaridi, Jomana (Endeavour Hills, AU)

    Abstract: According to the present invention, there is provided a range of new conotoxin derivatives and methods for synthesizing these analogues and other intramolecular dicarba bridge-containing peptides, including dicarba-disulfide bridge-containing peptides.

    Assignee: Polychip Pharmaceuticals Pty Ltd. (Toorak, AU) Monash University (Clayton, AU)

    RELATED APPLICATIONS: This application claims the benefit of the filing date of Australian Patent Application No. 2006900798, which was filed Feb. 17, 2006. The contents of that application are hereby incorporated by reference in their entirety.

    15 June 2010

    Orally active cyclic Conotoxin Vc1.1

    Clark RJ, Jensen J, Nevin ST, Callaghan BP, Adams DJ, Craik DJ. (2010) The Engineering of an Orally Active Conotoxin for the Treatment of Neuropathic Pain. Angew Chem Int Ed Engl. 122: 1-5 DOI:10.1002/anie.201000620

    Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072 (Australia), Fax: (+61) 7-3346-2101.

    Abstract: From killers to curers: Peptides from cone snail venoms are potential therapeutic agents for the treatment of neuropathic pain. Unfortunately, these peptides suffer from the disadvantage of short biological half-lives and poor activity when taken orally. A new orally active conotoxin was developed to solve these problems.

    Extract: "In summary, we have developed an orally active peptide based on a natural conotoxin, with exciting potential for the treatment of neuropathic pain. Our orally active peptide exhibits higher selectivity and potency for GABA(B) receptor-mediated N-type Ca2+ channel currents, the proposed target for analgesia, than the parent linear peptide...... By developing an orally active analogue of Vc1.1, we have substantially enhanced its potential as a new therapy for neuropathic pain."

    See also: Article by Jessica Hamzelou, Sea snail venom provides potent pain relief reported in NewScientist 10 June 2010 No. 2765, page 12.

    EXTRACT: "Sea snail venom could become the gold standard for the relief of nerve-related pain following the development of a pill that is 100 times as potent as leading treatments.
    Current treatements for neuropathic pain include morphine, which is highly addictive, and gabapentin, which both act on nerve receptors. Sea snail venom had been suggested as a good alternative because it consistes of a cocktail of peptides, known as conotoxins. These act to immobilize the snail's prey by blocking nerve-cell conduction, but in mammals the peptides are an effective analgesic.
    The only conotoxin-derived drug approved for human use is ziconotide. Unfortunately, it is susceptible to breakdown by enzymes in the saliva and gut, so it is administered by a pump surgically inserted into the abdominal wall, making it an invasive and expensive treatment.
    To solve this problem, David Craik and his team at the University of Queensland in Brisbane, Australia, have developed the first "orally active" conotoxin drug. They started with a synthetic version of conotoxin. Since the enzymes that break down the drug usually act at the ends of the conotoxin molecule, the team used a chain of amino-acids to join up these ends, to form a circular structure. They found this version to be resistant to enzymes in the body.
    Craik's team tested the conotoxin in rats with neruopathic pain. They found that a single oral dose significantly reduced pain using a standard test - how much pressure the rat could withstand before withdrawing its paw. Compared with gabapentin, conotoxin was judged to be 100 times as potent (Angewandte Chemie, DOI: 10.1002/anie.2010006.20).

    Because conotoxin is so potent only very small doses are needed to provide pain relief, reducing the risk of side effects, says Craik. His team has applied for approval from the US Food and Drug Administration for a trial in humans."

    See also: Article by Bethany Halford, Pain Relief From Snail Spit reported in Chemical & Engineering News 88 (30) 39-40, July 26, 2010

    31 May 2010

    Marine pharmaceuticals - Review

    Mayer AM, Glaser KB, Cuevas C, Jacobs RS, Kem W, Little RD, McIntosh JM, Newman DJ, Potts BC, Shuster DE (2010). The odyssey of marine pharmaceuticals: a current pipeline perspective. Trends Pharmacol Sci. 31: 255-265.

    Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA.

    Abstract:The global marine pharmaceutical pipeline consists of three Food and Drug Administration (FDA) approved drugs, one EU registered drug, 13 natural products (or derivatives thereof) in different phases of the clinical pipeline and a large number of marine chemicals in the preclinical pipeline. In the United States there are three FDA approved marine-derived drugs, namely cytarabine (Cytosar-U((R)), Depocyt((R))), vidarabine (Vira-A((R))) and ziconotide (Prialt((R))). The current clinical pipeline includes 13 marine-derived compounds that are either in Phase I, Phase II or Phase III clinical trials. Several key Phase III studies are ongoing and there are seven marine-derived compounds now in Phase II trials. The preclinical pipeline continues to supply several hundred novel marine compounds every year and those continue to feed the clinical pipeline with potentially valuable compounds. From a global perspective the marine pharmaceutical pipeline remains very active, and now has sufficient momentum to deliver several additional compounds to the marketplace in the near future; this review provides a current view of the pipeline. Copyright © 2010 Elsevier Ltd. All rights reserved.

    Extract: "Ziconotide (Prialt1) is the synthetic equivalent of a naturally occurring 25-amino acid peptide, w-conotoxin MVIIA (Figure 1), originally isolated from the venom of the fish-hunting marine snail Conus magus [7]. Ziconotide is a potent analgesic with a completely novel mechanism of action [8,9]. Various subtypes of voltage-gated calcium channels have been identified in the nervous system. Ziconotide reversibly blocks N-type calcium channels located on primary nociceptive afferent nerves in the superficial layers of the dorsal horn of the spinal cord. Binding of ziconotide to presynaptic N-type calcium channels reduces the release of excitatory neurotransmitter release from the primary afferent nerve terminals [10,11]. Tolerance to drug effects is a major limiting factor in opiate-based therapies; unlike opiates, ziconotide does not produce tolerance [12]. A recent search in PubMed (December 2009) using the search terms v-conotoxin MVIIA, SNX-111, ziconotide or Prialt1 retrieved 261 publications in the peer-reviewed literature. Ziconotide does not readily cross the blood–brain barrier and is therefore delivered intrathecally via an implantable pump or temporarily by an external microinfusion device [11,13,14]. Ziconotide received FDA approval in December 2004 and is currently labeled for the management of severe chronic pain in patients with cancer or AIDS [14,15] for whom intrathecal (IT) therapy is warranted, and who are intolerant of or refractory to other treatments, such as systemic analgesics, adjunctive therapies or IT morphine. Prialt1 is marketed by Elan Corporation, PLC (http://www.elan.com/therapies/products/prialt.asp). Ziconotide has also been approved by the EMEA [16]." Copyright © 2010 Elsevier Ltd. All rights reserved.

    For data on Ziconotide, see also: http://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?id=6893
    or Downlad the FDA offical download about Ziconotide

    29 May 2010

    Conantokin dimerization

    Cnudde SE, Prorok M, Castellino FJ, Geiger JH. (2010) Metal ion determinants of conantokin dimerization as revealed in the X-ray crystallographic structure of the Cd(2+)/Mg (2+)-con-T[K7gamma] complex. J Biol Inorg Chem.15: 667-675.

    Department of Chemistry, Michigan State University, East Lansing, MI, 48824, USA.

    Abstract: Predatory sea snails from the Conus family produce a variety of venomous small helical peptides called conantokins that are rich in gamma-carboxyglutamic acid (Gla) residues. As potent and selective antagonists of the N-methyl-D: -aspartate receptor, these peptides are potential therapeutic agents for a variety of neurological conditions. The two most studied members of this family of peptides are con-G and con-T. Con-G has Gla residues at sequence positions 3, 4, 7, 10, and 14, and requires divalent cation binding to adopt a helical conformation. Although both Ca(2+) and Mg(2+) can fulfill this role, Ca(2+) induces dimerization of con-G, whereas the Mg(2+)-complexed peptide remains monomeric. A variant of con-T, con-T[K7gamma] (gamma is Gla), contains Gla residues at the same five positions as in con-G and behaves very similarly with respect to metal ion binding and dimerization; each peptide binds two Ca(2+) ions and two Mg(2+) ions per helix. To understand the difference in metal ion selectivity, affinity, and the dependence on Ca(2+) for dimer formation, we report here the structure of the monomeric Cd(2+)/Mg(2+)-con-T[K7gamma] complex, and, by comparison with the previously published con-T[K7gamma]/Ca(2+) dimer structure, we suggest explanations for both metal ion binding site specificity and metal-ion-dependent dimerization.

    New way to screen alpha-conotoxins.

    Check out the report by Stu Borman in Chemical and Engineering News, March 2010 here

    A new way to synthesize snail-based peptides called alpha-conotoxins, fold them into their native structures, and screen them for biological activity has been developed by an Australian research group. The approach could lead to new medications for neurological disorders and other conditions.
    alpha-Conotoxins are one of many families of conotoxins—neurotoxic peptides made by predatory cone snails, which use them to immobilize their prey. Conotoxins have attracted considerable attention for their bioactivity, especially pain relief. One conotoxin, Prialt (ziconotide), is an approved drug for severe chronic pain, and a conotoxin analog called Xen2174 is in Phase II human clinical trials, also as a pain reliever.
    Although those two agents belong to other conotoxin families, alpha-conotoxins also relieve pain. But it has been difficult to synthesize alpha-conotoxins in their natively folded forms to assess their prospects as drug candidates. The peptides each have two disulfide links, and when they are synthesized as linear peptides, their disulfide-forming cysteines often combine with the “wrong” cysteine partners, yielding nonnative, misfolded alpha-conotoxins with impaired biological activity.

    Bioactive-peptide specialist Paul F. Alewood of the University of Queensland and coworkers have now found a workaround (J. Am. Chem. Soc., DOI: 10.1021/ja910602h). They synthesize alpha-conotoxins in which one pair of cysteine residues has been replaced by a pair of selenocysteines. The selenocysteines combine to form diselenides more readily than cysteines react to form disulfides. So when a linear alpha-conotoxin is oxidized, the diselenide forms first, preventing mismatches, and the disulfide forms later, yielding a correct structure.
    The diselenide analogs generally have conformations and bioactivities similar to those of the corresponding native alpha-conotoxins, and in some cases, the analogs’ bioactivities are better.
    Alewood and coworkers also report the first method for synthesizing selenocysteine analogs of alpha-conotoxins on solid-support beads and then folding them while they are still on the resin. Having the folded analogs on solid-support particles makes it possible to screen the compounds for biological activity more quickly and conveniently than has been previously possible.
    The use of diselenides as disulfide replacements in synthetic peptides was pioneered in the late 1990s by bioorganic chemist Luis Moroder of the Max Planck Institute of Biochemistry, in Martinsried, Germany. But the technique has only recently been applied to conotoxins — in one earlier study by the Alewood group and in another by conotoxin specialist Grzegorz Bulaj of the University of Utah and coworkers.
    Of the new work, Bulaj says: “Having selenoconotoxins on-resin is a very nice advance. The solid-support-based approach can probably be extended to other di­sulfide-bridged peptides as well. It’s an important step toward the discovery and development of cysteine-rich peptides as future drugs.”

    28 May 2010

    Image collection of shells of Carl Linnaeus (1707-1778).

    On May 24th 2010 the Linnean Society of London launched online the digitised images of the shell collection of the great Swedish naturalist Carl Linnaeus (1707-1778).The 3,054 new images represent the molluscan specimens held in the Linnean Society's strongroom collection. All major worldwide groups are represented, the coverage reflecting the stage of exploration that had been reached in the early 19th century; today of course, Linnaeus' species represent only a portion of the recent mollusc fauna now known but as the basis of all shell nomenclature the Linnaean specimens are invaluable to researchers across the globe. Molluscan taxonomy is in a constant state of revision and work continues on interpreting the Linnean collections.

    The direct link to the collection of mollusks in the Linnaean collection is:

    The direct link to the collection of cone shells in the Linnaean collection is:

    If you have any comments to the Curator of Zoological Collections, The Linnaean Society, London (and collections manager of Higher Invertebrates, Natural History Museum), Ms. Kathie Way, (congratulations to her for this great achievement), her email is: k.way@nhm.ac.uk

    An interesting query raised by Paul Monfils and answered on CONCH-L by Bill Fenzan relates to Conus aulicus:

    Subject: Re: [CONCH-L] Photos of the Linnaean mollusk collection online.

    A number of taxonomic questions came to mind in viewing the Linnaeus collection. Just a sample one. Perhaps an explanation on this one will help makes sense of some of my others. The shell labeled "Conus aulicus" is obviously not what we now know as Conus aulicus. If the pictured shell is the one Linnaeus assigned that name to, how did a different shell end up with that name?

    The direct link to the collection of Conus aulicus in the Linnaean collection is: http://www.linnean-online.org/16950/

    Bill Fenzan replied : There is information on your question in: Kohn, A. J. 1992. A Chronological Taxonomy of Conus, 1758-1840. Smithsonian Institution Press, Washington D. C., pp. 315.

    The short version is that Linnaeus listed references to illustrations of many different cones in the original description. Each subsequent reviser (Hanley in the 1850s & Dodge in the 1950s) noted this but did not take action to clarify the situation. Alan Kohn finally designated a lectotype in 1963 when he studied the Linnaean cones and realized that the species needed to be better defined. Ironically, the shell desigated as the lectotype for Conus aulicus is not in the Linnaean collection on the website, but in the Royal (MLU) collection which is preserved at the university in Uppsala, Sweden.

    Alan Kohn replied on 31 May 2010 in CONCH-L as follows:

    Dear Bill, Paul, et al.,
    Actually the lectotype of Conus aulicus is not in Sweden but in the Gualtieri Collection in the Museo di Storia Naturale, Universitŕ di Pisa, Italy, where Robert Moolenbeek discovered it, I believe around 1990. Here is the story: In his own copy of the Systema Naturae of 1758, Linnaeus indicated that he owned a specimen of C. aulicus in his collection. When I studied his collection 200 years later, I found no specimen of C. aulicus. Neither had Sylvanus Hanley a hundred years before (Ipsa Linnaei Conchylia, London, 1855). Thus in the absence of a type specimen, in my 1963 paper I selected as lectotype a specimen that Linnaeus had cited as representing his new species in his original description. This shell was described and illustrated by Gualtieri in 1742. Of course he could not have named it according to Linnaeus's system, which was not yet invented. However, Gualtieri described his specimen and illustrated it in a fine engraving that Linnaeus also cited. I reproduced Gualtieri's image as the representation of the lectotype of C. aulicus. At that time we did not know the whereabouts of Gualtieri's specimen either. However, as mentioned above Robert Moloenbeek located it in the Pisa Museum and kindly provided me with photographs of it. They appear, along with a reproduction of Gualtieri's original illustration, in my 1992 book that Bill cited.

    27 May 2010

    alpha-Conotoxin RgIA reveals a role for alpha-9 nAChRs in adrenal stress response in the rat

    Colomer C, Olivos-Oré LA, Vincent A, McIntosh JM, Artalejo AR, Guérineau NC (2010). Functional characterization of alpha9-containing cholinergic nicotinic receptors in the rat adrenal medulla: implication in stress-induced functional plasticity. J Neurosci. 30: 6732-6742.

    Institute of Functional Genomics, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5203, Institut National de la Santé et de la Recherche Médicale U661, University of Montpellier, 34094 Montpellier, France.

    Abstract:An increase in circulating adrenal catecholamine levels constitutes one of the mechanisms whereby organisms cope with stress. Accordingly, stimulus-secretion coupling within the stressed adrenal medullary tissue undergoes persistent remodeling. In particular, cholinergic synaptic neurotransmission between splanchnic nerve terminals and chromaffin cells is upregulated in stressed rats. Since synaptic transmission is mainly supported by activation of postsynaptic neuronal acetylcholine nicotinic receptors (nAChRs), we focused our study on the role of alpha9-containing nAChRs, which have been recently described in chromaffin cells. Taking advantage of their specific blockade by the alpha-conotoxin RgIA (alpha-RgIA), we unveil novel functional roles for these receptors in the stimulus-secretion coupling of the medulla. First, we show that in rat acute adrenal slices, alpha9-containing nAChRs codistribute with synaptophysin and significantly contribute to EPSCs. Second, we show that these receptors are involved in the tonic inhibitory control exerted by cholinergic activity on gap junctional coupling between chromaffin cells, as evidenced by an increased Lucifer yellow diffusion within the medulla in alpha-RgIA-treated slices. Third, we unexpectedly found that alpha9-containing nAChRs dominantly (>70%) contribute to acetylcholine-induced current in cold-stressed rats, whereas alpha3 nAChRs are the main contributing channels in unstressed animals. Consistently, expression levels of alpha9 nAChR transcript and protein are overexpressed in cold-stressed rats. As a functional relevance, we propose that upregulation of alpha9-containing nAChR channels and ensuing dominant contribution in cholinergic signaling may be one of the mechanisms whereby adrenal medullary tissue appropriately adapts to increased splanchnic nerve electrical discharges occurring in stressful situations.

    15 May 2010

    alpha-Conotoxin AuIB isomers. Action on nicotinic receptors

    Grishin AA, Wang C-IA, Muttenthaler M, Alewood PF, Lewis RJ and Adams DJ (2010) {alpha}-Conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity to stoichiometry of {alpha}3{beta}4 nAChRs. J. Biol. Chem. 285: 22254-22263

    RMIT University, Australia; University of Queensland, Australia Corresponding author; email: david.adams@rmit.edu.au

    Abstract: Non-native disulfide isomers of alpha-conotoxins are generally inactive although some unexpectedly demonstrate comparable or enhanced bioactivity. The actions of 'globular' and 'ribbon' isomers of alpha-conotoxin AuIB have been characterized on alpha3beta4 nAChRs heterologously expressed in Xenopus oocytes. Using two electrode voltage clamp recording, we showed that inhibitory efficacy of the ribbon isomer of AuIB is limited to ~50%. The maximal inhibition was stoichiometry-dependent since altering alpha3:beta4 RNA injection ratios either increased AuIB[ribbon] efficacy (10alpha:1beta) or completely abolished blockade (1alpha:10beta). In contrast, inhibition by AuIB[globular] was independent of injection ratios. ACh-evoked current amplitude was largest for 1:10 injected oocytes and smallest for 10:1 ratio. ACh concentration-response curves revealed high (HS, 1:10) and low (LS, 10:1) sensitivity alpha3beta4 nAChRs with corresponding EC50's of 22.6 uM and 176.9 uM, respectively. Increasing the agonist concentration antagonized the inhibition of LS alpha3beta4 nAChRs by AuIB[ribbon] whereas inhibition of HS and LS alpha3beta4 nAChRs by AuIB[globular] was unaffected. Inhibition of LS and HS alpha3beta4 nAChRs by AuIB[globular] was insurmountable and independent of membrane potential. Molecular docking simulation suggested that AuIB[globular] is likely to bind to both ?3?4 nAChR stoichiometries outside of the ACh-binding pocket whereas AuIB[ribbon] binds to the classical agonist-binding site of the LS alpha3beta4 nAChR only. In conclusion, the two isomers of AuIB differ in their inhibitory mechanisms such that AuIB[ribbon] inhibits only LS alpha3beta4 nAChRs competitively whereas AuIB[globular] inhibits alpha3beta4 nAChRs irrespective of receptor stoichiometry, primarily by a non-competitive mechanism.

    12 May 2010

    An omega conotoxin from Conus striatus is analgesic in rodents

    Yan LD, Liu YL, Zhang L, Dong HJ, Zhou PL, Su RB, Gong ZH, Huang PT. (2010) Spinal antinociception of synthetic omega-conotoxin SO-3, a selective N-type neuronal voltage-sensitive calcium channel blocker, and its effects on morphine analgesia in chemical stimulus tests in rodent. Eur J Pharmacol. 636): 73-81.

    Beijing Institute of Pharmacology and Toxicology, 27 Tai-Ping Road, Haidian District, Beijing 100850, China.

    Abstract: SO-3, a novel Omega-superfamily conotoxin derived from Conus striatus, selectively inhibits N-type neuronal voltage-sensitive calcium channels. In current study, antinociception of SO-3 compared with MVIIA or morphine and its effects on morphine analgesia were investigated in rodent chemical stimulus tests after acute or repeated intrathecal administration. In mice acetic acid writhing test, similar to MVIIA, SO-3 caused dose- and time-dependent spinal antinociception with ED(50) of 0.25 microg/kg and t(1/2) of 4h, which was more potent and longer-acting than morphine. In rat formalin test after intrathecal bolus injection, SO-3 produced dose- and time-dependent antinociception by suppressing acute (ED(50), 1.79 microg/kg) and tonic phases (ED(50), 0.41 microg/kg), which was similar to MVIIA and approximately 10-fold potency and twice longer-acting of morphine in blocking tonic phase responses. After repeated intrathecal injections twice daily for 5 consecutive days, SO-3 produced analgesia without loss of potency whereas morphine produced analgesia tolerance in rat formalin test; further, SO-3 still produced potent analgesia in morphine-tolerant rats. SO-3 co-administered with morphine left-shift the dose-response curve of morphine in mice acetic acid writhing test and significantly potentiated morphine analgesia in rat formalin test. No changes in motor function were seen in mice or rats receiving antinociceptive doses of SO-3 whereas MVIIA caused motor dysfunction at doses of 1.0-2.0 microg/kg in rats. This study showed that (1) novel SO-3 produced potent and long-acting spinal antinociception without observable motor dysfunction, (2) SO-3 significantly potentiated morphine analgesia, (3) After repeated intrathecal administration, SO-3 produced neither tolerance nor cross-tolerance to morphine analgesia.

    11 May 2010

    Differential expression of conotoxins along venom duct

    Tayo LL, Lu B, Cruz LJ, Yates JR 3rd. (2010) Proteomic analysis provides insights on venom processing in Conus textile. J Proteome Res. 9: 2292-2301

    School of Chemical Engineering and Chemistry, Mapua Institute of Technology, Muralla Street Intramuros, Manila 1002, Philippines.

    Abstract: Conus species of marine snails deliver a potent collection of toxins from the venom duct via a long proboscis attached to a harpoon tooth. Conotoxins are known to possess powerful neurological effects and some have been developed for therapeutic uses. Using mass-spectrometry based proteomics, qualitative and quantitative differences in conotoxin components were found in the proximal, central and distal sections of the Conus textile venom duct suggesting specialization of duct sections for biosynthesis of particular conotoxins. Reversed phase HPLC followed by Orbitrap mass spectrometry and data analysis using SEQUEST and ProLuCID identified 31 conotoxin sequences and 25 post-translational modification (PTM) variants with King-Kong 2 peptide being the most abundant. Several previously unreported variants of known conopeptides were found and this is the first time that HyVal is reported for a disulfide rich Conus peptide. Differential expression along the venom duct, production of PTM variants, alternative proteolytic cleavage sites, and venom processing enroute to the proboscis all appear to contribute to enriching the combinatorial pool of conopeptides and producing the appropriate formulation for a particular hunting situation. The complementary tools of mass spectrometry-based proteomics and molecular biology can greatly accelerate the discovery of Conus peptides and provide insights on envenomation and other biological strategies of cone snails.

    9 May 2010

    Book Review in The Cone Collector, April 2010

    John Tucker and Manuel Tenorio.(2009) Systematic Classification of Recent and Fossil Conoidean Gastropods.

    Conchbooks 2009

    Abstract: This book review by Paul Kersten.in The Cone Collector Vol. 14 (2010) provides an account of the the joint efforts of two well-known scientists and cone experts, John Tucker and Manuel Tenorio, that has resulted in a new classification of what we are used to call Conus species. The new scheme is based primarily on the radula, with morphology and other factors used (eg. fossils) when the radula is not known.

    Individual issues of "The Cone Collector" - a Newsletter devoted to Cones, can be downloaded from this site

    mu-conotoxins as analgesics

    Norton RS (2010) mu-conotoxins as leads in the development of new analgesics. Molecules 15:2825-2844

    Walter and Eliza Hall Institute of Medical Research, Victoria, Australia. ray.norton@wehi.edu.au

    Abstract: Voltage-gated sodium channels (VGSCs) contain a specific binding site for a family of cone shell toxins known as mu-conotoxins. As some VGSCs are involved in pain perception and mu-conotoxins are able to block these channels, mu-conotoxins show considerable potential as analgesics. Recent studies have advanced our understanding of the three-dimensional structures and structure-function relationships of the mu-conotoxins, including their interaction with VGSCs. Truncated peptide analogues of the native toxins have been created in which secondary structure elements are stabilized by non-native linkers such as lactam bridges. Ultimately, it would be desirable to capture the favourable analgesic properties of the native toxins, in particular their potency and channel sub-type selectivity, in non-peptide mimetics. Such mimetics would constitute lead compounds in the development of new therapeutics for the treatment of pain.

    7 May 2010

    Ziconotide for pain treatment - Review

    Schmidtko A, Lötsch J, Freynhagen R, Geisslinger G. (2009) Ziconotide for treatment of severe chronic pain. Lancet 375:1569-1577

    Pharmazentrum Frankfurt/ZAFES, Institut für Klinische Pharmakologie, Goethe-Universität, Frankfurt am Main, Germany.

    Abstract:Pharmacological management of severe chronic pain is difficult to achieve with currently available analgesic drugs, and remains a large unmet therapeutic need. The synthetic peptide ziconotide has been approved by the US Food and Drug Administration and the European Medicines Agency for intrathecal treatment of patients with severe chronic pain that is refractory to other treatment modalities. Ziconotide is the first member in the new drug class of selective N-type voltage-sensitive calcium-channel blockers. The ziconotide-induced blockade of N-type calcium channels in the spinal cord inhibits release of pain-relevant neurotransmitters from central terminals of primary afferent neurons. By this mechanism, ziconotide can effectively reduce pain. However, ziconotide has a narrow therapeutic window because of substantial CNS side-effects, and thus treatment with ziconotide is appropriate for only a small subset of patients with severe chronic pain. We provide an overview of the benefits and limitations of intrathecal ziconotide treatment and review potential future developments in this new drug class.

    Marine Pharmaceuticals - Review

    Mayer AM, Glaser KB, Cuevas C, Jacobs RS, Kem W, Little RD, McIntosh JM, Newman DJ, Potts BC, Shuster DE. (2010) The odyssey of marine pharmaceuticals: a current pipeline perspective. Trends Pharmacol Sci. 31: 255-265. .

    Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA.

    Abstract:The global marine pharmaceutical pipeline consists of three Food and Drug Administration (FDA) approved drugs, one EU registered drug, 13 natural products (or derivatives thereof) in different phases of the clinical pipeline and a large number of marine chemicals in the preclinical pipeline. In the United States there are three FDA approved marine-derived drugs, namely cytarabine (Cytosar-U((R)), Depocyt((R))), vidarabine (Vira-A((R))) and ziconotide (Prialt((R))). The current clinical pipeline includes 13 marine-derived compounds that are either in Phase I, Phase II or Phase III clinical trials. Several key Phase III studies are ongoing and there are seven marine-derived compounds now in Phase II trials. The preclinical pipeline continues to supply several hundred novel marine compounds every year and those continue to feed the clinical pipeline with potentially valuable compounds. From a global perspective the marine pharmaceutical pipeline remains very active, and now has sufficient momentum to deliver several additional compounds to the marketplace in the near future; this review provides a current view of the pipeline.

    Mechanisms of action of analgesics

    Tsukamoto M, Kiso T, Shimoshige Y, Aoki T, Matsuoka N. (2010) Spinal mechanism of standard analgesics: evaluation using mouse models of allodynia. Eur J Pharmacol. 634: 40-45.

    Pharmacology Research Laboratories, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan. mina.tsukamoto@jp.astellas.com

    Abstract: Spinal neurotransmission plays an important role in the perception of pain signaling. In the present study, we investigated the spinal anti-nociceptive mechanism of current standard analgesics in mouse models of tactile allodynia induced by intrathecal administration of N-methyl-D-aspartic acid (NMDA), prostaglandin E2 (PGE2), and bicuculline. NMDA-induced allodynia is induced by postsynaptic NMDA receptor activation, while PGE2-induced allodynia is triggered by the enhancement of presynaptic glutamate release via EP1 receptor activation. In contrast, bicuculline induces allodynia by the blockade of gamma-aminobutyric acid (GABA)A receptor-mediated inhibitory system. As the clinically available analgesics, pregabalin (alpha2delta-subunit calcium channel ligand), ziconotide (N-type calcium channel blocker), mexiletine (sodium channel blocker), and duloxetine (serotonin and norepinephrine reuptake inhibitors) were evaluated in these neurochemically-induced allodynia models. Pregabalin almost completely alleviated NMDA-, PGE2-, and bicuculline-induced allodynia. Despite being classified as an agent with a similar molecular target mechanism, ziconotide could only alleviate PGE2-induced allodynia, but not NMDA- or bicuculline-induced allodynia, as did mexiletine and duloxetine. These results taken together suggest that ziconotide, mexiletine, and duloxetine suppress spinal hyperactivity via the presynaptic site mechanism. In contrast, pregabalin could suppress via the downstream step during spinal hyperactivation such as postsynaptic NMDA activation or dysfunction of GABAergic control in addition to presynaptic mechanism. In conclusion, present findings provide implication that the spinal anti-nociceptive mechanistic site of pregabalin is different from that of ziconotide, mexiletine, and duloxetine, and pregabalin could have a broader anti-nociceptive mechanism other than N-type calcium channel blockade.

    Nicotinic agonists for relief of neuropathic pain

    Gao B, Hierl M, Clarkin K, Juan T, Nguyen H, Valk M, Deng H, Guo W, Lehto SG, Matson D, McDermott JS, Knop J, Gaida K, Cao L, Waldon D, Albrecht BK, Boezio AA, Copeland KW, Harmange JC, Springer SK, Malmberg AB, McDonough SI.(2010) Pharmacological effects of nonselective and subtype-selective nicotinic acetylcholine receptor agonists in animal models of persistent pain. Pain. 149: 33-49.

    Department of Neuroscience, Amgen, Inc., Thousand Oaks, CA 91320, USA.

    Abstract: Nicotinic acetylcholine receptors (nAChRs) are longstanding targets for a next generation of pain therapeutics, but the nAChR subtypes that govern analgesia remain unknown. We tested a series of nicotinic agonists, including many molecules used or tried clinically, on a panel of cloned neuronal nAChRs for potency and selectivity using patch-clamp electrophysiology and a live cell-based fluorescence assay. Nonselective nicotinic agonists as well as compounds selective either for alpha4beta2 or for alpha7 nAChRs were then tested in the formalin and complete Freund's adjuvant models of pain. Nonselective nAChR agonists ABT-594 and varenicline were effective analgesics. By contrast, the selective alpha4beta2 agonist ispronicline and a novel alpha4beta2-selective potentiator did not appear to produce analgesia in either model. alpha7-selective agonists reduced the pain-related endpoint, but the effect could be ascribed to nonspecific reduction of movement rather than to analgesia. Neither selective nor nonselective alpha7 nicotinic agonists affected the release of pro-inflammatory cytokines in response to antigen challenge. Electrophysiological recordings from spinal cord slice showed a strong nicotine-induced increase in inhibitory synaptic transmission that was mediated partially by alpha4beta2 and only minimally by alpha7 subtypes. Taken with previous studies, the results suggest that agonism of alpha4beta2 nAChRs is necessary but not sufficient to produce analgesia, and that the spinal cord is a key site where the molecular action of nAChRs produces analgesia.

    2 May 2010

    Conus glorioceanus Poppe & Tagaro, 2009

    Guido T. Poppe announced via the newsgroup CONCH-L the discovery of a second specimen of Conus glorioceanus. As reported, "The shell measures 41.1 mm in length, is fresh dead and has a defect on the ventral side. Definitely fresh but dead taken with a hermit crab in a fish trap on the same place as the holotype: Zamboanga, between Recodo and Perlas Island in about 100 m depth. This C. glorioceanus is adult, but 8.5 mm smaller than the holotype.

    The shell confirms the size and conchological characteristics of the holotype as to be stable. Indeed, the description, based on a single specimen raised rumors as this should be a freak from another Conus-species. Despite a triple expertise of Gabriella Raybaudi, Sheila Tagaro and myself who all agreed that the species was new.

    Dozens of local fishermen got photos from the holotype and the sums offered locally are high enough so that we can positively expect more shells to reach Cebu in the future. Getting a shell out of 100 m deep may sound simple, in reality it is not. It is intensive in work and high in investment: one needs a boat, 160 m of rope, nets and a lot of muscles and time to get the gear to the surface daily.

    The topotype is now in the collection of Conchology, Inc., the holotype indeed belongs to the National Museum of the Philippines, Manila."

    You can view the shell in the Encyclopedia

    17 April 2010

    Bioactives from marine sponges

    Ebada SS, Lin W, Proksch P. (2010) Bioactive sesterterpenes and triterpenes from marine sponges: occurrence and pharmacological significance. Mar Drugs. 8:313-346.

    Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine University, Duesseldorf, Germany. sherif.Elsayed@uni-duesseldorf.de

    Abstract:Marine ecosystems (>70% of the planet's surface) comprise a continuous resource of immeasurable biological activities and immense chemical entities. This diversity has provided a unique source of chemical compounds with potential bioactivities that could lead to potential new drug candidates. Many marine-living organisms are soft bodied and/or sessile. Consequently, they have developed toxic secondary metabolites or obtained them from microorganisms to defend themselves against predators [1]. For the last 30-40 years, marine invertebrates have been an attractive research topic for scientists all over the world. A relatively small number of marine plants, animals and microbes have yielded more than 15,000 natural products including numerous compounds with potential pharmaceutical potential. Some of these have already been launched on the pharmaceutical market such as Prialt (ziconotide; potent analgesic) and Yondelis (trabectedin or ET-743; antitumor) while others have entered clinical trials, e.g., alpidin and kahalalide F. Amongst the vast array of marine natural products, the terpenoids are one of the more commonly reported and discovered to date. Sesterterpenoids (C(25)) and triterpenoids (C(30)) are of frequent occurrence, particularly in marine sponges, and they show prominent bioactivities. In this review, we survey sesterterpenoids and triterpenoids obtained from marine sponges and highlight their bioactivities.

    alpha-conotoxin from Conus litteratus

    Luo S, Akondi KB, Zhangsun D, Wu Y, Zhu X, Hu Y, Christensen S, Dowell C, Daly NL, Craik DJ, Wang CI, Lewis RJ, Alewood PF, Michael McIntosh J. (2010) Atypical alpha-Conotoxin LtIA from Conus litteratus targets a novel microsite of the alpha3beta2 nicotinic receptor. J Biol Chem. 285:12355-12366.

    Key Laboratory of Tropical Biological Resources, Ministry of Education, Ocean College, College of Materials and Chemical Engineering, Center for Experimental Biotechnology, Hainan University, Haikou Hainan 570228, China. luosulan2003@163.com

    Abstract: Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. alpha-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of alpha-conotoxins may serve as a platform for development of new therapies. Previously characterized alpha-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel alpha-conotoxin LtIA, which lacks this highly conserved motif but potently blocked alpha3beta2 nAChRs with a 9.8 nm IC(50) value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing alpha-conotoxin MII. Nevertheless, pre-block of alpha3beta2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR beta subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for alpha3beta2 versus alpha3beta4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC(50) value on alpha3beta2(F119Q) versus wild type nAChRs, whereas T59K and V111I beta2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the alpha3beta2 nAChR that includes beta2 Lys-79. The K79A mutant disrupted LtIA binding but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.

    Evolution of Conus peptide genes

    Puillandre N, Watkins M, Olivera BM. (2010) Evolution of Conus Peptide Genes: Duplication and Positive Selection in the A-Superfamily. J Mol Evol. 2010 Feb 9. [Epub ahead of print]

    Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT, 84112, USA, puillandre@biology.utah.edu.

    Abstract: A remarkable diversity of venom peptides is expressed in the genus Conus (known as "conotoxins" or "conopeptides"). Between 50 and 200 different venom peptides can be found in a single Conus species, each having its own complement of peptides. Conopeptides are encoded by a few gene superfamilies; here we analyze the evolution of the A-superfamily in a fish-hunting species clade, Pionoconus. More than 90 conopeptide sequences from 11 different Conus species were used to build a phylogenetic tree. Comparison with a species tree based on standard genes reveals multiple gene duplication events, some of which took place before the Pionoconus radiation. By analysing several A-conopeptides from other Conus species recorded in GenBank, we date the major duplication events after the divergence between fish-hunting and non-fish-hunting species. Furthermore, likelihood approaches revealed strong positive selection; the magnitude depends on which A-conopeptide lineage and amino-acid locus is analyzed. The four major A-conopeptide clades defined are consistent with the current division of the superfamily into families and subfamilies based on the Cys pattern. The function of three of these clades (the kappaA-family, the alpha4/7-subfamily, and alpha3/5-subfamily) has previously been characterized. The function of the remaining clade, corresponding to the alpha4/4-subfamily, has not been elucidated. This subfamily is also found in several other fish-hunting species clades within Conus. The analysis revealed a surprisingly diverse origin of alpha4/4 conopeptides from a single species, Conus bullatus. This phylogenetic approach that defines different genetic lineages of Conus venom peptides provides a guidepost for identifying conopeptides with potentially novel functions.


    Kaas Q, Westermann JC, Craik DJ. (2010) Conopeptide characterization and classifications: An analysis using ConoServer. Toxicon. 55: 1491-1509

    The University of Queensland, Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, Brisbane, 4072 QLD Australia.

    Abstract: Cone snails are carnivorous marine gastropods that have evolved potent venoms to capture their prey. These venoms comprise a rich and diverse cocktail of peptide toxins, or conopeptides, whose high diversity has arisen from an efficient hypermutation mechanism, combined with a high frequency of post-translational modifications. Conopeptides bind with high specificity to distinct membrane receptors, ion channels, and transporters of the central and muscular nervous system. As well as serving their natural function in prey capture, conopeptides have been utilized as versatile tools in neuroscience and have proven valuable as drug leads that target the nervous system in humans. This paper examines current knowledge on conopeptide sequences based on an analysis of gene and peptide sequences in ConoServer (http://www.conoserver.org), a specialized database of conopeptide sequences and three-dimensional structures. We describe updates to the content and organization of ConoServer and discuss correlations between gene superfamilies, cysteine frameworks, pharmacological families targeted by conopeptides, and the phylogeny, habitat, and diet of cone snails. The study identifies gaps in current knowledge of conopeptides and points to potential directions for future research. Copyright © 2010 Elsevier Ltd. All rights reserved.

    kappaM-conotoxin RIIJ from Conus radiatus

    Chen P, Dendorfer A, Finol-Urdaneta RK, Terlau H, Olivera BM. (2010) Biochemical characterization of kM-RIIIJ, a KV1.2 channel blocker: Evaluation of cardioprotective effects of kM-conotoxins. J Biol Chem. 285:14882-14889;

    University of Utah, United States;

    Abstract: Conus snail (Conus) venoms are a valuable source of pharmacologically active compounds; some of the peptide toxin families from the snail venoms are known to interact with K channels. We report the purification, synthesis and characterization of kappaM-conotoxin RIIIJ from the venom of a fish-hunting species, Conus radiatus. This conopeptide, like a previously characterized peptide in the same family, kappaM-RIIIK, inhibits the homotetrameric human Kv1.2 channels. When tested in Xenopus oocytes, kappaM-RIIIJ has an order of magnitude higher affinity (IC50 = 33 nM) to Kv1.2 than kappaM-RIIIK (IC50 = 352 nM). Chimeras of RIIIK and RIIIJ tested on the human Kv1.2 channels revealed that Lys-9 from kappaM-RIIIJ is a determinant of its higher potency against hKv1.2. However, when compared in a model of ischemia/reperfusion, kappaM-RIIIK (100 mug/kg b.w.), administered just before reperfusion, significantly reduces the infarct size in rat hearts in vivo without influencing hemodynamics, providing a potential compound for cardioprotective therapeutics. In contrast, kappaM-RIIIJ does not exert any detectable cardioprotective effect. kappaM-RIIIJ shows more potency for Kv1.2-Kv1.5 and Kv1.2-Kv1.6 heterodimers than kappaM-RIIIK; whereas the affinity of kappaM-RIIIK to Kv1.2-Kv1.7 heterodimeric channels is higher (IC50 = 680 nM) than that of kappaM-RIIIJ (IC50 = 3.15 muM). Thus, the cardioprotection seems to correlate to antagonism to heteromultimeric channels, involving the Kv1.2 alpha-subunit, rather than antagonism to Kv1.2 homotetramers. Furthermore, kappaM-RIIIK and kappaM-RIIIJ provide a valuable set of probes for understanding the underlying mechanism of cardioprotection.

    Treatment of trigeminal neuropathy with Ziconotide

    Lux EA. (2010) Case report: successful treatment of a patient with trigeminal neuropathy using ziconotide Anesth Analg. 110: 1195-1197.

    Clinic of Pain and Palliative Medicine, St.-Marien-Hospital, Lünen, Germany. drlux@web.de

    Abstract:A 50-year-old female patient with chronic neuropathic pain in the distribution of the second branch of the trigeminal nerve was unsuccessfully treated over several years. Intrathecal therapy with ziconotide was administered at an initial dose of 0.33 microg/d, which was gradually increased by 0.7 microg/d. Subjective pain on the numeric rating scale was reduced from 9/10 to 3-4/10 at a dose of 6.3 microg/d. Long-term treatment was continued at a ziconotide concentration of 12.5 microg/mL and a daily dose of 3.9 microg/d by a SynchroMed Infusion System (Medtronic, Minneapolis, MN). Pain reduction was maintained in the follow-up observation period over 5 months.

    6 April 2010

    Defining a clade for Conus praecellens

    Biggs JS, Watkins M, Cornell PS and Olivera BM (2010). Defining a clade by morphological, molecular, and toxinological criteria: distinctive forms related to Conus praecellens (Gastropoda: Conidae): The Nautilus 124: 1-19

    21 February 2010

    Cone shells from Victoria, Australia

    Conus anemone: A common species in dredgings at Western Port and living on the shore in crevices of rocks exhibiting much variation in colour. A pure white form invested with a creamy epidermis appears in life on the rocks at Back Beach Portsea and nearby frequently seen on the shores. This is merely an albino of the above and is sometimes known under the name of C. singletoni.

    Conus segravei: A rare species in Western Port dredgings and occasionally found in the shores at Shoreham and Flinders. This is a distinct form named by the late Mr. J.H. Gatliff. It is beaurifully coloured and may be separated from anemone by its smoothness and roseate interior.

    Conus rutilus: A small charming little species about 1/2 inch. Dredged in Western Port and easily recognized by its pyriform shape and brilliant colouring.

    Source: "Forty Years' Dredging in Victorian Waters" An address by Mr. C.J. Gabriel, Hon. Assoc. Conchologist, National Museum of Victoria to the Malacological Club of Victoria on the 30th of March 1953. Reprinted in The Malacological Society of Australasia Inc. Victorian Branch Bulletin, VIC. BR. BULL. NO. 252 pp.4-8, 2010.

    Selenium-directed synthesis of potent alpha-conotoxins

    Muttenthaler M, Nevin ST, Grishin AA, Ngo ST, Choy PT, Daly NL, Hu SH, Armishaw CJ, Wang CI, Lewis RJ, Martin JL, Noakes PG, Craik DJ, Adams DJ, Alewood PF. (2010) Solving the alpha-Conotoxin Folding Problem: Efficient Selenium-Directed On-Resin Generation of More Potent and Stable Nicotinic Acetylcholine Receptor Antagonists. J Am Chem Soc. 132: 3514-3522

    Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, The University of Queensland, Brisbane, Queensland 4072, Australia; Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia; and School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia.

    Abstract: alpha-Conotoxins are tightly folded miniproteins that antagonize nicotinic acetylcholine receptors (nAChR) with high specificity for diverse 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 amphiphilic 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 alpha1beta1deltagamma 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.

    Harvesting conoidean venoms

    Seronay RA, Fedosov AE, Astilla MA, Watkins M, Saguil N, Heralde FM 3rd, Tagaro S, Poppe GT, Alińo PM, Oliverio M, Kantor YI, Concepcion GP, Olivera BM. (2010) Accessing novel conoidean venoms: Biodiverse lumun-lumun marine communities, an untapped biological and toxinological resource. Toxicon. 2009 Dec 11. [Epub ahead of print]

    Marine Science Institute, University of the Philippines, Diliman, Quezon City, 1101, Philippines; Northern Mindanao State Institute of Science and Technology (NORMISIST), Ampayon, Butuan City, Philippines.

    Abstract: Cone snail venoms have yielded pharmacologically active natural products of exceptional scientific interest. However, cone snails are a small minority of venomous molluscan biodiversity, the vast majority being tiny venomous morphospecies in the family Turridae. A novel method called lumun-lumun opens access to these micromolluscs and their venoms. Old fishing nets are anchored to the sea bottom for a period of 1-6months and marine biotas rich in small molluscs are established. In a single lumun-lumun community, we found a remarkable gastropod biodiversity (155 morphospecies). Venomous predators belonging to the superfamily Conoidea (36 morphospecies) were the largest group, the majority being micromolluscs in the family Turridae. We carried out an initial analysis of the most abundant of the turrid morphospecies recovered, Clathurella (Lienardia) cincta (Dunker, 1871). In contrast to all cDNA clones characterized from cone snail venom ducts, one of the C. cincta clones identified encoded two different peptide precursors presumably translated from a single mRNA. The prospect of easily accessing so many different morphospecies of venomous marine snails raises intriguing toxinological possibilities: the 36 conoidean morphospecies in this one net alone have the potential to yield thousands of novel pharmacologically active compounds.

    20 February 2010

    Conus dorotheae

    Visaya Vol.2 No. 6. has been published

    Featuring an article by Eric Monnier and Loďc Limpalaër on a new species of cone shell, Conus dorotheae n. sp. Monnier & Limpalaër, 2010

    Successful treatment with Ziconotide

    Lux EA (2010) Successful Treatment of a Patient with Trigeminal Neuropathy Using Ziconotide. Anesth Analg. 110: 1195-1197.

    The St.-Marien-Hospital, Lünen, Germany.

    Abstract: A 50-year-old female patient with chronic neuropathic pain in the distribution of the second branch of the trigeminal nerve was unsuccessfully treated over several years. Intrathecal therapy with ziconotide was administered at an initial dose of 0.33 mug/d, which was gradually increased by 0.7 mug/d. Subjective pain on the numeric rating scale was reduced from 9/10 to 3-4/10 at a dose of 6.3 mug/d. Long-term treatment was continued at a ziconotide concentration of 12.5 mug/mL and a daily dose of 3.9 mug/d by a SynchroMed(R) Infusion System (Medtronic, Minneapolis, MN). Pain reduction was maintained in the follow-up observation period over 5 months. PMID: 20142352 [PubMed - as supplied by publisher]

    Methodology for trialing Ziconotide

    Burton AW, Deer TR, Wallace MS, Rauck RL, Grigsby E.(2010) Considerations and methodology for trialing ziconotide.Pain Physician. 13: 23-33

    University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. awburton@mdanderson.org

    Abstract: BACKGROUND: Before long-term intrathecal analgesic therapy is initiated, patients often undergo a spinal analgesia trial. Ziconotide is a nonopioid intrathecal analgesic used to manage severe chronic pain, and a variety of methods have been used to trial ziconotide. OBJECTIVES: The purpose of this review is to compare and discuss the different methods of ziconotide trialing. METHODS: Various databases (i.e., PubMed, Excerpta Medica, Cumulative Index to Nursing and Allied Health Literature, Biological Abstracts, Cochrane Database of Systematic Reviews, EMBASE, International Pharmaceutical Abstracts, and Google Scholar) and association meeting abstracts were searched with the use of the terms ziconotide, Prialt, trial, and trialing. In addition, a search was conducted for abstracts/posters presented at a variety of association meetings. RESULTS: Nine sources, including one expert opinion piece, were identified. Three methods of ziconotide trialing were discovered: continuous infusion, limited-duration infusion, and bolus injection. Results indicate that patients often achieve analgesia during trialing, regardless of the trialing method. Adverse events reported during ziconotide trialing studies were similar to those reported during ziconotide clinical trials. Preliminary evidence suggests that both effectiveness and safety may be dose-related. In 3 studies the value of ziconotide trialing in predicting long-term patient response to ziconotide therapy was investigated; however, the results were preliminary. The expert opinion piece from 2008 recommended trialing ziconotide via continuous infusion, using a starting dose of 1.2 mcg/d and dose increases of 1.2 mcg/d every 12 to 24 hours, for up to 3 days; the trial may be extended in some cases. LIMITATIONS: Given the small samples size and lack of controlled ziconotide trialing studies, it is currently not possible to determine the relative safety and effectiveness of different methods of ziconotide trialing, nor is it possible to determine if trialing is predictive of patient response to long-term ziconotide therapy. CONCLUSIONS: All 3 methods of ziconotide trialing appear to be viable options, and no method can be considered superior on the basis of the evidence presented in this review. Controlled studies comparing ziconotide trialing methods may be warranted.

    Novel peptides from Conus marmoreus

    Zhang L, Shao X, Chi C, Wang C. (2010) Two short D-Phe-containing cysteine-free conopeptides from Conus marmoreus. Peptides.31:177-179.

    Institute of Protein Research, Tongji University, Shanghai, China.

    Abstract: The cysteine-free conopeptides are naturally occurring components of the venom of cone snails and have been relatively less investigated than the cysteine-containing conopeptides. In this work, we used thiol-exchange chromatography to isolate cysteine-free conopeptides from the venom of Conus marmoreus. The full-length previously reported conomarphin and two novel shortened forms of it were found in the cysteine-free conopeptide fraction. The two shortened conomarphins also contain a D-Phe and a hydroxylated proline residue, suggesting complex post-translational modifications of these conopeptides. In particular, the shorter conomarphin fragment shares significant sequence similarity with conophans. This work demonstrated that thiol-exchange chromatography is a useful approach to isolate cysteine-free conopeptides from venom cocktail. The newly identified conopeptides further revealed the complexity of post-translational processing and the high diversity of conotoxins.

    12 February 2010

    Oxidative folding of conotoxins

    Safavi-Hemami H, Bulaj G, Olivera BM, Williamson NA, Purcell AW.(2010) Identification of Conus peptidyl Prolyl cis-trans isomerases (PPIases) and assessment of their role in the oxidative folding of conotoxins. J Biol Chem. 285: 12735-12746.

    University of Melbourne, Australia;

    Abstract: Peptidyl prolyl cis-trans isomerases (PPIases) are ubiquitous proteins that catalyse the cis-trans isomerisation of prolines. A number of proteins have been identified as endogenous substrates for PPIases such as Drosophila rhodopsin and the human immunodeficiency viral protein HIV-1 Gag, however very little is known about the interaction of PPIases with small, disulfide-rich peptides. Marine cone snails synthesise a wide array of cysteine-rich peptides, called conotoxins, many of which contain one or more prolines or hydroxyprolines. To identify whether PPIase-associated cis-trans isomerisation of these residues affects the oxidative folding of conotoxins we identified, sequenced and expressed three functionally active isoforms of PPIase from the venom gland of Conus novaehollandiae and characterised their ability to facilitate oxidative folding of conotoxins in vitro. Three conotoxins, namely mu-GIIIA, mu-SIIIA and omega-MVIIC, derived from two distinct toxin gene families were assayed. Conus PPIase significantly increased the rate of appearance of the native form of mu-GIIIA, a peptide containing three hydroxyprolines. In contrast, the presence of PPIase had no effect on the folding of mu-SIIIA and omega-MVIIC, peptides containing no or one proline residue respectively. We further show that an ER-resident PPIase isoform facilitates folding of mu-GIIIA more efficiently than two cytosolic isoforms. This is the first study to demonstrate PPIase-assisted folding of conotoxins, small disulfide-rich peptides with unique structural properties.

    alpha-Conotoxin Lt IA from Conus litteratus

    Luo S, Bharati AK, Zhangsun D, Wu Y, Zhu X, Hu Y, Christensen S, Dowell C, Daly NL, Craik DJ, Wang CI, Lewis RJ, Alewood PF, McIntosh JM. (2010) The atypical alpha-conotoxin LtIA from Conus litteratus targets a novel microsite of the alpha3beta2 nicotinic receptor. J Biol Chem. 285: 12355-12366

    Hainan University, China;

    Abstract: Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states including Parkinson's and nicotine addiction. Alpha-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function and receptor interaction of alpha-conotoxins may serve as a platform for development of new therapies. Previously characterized alpha-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. The current study characterized the novel alpha-conotoxin LtIA, which lacks this highly conserved motif but potently blocked alpha3beta2 nAChRs with a 9.8 nM IC50. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing alpha-conotoxin MII. Nevertheless, pre-block of alpha3beta2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR beta subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for alpha3beta2 vs. alpha3beta4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC50 on alpha3beta2(F119Q) versus wildtype nAChRs, whereas T59K and V111I beta2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the alpha3beta2 nAChR that includes beta2 K79. The K79A mutant disrupted LtIA binding, but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.

    Evolution of Conus Peptide Genes

    Puillandre N, Watkins M, Olivera BM.(2010) Evolution of Conus Peptide Genes: Duplication and Positive Selection in the A-Superfamily. J Mol Evol. 2010 Feb 9. [Epub ahead of print]

    Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT, 84112, USA, puillandre@biology.utah.edu.

    Abstract: A remarkable diversity of venom peptides is expressed in the genus Conus (known as "conotoxins" or "conopeptides"). Between 50 and 200 different venom peptides can be found in a single Conus species, each having its own complement of peptides. Conopeptides are encoded by a few gene superfamilies; here we analyze the evolution of the A-superfamily in a fish-hunting species clade, Pionoconus. More than 90 conopeptide sequences from 11 different Conus species were used to build a phylogenetic tree. Comparison with a species tree based on standard genes reveals multiple gene duplication events, some of which took place before the Pionoconus radiation. By analysing several A-conopeptides from other Conus species recorded in GenBank, we date the major duplication events after the divergence between fish-hunting and non-fish-hunting species. Furthermore, likelihood approaches revealed strong positive selection; the magnitude depends on which A-conopeptide lineage and amino-acid locus is analyzed. The four major A-conopeptide clades defined are consistent with the current division of the superfamily into families and subfamilies based on the Cys pattern. The function of three of these clades (the kappaA-family, the alpha4/7-subfamily, and alpha3/5-subfamily) has previously been characterized. The function of the remaining clade, corresponding to the alpha4/4-subfamily, has not been elucidated. This subfamily is also found in several other fish-hunting species clades within Conus. The analysis revealed a surprisingly diverse origin of alpha4/4 conopeptides from a single species, Conus bullatus. This phylogenetic approach that defines different genetic lineages of Conus venom peptides provides a guidepost for identifying conopeptides with potentially novel functions.

    Conotoxin therapeutics: A Review

    Lewis RJ.(2009) Conotoxin venom Peptide therapeutics. Adv Exp Med Biol. 655: 44-48.

    Xenome Ltd, Indooroopilly 4068 and Institute for Molecular Biosciences, The University of Queensland, 4072, Brisbane, Australia, r.lewis@imb.uq.edu.au.

    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.

    chi-conopeptide MrIA (XEN2174), a NET inhibitor for pain

    Brust A, Palant E, Croker DE, Colless B, Drinkwater R, Patterson B, Schroeder CI, Wilson D, Nielsen CK, Smith MT, Alewood D, Alewood PF, Lewis RJ (2010). chi-Conopeptide pharmacophore development: toward a novel class of norepinephrine transporter inhibitor (Xen2174) for pain. J Med Chem. 52:6991-7002.

    Xenome Ltd., 120 Meiers Road, Indooroopilly, Queensland, Australia 4068. andreas.brust@xenome.com

    Abstract: Norepinephrine (NE) amplifies the strength of descending pain inhibition, giving inhibitors of spinal NET clinical utility in the management of pain. chi-MrIA isolated from the venom of a predatory marine snail noncompetitively inhibits NET and reverses allodynia in rat models of neuropathic pain. An analogue of chi-MrIA has been found to be a suitable drug candidate. On the basis of the NMR solution structure of this related peptide, Xen2174 (3), and structure-activity relationships of analogues, a pharmacophore model for the allosteric binding of 3 to NET is proposed. It is shown that 3 interacts with NET predominantly through amino acids in the first loop, forming a tight inverse turn presenting amino acids Tyr7, Lys8, and Leu9 in an orientation allowing for high affinity interaction with NET. The second loop interacts with a large hydrophobic pocket within the transporter. Analogues based on the pharmacophore demonstrated activities that support the proposed model. On the basis of improved chemical stability and a wide therapeutic index, 3 was selected for further development and is currently in phase II clinical trials.
    This publication is dedicated to Dianne Alewood: gone to early and dearly missed by us all

    Control of disulfide bond isomers in conotoxin ImI

    Armishaw CJ, Dutton JL, Craik DJ, Alewood PF.(2010) Establishing regiocontrol of disulfide bond isomers of alpha-conotoxin ImI via the synthesis of N-to-C cyclic analogues. Biopolymers 94(3):307-313

    Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, 4072, Australia.

    Abstract: alpha-Conotoxins are multiple disulfide bond containing peptides that are isolated from venomous marine cone snails. They display remarkable selectivity for different subtypes of nicotinic acetylcholine receptors (nAChRs). While alpha-conotoxins display poor resistance to in vivo degradation by proteases which limits their use as drug leads, N-to-C cyclization via an oligopeptide spacer unit has been previously shown to improve stability. However the effect of N-to-C cyclisation on the formation of the disulfide bond framework is not fully understood. Four N-to-C cyclic analogues of alpha-conotoxin ImI; cImI-A, cImI-betaA, cImI-AG and cImI-AGG were synthesised to evaluate the effect of oligopeptide spacer length on disulfide bond selectivity and stability to proteolysis. Different ratios of disulfide bond isomers were obtained for each analogue using a non-selective random disulfide bond forming strategy, which was dependent on the length of the spacer. In order to identify each isomer obtained using the random strategy, and to gain access to disulfide bond isomers otherwise unattainable using the random strategy, both the native (globular) and ribbon isomers were synthesised in good yield and purity using a selective orthogonal cysteine protecting group strategy. As such, a random oxidation strategy showed a clear preference for the ribbon isomer in cImI-A. The cyclic globular isomers showed a high resistance to enzymatic degradation compared to the ribbon isomers, with the cImI-A and cImI-AG globular isomers demonstrating the highest stability. These results suggest that cyclisation can improve the biochemical stability of conotoxins with potential applications in the development of drugs.

    More new material will be added here soon, updating from 31 October 2009

    Analgesic omega-conotoxins from Conus catus

    Berecki G, Motin L, Haythornthwaite A, Vink S, Bansal P, Drinkwater R, Wang CI, Moretta M, Lewis RJ, Alewood PF, Christie MJ, Adams DJ (2009). Analgesic{omega}-conotoxins CVIE and CVIF selectively and voltage dependently block recombinant and native N-type calcium channels. Mol Pharmacol. 77:139-148.

    1 University of Queensland;

    Abstract: 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 oocytes, CVIE and CVIF potently and selectively inhibited depolarization-activated Ba(2+) currents through recombinant N-type (alpha(1B-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 diversity 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.

    Conotoxins Vc1.1 and RgIA are analgesic in alpha9 KO mice

    Callaghan, B and Adams, DJ (2010) Analgesic alpha-conotoxins Vc1.1 and RgIA inhibit N-type calcium channels in sensory neurons of alpha9 nicotinic receptor knockout mice. Channels 4: 51-54. (This is an Addendum to Callaghan et al 2008, see below)

    Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia.

    Abstract: Alpha-conotoxins Vc1.1 and RgIA are peptides from the venom of marine Conus snails that are currently in development as a treatment for neuropathic pain. We have reported previously that the alpha9alpha10 nicotinic acetylcholine receptor (nAChR) selective-conotoxins Vc1.1 and RgIA potently and selectively inhibit high voltage-activated (HVA) N-type calcium channel currents in dissociated neurons from rat dorsal root ganglia (DRG). Our data indicated that Vc1.1 does not interact directly with N-type Ca(2+) channels but inhibits them via GABA(B) receptor activation. The present study investigated Vc1.1 and RgIA inhibition of N-type Ca(2+) channels currents in DRG neurons of wild-type and alpha9 knockout (KO) mice to determine if the alpha9 nAChR was necessary for inhibition of the Ca(2+) channel current. Application of Vc1.1 (100 nM) inhibited N-type Ca(2+) channel currents to 69.2 +/- 3.5% of control in DRG neurons isolated from wild-type mice. In >70% of DRG neurons isolated from the alpha9 KO mice, both Vc1.1 and RgIA selectively inhibited N-type Ca(2+) channel currents with an IC(50) of 24.6 nM and 22.4 nM, respectively. The GABA(B) receptor antagonist CGP55845 (1 microM) antagonized the effect of Vc1.1 and RgIA on the N-type calcium channels in alpha9 KO mice. RT-PCR and western blot analysis confirmed the absence of the alpha9 nAChR in mice carrying a null mutation for the nAChR alpha9 gene. These results demonstrate that the inhibition of N-type Ca(2+) channel channels by Vc1.1 and RgIA is not mediated by the expression of alpha9alpha10 nAChRs in DRG neurons.

    Omega conotoxins as drugs: A Review

    Bingham JP, Mitsunaga E, Bergeron ZL (2010). Drugs from slugs--past, present and Future perspectives of omega-conotoxin research. Chem Biol Interact. 183: 1-18.

    Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, HI 96822, USA. jbingham@hawaii.edu

    Abstract: Peptides from the venom of carnivorous cone shells have provided six decades of intense research, which has led to the discovery and development of novel analgesic peptide therapeutics. Our understanding of this unique natural marine resource is however somewhat limited. Given the past pharmacological record, future investigations into the toxinology of these highly venomous tropical marine snails will undoubtedly yield other highly selective ion channel inhibitors and modulators. With over a thousand conotoxin-derived sequences identified to date, those identified as ion channel inhibitors represent only a small fraction of the total. Here we discuss our present understanding of conotoxins, focusing on the omega-conotoxin peptide family, and illustrate how such a seemingly simple snail has yielded a highly effective clinical drug.

    An omega conotoxin given intravenously is effective against neuropathic pain

    Kolosov A, Goodchild CS, Cooke I. (2010) CNSB004 (Leconotide) Causes Antihyperalgesia Without Side Effects When Given Intravenously: A Comparison with Ziconotide in a Rat Model of Diabetic Neuropathic Pain. Pain Med. 11: 262-273..

    Centre for Pain Medicine and Palliative Care, Monash Institute of Medical Research, Monash University, Clayton, Victoria.

    Abstract: Leconotide is an omega-conotoxin that blocks neuronal voltage sensitive calcium channels. This study compared the antihyperalgesic potencies of leconotide and ziconotide given intravenously alone and in combinations with a potassium channel modulator flupirtine, given intraperitoneally, in a rat model of diabetic neuropathic pain. Design. Rats were given streptozotocin (150 mg/kg ip) to induce diabetic neuropathy and hyperalgesia. Experiments were performed on diabetic rats with >/=30% hyperalgesia to noxious heat. Rats were given each conopeptide alone and with flupirtine. Open field activity monitoring and non-invasive blood pressure measurements were used to define the maximum doses and combinations that caused no side effects. Doses in a range up to maximum no side effect doses were tested for antihyperalgesic effects in rats with hyperalgesia. Results. The maximum no side effect dose of leconotide (2 mg/kg intravenously) caused 51.7% reversal of hyperalgesia compared with 0.4% for the highest no side effect dose of ziconotide (0.02 mg/kg; P < 0.001, one-way anova). Leconotide caused dose-related antihyperalgesic effects that were potentiated by coadministration with flupirtine at doses that were ineffective when given alone. Leconotide (0.02 mg/kg) and flupirtine (5 mg/kg) caused 25.3 +/- 7.6 and -6 +/- 9.5% reversal of hyperalgesia, respectively when given alone but in combination they caused 84.1 +/- 7.2% reversal of hyperalgesia (P < 0.01; one-way anova). No such interaction occurred with ziconotide. Conclusion. Leconotide could have wider clinical applications than ziconotide. Unlike ziconotide, powerful antihyperalgesia without side effects can be achieved by intravenous administration of leconotide thus avoiding the need for an intrathecal injection.

    1 January, 2010

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