What's New in 2009

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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)

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    31 December, 2009

    Halai R, Clark RJ, Nevin ST, Jensen JE, Adams DJ, Craik DJ. (2009) Scanning mutagenesis of alpha-conotoxin Vc1.1 reveals residues crucial for activity at the alpha9alpha10 nicotinic acetylcholine receptor. J Biol Chem 284:20275-20284.

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

    Abstract: Vc1.1 is a disulfide-rich peptide inhibitor of nicotinic acetylcholine receptors that has stimulated considerable interest in these receptors as potential therapeutic targets for the treatment of neuropathic pain. Here we present an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp, or Lys. The effect on acetylcholine (ACh)-evoked membrane currents at the alpha9alpha10 nicotinic acetylcholine receptor (nAChR), which has been implicated as a target in the alleviation of neuropathic pain, was then observed. The analogs were characterized by NMR spectroscopy to determine the effects of mutations on structure. The structural fold was found to be preserved in all peptides except where Pro was substituted. Electrophysiological studies showed that the key residues for functional activity are Asp(5)-Arg(7) and Asp(11)-Ile(15), because changes at these positions resulted in the loss of activity at the alpha9alpha10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. A second generation of mutants was synthesized, namely N9G, N9I, N9L, S4R, and S4K+N9A, all of which were more potent than Vc1.1 at both the rat alpha9alpha10 and the human alpha9/rat alpha10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the alpha3beta2, alpha3beta4, and alpha7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the alpha9alpha10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the alpha9alpha10 nAChR subtype and will help in the further development of analogs of Vc1.1 as analgesic drugs.

    Peraud O, Biggs JS, Hughen RW, Light AR, Concepcion GP, Olivera BM, Schmidt EW. (2009) Microhabitats within venomous cone snails contain diverse actinobacteria. Appl Environ Microbiol. 75) :6820-6826.

    Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.

    Abstract: Actinomycetes can be symbionts in diverse organisms, including both plants and animals. Some actinomycetes benefit their host by producing small molecule secondary metabolites; the resulting symbioses are often developmentally complex. Actinomycetes associated with three cone snails were studied. Cone snails are venomous tropical marine gastropods which have been extensively examined because of their production of peptide-based neurological toxins, but no microbiological studies have been reported on these organisms. A microhabitat approach was used in which dissected tissue from each snail was treated as an individual sample in order to explore bacteria in the tissues separately. Our results revealed a diverse, novel, and highly culturable cone snail-associated actinomycete community, with some isolates showing promising bioactivity in a neurological assay. This suggests that cone snails may represent an underexplored reservoir of novel actinomycetes of potential interest for drug discovery.

    McIntosh JM, Absalom N, Chebib M, Elgoyhen AB, Vincler M.(2009) Alpha9 nicotinic acetylcholine receptors and the treatment of pain. Biochem Pharmacol. 2009 Oct 1;78(7):693-702. Epub 2009 May 27

    Department of Psychiatry, University of Utah, Salt Lake City, UT 84132, USA. mcintosh.mike@gmail.com

    Abstract: Chronic pain is a vexing worldwide problem that causes substantial disability and consumes significant medical resources. Although there are numerous analgesic medications, these work through a small set of molecular mechanisms. Even when these medications are used in combination, substantial amounts of pain often remain. It is therefore highly desirable to develop treatments that work through distinct mechanisms of action. While agonists of nicotinic acetylcholine receptors (nAChRs) have been intensively studied, new data suggest a role for selective antagonists of nAChRs. alpha-Conotoxins are small peptides used offensively by carnivorous marine snails known as Conus. A subset of these peptides known as alpha-conotoxins RgIA and Vc1.1 produces both acute and long lasting analgesia. In addition, these peptides appear to accelerate the recovery of function after nerve injury, possibly through immune mediated mechanisms. 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).

    11 November, 2009

    Filipino scientist wins P4.6-M prize for snail toxin work

    MANILA - A local scientist who helped discover a snail toxin a thousand times more powerful than morphine, was chosen as the first Filipino winner of the 2010 L'oréal-UNESCO "For Women In Science Awards." Dr. Lourdes Jansuy Cruz, 67, a National Scientist based at the University of the Philippines- Diliman, was recognized for her role in discovering Conotoxins (or toxins from marine snails) during the 1970s to 80s. See story here. Cruz was directly involved in isolating peptides (a chain of amino acids which are the building blocks of proteins) from the venom of Conus (cone) snails found in the Philippines. One of these peptides was developed by U.S. biotechnology firm Cognetix Corp. (later bought by Elan Pharmaceuticals) to produce the non-addictive drug Ziconotide / Prialt (Primary alternative to morphine).

    31 October, 2009

    Evolution of Conus in the Cape Verde Islands

    Regina Lopes da Cunha (2009) Tempo and mode of evolution of the genus Conus in the Cape Verde Islands. 12th Annual Molluscan Forum, Thursday 12th November 2009, The Dorothea Bate Room, Natural History Museum, London, UK

    20 October, 2009

    Novel I(2)-superfamily conotoxins from Conus spurius, Gulf of Mexico

    Zamora-Bustillos R, Aguilar MB, Falcón A. (2009) Identification, by molecular cloning, of a novel type of I(2)-superfamily conotoxin precursor and two novel I(2)-conotoxins from the worm-hunter snail Conus spurius from the Gulf of Méxic. Peptides. 2009 Oct 14. [Epub ahead of print]

    Laboratorio de Neurofarmacología Marina, Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro 76230, México.

    Abstract: cDNA was prepared from the venom duct of a single Conus spurius specimen collected near the coast of Campeche, México. From it, PCR products were generated aiming to clone I-conotoxin precursors. Thirty clones were sequenced and predicted to encode ten distinct precursors: seven of I(2)-conotoxins and three of I(2)-like-conotoxins. These precursors contain three different, mature toxins, sr11a, sr11b and sr11c, of which two are novel and one (sr11a) has been previously purified and characterized from the venom of this species. The precursors include a 26- (I(2)) or 23- residue signal peptide (I(2)-like), a 31-residue "pro" region (I(2)-like), and a 32-residue mature toxin region (I(2) and I(2)-like). In addition, all the precursors have a 13-residue "post" region which contains a gamma-carboxylation recognition sequence that directs the gamma-carboxylation of Glu-9 and Glu-10 of toxin sr11a and, possibly, Glu-13 of toxin sr11b and Glu-9 of toxin sr11c. This is the first time that a "post" region has been found in precursors of I-conotoxins that also contain a "pro" region. The "post" peptide is enzymatically processed to yield the amidated mature toxin sr11a, which implies that gamma-carboxylation occurs before amidation. Phylogenetic analysis at the whole precursor level indicates that the I(2)-like-conotoxins of C. spurius are more related to I(2)-conotoxins than to I(1)- and I(3)-conotoxins from other species, and that they might represent a new subgroup of the I(2)-superfamily. The three I-conotoxins from C. spurius have charge differences at seven to nine positions, suggesting that they might have different molecular target types or subtypes.

    17 October, 2009

    Human envenomation by Conus regius

    Haddad, V. Jr., Coltro, M and Simone, LRL (2009) Report of a human accident caused by Conus regius (Gastropoda, Conidae). Revista da Sociedade Brasileira de Medicina Tropical 42: 446-448.[Article.pdf]

    Botuscatu School of Medicine, Sao Paulo State Universiy, Botucatu, SP, Brazil; Vital Brasil Hospital, Butantan Institute, Sao Paulo, SP, Brazil; Femorale Shells, Sao Paulo, SP, Brazil. Address correspondence to Prof. Vidal Haddad Junior, Caixa Postal 557, 18618-000 Botucatu, SP, Brazil

    Abstract: Conus regius is a venomous molluc in the Conidae family, which includes species responsible for severe or even fatal accidents affecting human beings. This is the first report of a clinical case involving this species. It consisted a puncture in the right hand of a diver who presented parashesia and movement diffikculty in the whole limb. The manifestations disappeared after around twelve hours, without sequelae.

    Marine pharmacology - a REVIEW

    Glaser KB, Mayer AMS (2009) A renaissance in marine pharmacology: from preclinical curiosity to clinical reality Biochem Pharmacol 2009,Sep,01;78(5):440-448

    Cancer Research R47J-AP9, Abbott Laboratories, Abbott Park, IL 60064-6121, USA. keith.glaser@abbott.com

    Abstract: Marine pharmacology, the pharmacology of marine natural products, has been for some time more associated with marine natural products chemistry rather than mainstay pharmacology. However, in recent years a renaissance has occurred in this area of research, and has seen the US Food & Drug Administration (FDA) approval in 2004 of Prialt (ziconotide, omega-conotoxin MVIIA) the synthetic equivalent of a conopeptide found in marine snails, used for the management of severe chronic pain. Furthermore Yondelis) (trabectedin, ET-743) an antitumor agent scovered in a marine colonial tunicate, and now produced synthetically, receiving Orphan Drug designation from the European Commission (EC) and FDA for soft tissue sarcomas and ovarian cancer and its registration in 2007 in the EU for the treatment of soft tissue sarcoma. The approval/marketing of so few marine natural products has come after many years of research primarily by the academic community and the sporadic involvement of major pharmaceutical companies. This commentary, through the opinions provided by several leaders in the marine natural products field, will examine the potential reasons and perceptions from both the academic and pharmaceutical communities regarding the development of marine natural products as viable therapeutic entities.

    Some recent cone shell and conopeptide papers

    More to come as now back on line and updating from 10 April 2008

    MS identification of conotoxin Vc1.1 and conopeptides along venom duct of Conus victoriae

    Townsend, A., Livett, BG, Bingham, J-P, Truong, H-T, Karas, JA, O’Donnell, P, Williamson, NA, Purcell, AW and Scanlon D (2009) Mass spectral identification of Vc1.1 and differential distribution of conopeptides in the venom duct of Conus victoriae. Effect of post-translational modifications and disulfide isomerisation on bioactivity. Int. J. Peptide Res and Therap. 15 (3): 195-20 [Abstract], [ Article.pdf]

    Dept. Biochemistry & Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3010, Australia; Metabolic Pharmaceuticals Ltd., Level 3, 509 St. Kilda Rd. Melbourne, VIC. 3004, Australia; Dept Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, HI 96822, USA

    Abstract: Molluscs of the genus Conus (cone shells) are carnivorous, feeding on marine worms, small fish and other marine molluscs. They capture their prey by injecting venom containing hundreds of neurally active peptide components. These peptides are classed as conotoxins and consist of small disulfide-bonded peptides exhibiting a high degree of post-translational modifications (PTMs). The functional roles of these modifications remain largely unknown. Two of the most frequently observed modifications are gamma-carboxylation of glutamate and hydroxylation of proline (Buczek et al. Cell Mol Life Sci 62:3067, 2005). Vc1.1 is an alpha-conotoxin from Conus victoriae (Sandall et al. Biochemistry 42(22):6904–6911, 2003) and the only form of this peptide which has been detected in the venom is the gamma-glutamate and hydroxyproline (Vc1.1.P6O:E14Gla) version of the molecule (Jakubowski et al. Toxicon 47(6):688–699, 2006). In order to investigate the role of PTMs, we did mass spectral profiling of the venom duct of C. victoriae looking at changes in mass and the number of peptides detected. We synthesised a number of predicted Vc1.1-PTM peptides together with the three possible disulfide isoforms of Vc1.1 and assessed the possible functional role of the PTM conopeptides by measuring the in vitro activity at the cognate neuronal nicotinic acetylcholine receptors (nAChRs). In addition we looked for their presence Vc1.1 venom by mass spectrometry and by this approach we were able to detect unmodified Vc1.1 in C. victoriae venom for the first time.

    Dicarba analogues of alpha-Conotoxin ImI

    MacRaild CA, Illesinghe J, van Lierop BJ, Townsend AL, Chebib M, Livett BG, Robinson AJ, Norton RS (2009) Structure and activity of (2,8)-dicarba-(3,12)-cystino alpha-ImI, an alpha-conotoxin containing a nonreducible cystine analogue. J Med Chem. 52 : 755-762. [Abstract], [ Article.pdf], [ SUPPORTING INFO.pdf]

    The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville 3050, Victoria, Australia.

    Abstract: The alpha-conotoxins are potent and selective antagonists of nicotinic acetylcholine receptors (nAChR). Exploitation of these and other peptides in research and clinical settings has been hampered by the lability of the disulfide bridges that are essential for toxin structure and activity. One solution to this problem is replacement of cystine bridges with nonreducible dicarba linkages. We explore this approach by determining the solution structure and functional characteristics of a dicarba analogue of the alpha-conotoxin alpha-ImI, (2,8)-dicarba-(3,12)-cystino alpha-ImI. The structure of the dicarba analogue was similar to that of native alpha-ImI, with differences attributable to the different covalent geometry of the disulfide and dicarba bridges. Dicarba-alpha-ImI maintained inhibitory activity of nAChR comparable to that of native alpha-ImI in two in vitro assays. These findings confirm the potential of the dicarba linkage to improve stability while maintaining alpha-conotoxin function.

    Dicarba analogues of alpha-Conotoxin RgIA

    Robinson AJ, van Lierop BJ, Garland RD, Teoh E, Elaridi J, Illesinghe JP and Jackson WR (2009) Regioselective formation of interlocked dicarba bridges in naturally occurring cyclic peptide toxins using olefin metathesis. Chem. Commun. (Camb). 2009 Jul 28:(28)4293-4295.
    Supplementary Information on experimental details: MS and HPLC chromatograms for dicarba peptides, and IR data is available here.

    School of Chemistry, Centre for Green Chemistry, Monash University, Clayton 3800, Victoria, Australia.

    Abstract: Bis-dicarba analogues of native dicystine-containing-conotoxin Rg1A, an analgesic peptide isolated from cone snail venom, were constructed on resin using a regioselective metathesis–hydrogenation strategy.

    Conus anemone vs Conus novaehollandiae

    John K. Tucker (2009) Floraconus anemone: An Example of Circular Overlap? The Cone Collector 11: 8-24

    Great Rivers Field Station, Illinois Natural History Survey, USA

    Abstract: This article in The Cone Collector Vol. 11 (2009) provides an extensive account of the naming and distribution of Conus anemone and variants around the coast of Australia

    Conus anemone
    (Creator: Keith Davey © Department of the Environment, Water, Heritage and the Arts, Australia) Distribution

    Discrimination of alpha-conotoxins for subtypes of nicotinic receptors

    Turner M, Eidemiller S, Martin B, Narver A, Marshall J, Zemp L, Cornell KA, McIntosh JM, McDougal OM. (2009) Structural basis for alpha-conotoxin potency and selectivity. Bioorg Med Chem. 2009 Aug 15;17(16):5894-5899

    Department of Chemistry and Biochemistry, Boise State University, ID 83725-1520, USA.

    Abstract: Parkinson's disease is a debilitating movement disorder characterized by altered levels of alpha(6)beta(2) * ( * indicates the possible presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) localized on presynaptic striatal catecholaminergic neurons. alpha-Conotoxin MII (alpha-CTx MII) is a highly useful ligand to probe alpha(6)beta(2) nAChRs structure and function, but it does not discriminate among closely related alpha(6) * nAChR subtypes. Modification of the alpha-CTx MII primary sequence led to the identification of alpha-CTx MII[E11A], an analog with 500-5300-fold discrimination between alpha(6) * subtypes found in both human and non-human primates. alpha-CTx MII[E11A] binds most strongly (femtomolar dissociation constant) to the high affinity alpha(6) nAChR, a subtype that is selectively lost in Parkinson's disease. Here, we present the three-dimensional solution structure for alpha-CTx MII[E11A] as determined by two-dimensional (1)H NMR spectroscopy to 0.13+/-0.09A backbone and 0.45+/-0.08A heavy atom root-mean-square deviation from mean structure. Structural comparisons suggest that the increased hydrophobic area of alpha-CTx MII[E11A] relative to other members of the alpha-CTx family may be responsible for its exceptionally high affinity for alpha6alpha4beta2 * nAChR as well as discrimination between alpha(6)beta(2) and alpha(3)beta(2) containing nAChRs. This finding may enable the rational design of novel peptide analogs that demonstrate enhanced specificity for alpha(6) * nAChR subunit interfaces and provide a means to better understand nAChR structural determinants that modulate brain dopamine levels and the pathophysiology of Parkinson's disease.

    Conotoxins as pain killers - REVIEW

    Zhao CJ, Dai QY. (2009) [Recent advances in study of antinociceptive conotoxins] [Article in Chinese] Yao Xue Xue Bao. 2009 Jun;44(6):561-565.

    Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China.

    Abstract: The Conus venom is secreted by the duct and theca of venom. Most of conotoxins are composed of 10-40 amino acid residues with several disulfide bridges. They can specifically target neurotransmitter receptors including nAChRs, calcium ion channels, sodium ion channels and potassium ion channels, etc. Some conotoxins, such as that target N-Ca2+ channels, nAChR alpha9alpha10 subtype, TTX-R Na+ channels or NMDA receptors, have potent antinociceptive activities, omega-MVIIA, an Ca2+ channels blocker was approved by FDA in December, 2004 for marketing. Because of lower molecular weight and high specificity, conotoxins are the powerful pharmacology tools and potent analgesics without addiction. This review briefly summarizes the research progress of antinociceptive conotoxins and addresses on their targets and structure-activity relationships.

    Omega toxins - REVIEW

    Bingham JP, Mitsunaga E, Bergeron ZL(2009) Drugs from Slugs - Past, Present and Future Perspectives of omega-Conotoxin Research. Chem Biol Interact. 2009 Oct 1. [Epub ahead of print]

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

    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.

    Novel alpha conotoxin from Conus quercinus

    Peng C, Chen W, Han Y, Sanders T, Chew G, Liu J, Hawrot E, Chi C, Wang C (2009).

    Characterization of a novel alpha4/4-conotoxin, Qc1.2, from vermivorous Conus quercinus.Acta Biochim Biophys Sin (Shanghai). 2009 Oct;41(10):858-864.

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

    Abstract: As part of continuing studies of the identification of gene organization and cloning of novel alpha-conotoxins, the first alpha4/4-conotoxin identified in a vermivorous Conus species, designated Qc1.2, was originally obtained by cDNA and genomic DNA cloning from Conus quercinus collected in the South China Sea. The predicted mature toxin of Qc1.2 contains 14 amino acid residues with two disulfide bonds (I-III, II-IV connectivity) in a native globular configuration. The mature peptide of Qc1.2 is supposed to contain an N-terminal post-translationally processed pyroglutamate residue and a free carboxyl C-terminus. This peptide was chemically synthesized and refolded for further characterization of its functional properties. The synthetic Qc1.2 has two interconvertible conformations in aqueous solution, which may be due to the cis-trans isomerization of the two successive Pro residues in its first Cys loop. Using the Xenopus oocyte heterologous expression system, Qc1.2 was shown to selectively inhibit both rat neuronal alpha3beta2 and alpha3beta4 subtypes of nicotinic acetylcholine receptors with low potency. A block of about 63% and 37% of the ACh-evoked currents was observed, respectively, and the toxin dissociated rapidly from the receptors. Compared with other characterized alpha-conotoxin members, the unusual structural features in Qc1.2 that confer to its receptor recognition profile are addressed.

    Neuroprotective effects of SNX185

    Shahlaie K, Lyeth B, Gurkoff GG, Muizelaar JP, Berman RF (2009) Neuroprotective Effects of Selective N-Type VGCC Blockade on Stretch Injury-Induced Calcium Dynamics in Cortical Neurons. J Neurotrauma. 2009 Sep 22. [Epub ahead of print]

    University of California, Davis, Neurological Surgery, 4860 Y Street, Suite 3740, Sacramento, California, United States, 95817, 916-734-3071, 916-452-2580; krshahlaie@ucdavis.edu.

    Abstract: Acute elevation in intracellular calcium ([Ca2+]i) following traumatic brain injury (TBI) can trigger cellular mechanisms leading to neuronal dysfunction and death. The mechanisms underlying these processes are incompletely understood, but calcium influx through N-type VGCCs appears to play a central role. The present study examined the time course of [Ca2+]i flux, glutamate release, and loss of cell viability following injury using an in vitro neuronal-glial cortical cell culture model of TBI. The effects of N-channel blockade with SNX185 (e.g., omega-conotoxin TIVA) before or after injury were also examined. Neuronal injury produced a transient elevation in [Ca2+]i, increased glutamate release, and resulted in neuronal and glial death. SNX185 administered before or immediately after cell injury reduced glutamate release and increased survival of neurons and astrocytes, whereas delayed treatment did not improve cell survival but significantly facilitated the return of [Ca2+]i to baseline levels. The new findings that N-type VGCCs are critically involved in injury-induced glutamate release and recovery of [Ca2+]i argue for continued investigation of this treatment strategy for clinical management of TBI. In particular, SNX-185 may represent an effective class of drugs that can significantly protect injured neurons from secondary insults that commonly occur after TBI.

    Role of the Ca(V)2.3subtype in nociception

    Yang L, Stephens GJ(2009) Effects of neuropathy on high-voltage-activated Ca(2+) current in sensory neurones.Cell Calcium. 2009 Aug 31. [Epub ahead of print]

    School of Pharmacy, University of Reading, PO Box 228, Whiteknights, Reading RG6 6AJ, United Kingdom.

    Abstract: Voltage-dependent Ca(2+) channels (VDCCs) have emerged as targets to treat neuropathic pain; however, amongst VDCCs, the precise role of the Ca(V)2.3 subtype in nociception remains unproven. Here, we investigate the effects of partial sciatic nerve ligation (PSNL) on Ca(2+) currents in small/medium diameter dorsal root ganglia (DRG) neurones isolated from Ca(V)2.3(-/-) knock-out and wild-type (WT) mice. DRG neurones from Ca(V)2.3(-/-) mice had significantly reduced sensitivity to SNX-482 versus WT mice. DRGs from Ca(V)2.3(-/-) mice also had increased sensitivity to the Ca(V)2.2 VDCC blocker omega-conotoxin. In WT mice, PSNL caused a significant increase in omega-conotoxin-sensitivity and a reduction in SNX-482-sensitivity. In Ca(V)2.3(-/-) mice, PSNL caused a significant reduction in omega-conotoxin-sensitivity and an increase in nifedipine sensitivity. PSNL-induced changes in Ca(2+) current were not accompanied by effects on voltage-dependence of activation in either Ca(V)2.3(-/-) or WT mice. These data suggest that Ca(V)2.3 subunits contribute, but do not fully underlie, drug-resistant (R-type) Ca(2+) current in these cells. In WT mice, PSNL caused adaptive changes in Ca(V)2.2- and Ca(V)2.3-mediated Ca(2+) currents, supporting roles for these VDCCs in nociception during neuropathy. In Ca(V)2.3(-/-) mice, PSNL-induced changes in Ca(V)1 and Ca(V)2.2 Ca(2+) current, consistent with alternative adaptive mechanisms occurring in the absence of Ca(V)2.3 subunits.

    Alpha-conotoxin ImII and its ribbon isomer (ImIIiso)both block human alpha7 and muscle nAChRs equivalently

    Kasheverov IE, Zhmak MN, Fish A, Rucktooa P, Khruschov AY, Osipov AV, Ziganshin RH, D'hoedt D, Bertrand D, Sixma TK, Smit AB, Tsetlin VI(2009) Interaction of alpha-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor. J Neurochem. 2009 Aug 27. [Epub ahead of print]

    Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.

    Abstract: alpha-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. alpha-Conotoxins blocking muscle-type or alpha7 nAChRs compete with alpha-bungarotoxin. However, alpha-conotoxin ImII, a close homolog of the alpha7 nAChR-targeting alpha-conotoxin ImI, blocked alpha7 and muscle nAChRs without displacing alpha-bungarotoxin (Ellison et al. 2003, 2004), suggesting binding at a different site. We synthesized alpha-conotoxin ImII, its ribbon isomer (ImIIiso), 'mutant' ImII(W10Y) and found similar potencies in blocking human alpha7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [(125)I]-alpha-bungarotoxin from human alpha7 nAChRs in the cell line GH(4)C(1) (IC(50) 17 and 23 muM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC(50) 2.0-9.0 muM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized alpha-bungarotoxin (K(d) and IC(50) 2.5-8.2 muM). On Torpedo nAChR, alpha-conotoxin [(125)I]-ImII(W10Y) revealed specific binding (K(d) 1.5-6.1 muM) and could be displaced by alpha-conotoxin ImII, ImIIiso and ImII(W10Y) with IC(50) 2.7, 2.2 and 3.1 muM, respectively. As alpha-cobratoxin and alpha-conotoxin ImI displaced [(125)I]-ImII(W10Y) only at higher concentrations (IC(50)>/= 90 muM), our results indicate that alpha-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.

    Safety and Efficacy of intrathecal Ziconotide (PRIALT)

    Smith HS, Deer TR(2009) Safety and efficacy of intrathecal ziconotide in the management of severe chronic pain. Ther Clin Risk Manag. 2009 Jun;5(3):521-34. Epub 2009 Jul 12.

    Albany Medical College, Department of Anesthesiology, Albany, New York, USA.

    Abstract: Ziconotide is a conopeptide intrathecal (IT) analgesic which is approved by the US Food and Drug Administration (FDA) for the management of severe chronic pain. It is a synthetic equivalent of a naturally occurring conopeptide found in the venom of the fish-eating marine cone snail and provides analgesia via binding to N-type voltage-sensitive calcium channels in the spinal cord. As ziconotide is a peptide, it is expected to be completely degraded by endopeptidases and exopeptidases (Phase I hydrolytic enzymes) widely located throughout the body, and not by other Phase I biotransformation processes (including the cytochrome P450 system) or by Phase II conjugation reactions. Thus, IT administration, low plasma ziconotide concentrations, and metabolism by ubiquitous peptidases make metabolic interactions of other drugs with ziconotide unlikely. Side effects of ziconotide which tend to occur more commonly at higher doses may include: nausea, vomiting, confusion, postural hypotension, abnormal gait, urinary retention, nystagmus/amblyopia, drowsiness/somnolence (reduced level of consciousness), dizziness or lightheadedness, weakness, visual problems (eg, double vision), elevation of serum creatine kinase, or vestibular side effects. Initially, when ziconotide was first administered to human subjects, titration schedules were overly aggressive and led to an abundance of adverse effects. Subsequently, clinicians have gained appreciation for ziconotide's relatively narrow therapeutic window. With appropriate usage multiple studies have shown ziconotide to be a safe and effective intrathecal analgesic alone or in combination with other intrathecal analgesics.

    M-Superfamily of Conotoxins - REVIEW

    Jacob RB, McDougal OM(2009) The M-superfamily of conotoxins: a review. Cell Mol Life Sci. 2009 Aug 25. [Epub ahead of print]

    Department of Chemistry and Biochemistry, Boise State University, 1910 University Drive, Boise, ID, 83725-1520, USA.

    Abstract: The focus of this review is the M-superfamily of Conus venom peptides. Disulfide rich peptides belonging to the M-superfamily have three loop regions and the cysteine arrangement: CC-C-C-CC, where the dashes represent loops one, two, and three, respectively. Characterization of M-superfamily peptides has demonstrated that diversity in cystine connectivity occurs between different branches of peptides even though the cysteine pattern remains consistent. This superfamily is subdivided into five branches, M-1 through M-5, based on the number of residues in the third loop region, between the fourth and fifth cysteine residues. M-superfamily peptides appear to be ubiquitous in Conus venom. They are largely unexplained in indigenous biological function, and they represent an active area of research within the scientific community.

    Nicotinic receptors that mediate alpha-CtxMII-resistant dopamine release

    Grady SR, Salminen O, McIntosh JM, Marks MJ, Collins AC(2009) Mouse Striatal Dopamine Nerve Terminals Express alpha4alpha5beta2 and Two Stoichiometric Forms of alpha4beta2*-Nicotinic Acetylcholine Receptors. J Mol Neurosci. 2009 Aug 20. [Epub ahead of print]

    Institute for Behavioral Genetics, University of Colorado, Boulder, CO, USA.

    Abstract: Wild-type and alpha5 null mutant mice were used to identify nicotinic cholinergic receptors (nAChRs) that mediate alpha-conotoxin MII (alpha-CtxMII)-resistant dopamine (DA) release from striatal synaptosomes. Concentration-effect curves for ACh-stimulated release (20 s) were monophasic when wild-type synaptosomes were assayed but biphasic with synaptosomes from the alpha5 null mutant. Deleting the alpha5 gene also resulted in decreased maximal ACh-stimulated alpha-CtxMII-resistant DA release. When a shorter perfusion time (5 s) was used, biphasic curves were detected in both wild-type and alpha5 null mutants, indicative of high- and low-sensitivity (HS and LS) activity. In addition, DHbetaE-sensitive (HS) and DHbetaE-resistant (LS) components were found in both genotypes. These results indicate that alpha-CtxMII-resistant DA release is mediated by alpha4alpha5beta2, (alpha4)(2)(beta2)(3) (HS), and (alpha4)(3)(beta2)(2) (LS) nAChRs.

    Omega-conotoxin GVIA mimetics

    Andersson A, Baell JB, Duggan PJ, Graham JE, Lewis RJ, Lumsden NG, Tranberg CE, Tuck KL, Yang A(2009) Omega-conotoxin GVIA mimetics based on an anthranilamide core: effect of variation in ammonium side chain lengths and incorporation of fluorine. Bioorg Med Chem. 2009 Sep 15;17(18):6659-70. Epub 2009 Jul 30.

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

    Abstract: A number of omega-conotoxin GVIA mimetics based on an anthranilamide core were prepared and tested for their affinity for rat brain Ca(v)2.2 channels. Features such as the presence of hydroxyl and fluoro substituents on the tyrosine side chain mimic, the length of the chains on the lysine/arginine side chain mimics and the use of diguanidino and diamino substituents rather than mono-guanidine/mono-amine substitution were examined. The diguanidinylated compounds proved to be the most active and deletion of the hydroxyl substituent had a limited influence on activity. The SAR associated with variation in the lysine/arginine side chain mimics was not strong. The introduction of a fluoro substituent into the tyrosine mimic produced the most active compound prepared in this study (2g), with an EC(50) at rat brain Ca(v)2.2 channels of 6 microM.

    Rapid detection of peptide toxins in serum

    Fang J, Dong F, Wang N, He K, Liu B, Wu S, Li A, Zhang X(2009) Rapid detection of conotoxin SO(3) in serum using Cu-chelated magnetic beads coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J Anal Toxicol. 2009 Jun;33(5):272-277.

    National Center of Biomedical Analysis, Beijing 100039, China.

    Abstract: A novel method based on Cu-chelated magnetic beads (Cu-Magbeads) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was developed for the rapid detection of peptide toxins in serum. The peptides in the serum were efficiently adsorbed by the Cu-Magbeads, eluted with methanol solution, and assayed by MALDI-TOF-MS. Specific peptides were identified according to their characteristic mass-to-charge ratio values. Conotoxin SO(3), a synthesized peptide, was used as a model to evaluate the method. Conotoxin SO(3) was detected in human serum, as well as bovine and murine serum, with a detection sensitivity in the low femtomole range. The assay was performed within 40 min, without the need for a specific antibody or an expensive reagent. It shows potential for future use in clinical and emergency rescue practice because of its simplicity, high speed, and high sensitivity.

    Protective effects of omega-conotoxin GVIA in optic neuritis

    Gadjanski I, Boretius S, Williams SK, Lingor P, Knöferle J, Sättler MB, Fairless R, Hochmeister S, Sühs KW, Michaelis T, Frahm J, Storch MK, Bähr M, Diem R(2009) Role of N-type voltage-dependent calcium channels in autoimmune optic neuritis. Ann Neurol. 66(1): 81-93

    Department of Neurology, Georg-August University, Göttingen, Germany.

    Abstract: OBJECTIVE: The aim of this study was to investigate the role of voltage-dependent calcium channels (VDCCs) in axon degeneration during autoimmune optic neuritis. METHODS: Calcium ion (Ca(2+)) influx into the optic nerve (ON) through VDCCs was investigated in a rat model of optic neuritis using manganese-enhanced magnetic resonance imaging and in vivo calcium imaging. After having identified the most relevant channel subtype (N-type VDCCs), we correlated immunohistochemistry of channel expression with ON histopathology. In the confirmatory part of this work, we performed a treatment study using omega-conotoxin GVIA, an N-type specific blocker. RESULTS: We observed that pathological Ca(2+) influx into ONs during optic neuritis is mediated via N-type VDCCs. By analyzing the expression of VDCCs in the inflamed ONs, we detected an upregulation of alpha(1B), the pore-forming subunit of N-type VDCCs, in demyelinated axons. However, high expression levels were also found on macrophages/activated microglia, and lower levels were detected on astrocytes. The relevance of N-type VDCCs for inflammation-induced axonal degeneration and the severity of optic neuritis was corroborated by treatment with omega-conotoxin GVIA. This blocker led to decreased axon and myelin degeneration in the ONs together with a reduced number of macrophages/activated microglia. These protective effects were confirmed by analyzing the spinal cords of the same animals. INTERPRETATION: We conclude that N-type VDCCs play an important role in inflammation-induced axon degeneration via two mechanisms: First, they directly mediate toxic Ca(2+) influx into the axons; and second, they contribute to macrophage/microglia function, thereby promoting secondary axonal damage. Ann Neurol 2009;66:81-93.

    Flourescent alpha-conotoxin

    Hone AJ, Whiteaker P, Christensen S, Xiao Y, Meyer EL, McIntosh JM(2009) A novel fluorescent alpha-conotoxin for the study of alpha7 nicotinic acetylcholine receptors. J Neurochem. 11: 80-89.

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

    Abstract: Homomeric alpha7 nicotinic acetylcholine receptors are a well-established, pharmacologically distinct subtype. The more recently identified alpha9 subunit can also form functional homopentamers as well as alpha9alpha10 heteropentamers. Current fluorescent probes for alpha7 nicotinic ACh receptors are derived from alpha-bungarotoxin (alpha-BgTx). However, alpha-BgTx also binds to alpha9* and alpha1* receptors which are coexpressed with alpha7 in multiple tissues. We used an analog of alpha-conotoxin ArIB to develop a highly selective fluorescent probe for alpha7 receptors. This fluorescent alpha-conotoxin, Cy3-ArIB[V11L;V16A], blocked ACh-evoked alpha7 currents in Xenopus laevis oocytes with an IC(50) value of 2.0 nM. Observed rates of blockade were minute-scale with recovery from blockade even slower. Unlike FITC-conjugated alpha-BgTx, Cy3-ArIB[V11L;V16A] did not block alpha9alpha10 or alpha1beta1deltaepsilon receptors. In competition binding assays, Cy3-ArIB[V11L;V16A] potently displaced [(125)I]-alpha-BgTx binding to mouse hippocampal membranes with a K(i) value of 21 nM. Application of Cy3-ArIB[V11L;V16A] resulted in specific punctate labeling of KXalpha7R1 cells but not KXalpha3beta2R4, KXalpha3beta4R2, or KXalpha4beta2R2 cells. This labeling could be abolished by pre-treatment with alpha-cobratoxin. Thus, Cy3-ArIB[V11L;V16A] is a novel and selective fluorescent probe for alpha7 receptors.

    Selective alpha6 antagonist, alpha-conotoxin MII[H9A;L15A]

    Jackson KJ, McIntosh JM, Brunzell DH, Sanjakdar SS, Damaj MI(2009) The role of {alpha}6-containing nicotinic acetylcholine receptors in nicotine reward and withdrawal. J Pharmacol Exp Ther. 331: 547-554

    Virginia Commonwealth University.

    Abstract: The alpha6 nicotinic acetylcholine receptor (nAChR) subunit is involved in nicotine-stimulated dopamine release in the striatum. It is expressed in brain regions and co-expressed with nAChR subtypes implicated in nicotine dependence behaviors; hence, this subunit may play a role in nicotine dependence. Using the alpha6-selective antagonist, alpha-conotoxin H9A; L15A (MII[H9A;L15A]), we determined the role of alpha6* nAChRs in the pharmacological and behavioral effects of nicotine. We measured effects of pre-treatment with MII[H9A;L15A] on analgesia, locomotion, and body temperature following a single injection of nicotine. Effects of MII[H9A;L15A] on nicotine reward were measured using the conditioned place preference (CPP) paradigm. We further measured physical (somatic signs, hyperalgesia) and affective (anxiety-related behavior, conditioned place aversion (CPA)) nicotine withdrawal behaviors following extended nicotine exposure. Results showed that MII[H9A;L15A] did not block acute nicotine effects on the behaviors measured. Conversely, MII[H9A:l15A] blocked the expression of nicotine CPP, as well as withdrawal-associated CPA and anxiety-related behavior in the elevated plus maze, but not withdrawal-induced somatic signs or hyperalgesia. These results suggest a role for the alpha6 nAChR subunit in nicotine reward and affective nicotine withdrawal, but not acute nicotine-induced or physical withdrawal behaviors.

    Conotoxins as drug leads - REVIEW

    Halai R, Craik DJ.(2009) Conotoxins: natural product drug leads. Nat Prod Rep. 26:526-536

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

    Abstract: Venomous marine cone snails harbour a variety of small disulfide-rich peptides called conotoxins, which target a broad range of ion channels, membrane receptors, and transporters. More than 700 species of Conus are thought to exist, each expressing a wide array of different peptides. Within this large repertoire of toxins, individual conotoxins are able to discriminate between different subtypes and isoforms of ion channels, making them valuable pharmacological probes or leads for drug design. This review gives a brief background to the discovery of conotoxins and describes their sequences, biological activities, and applications in drug design.

    Conus ebreus - variations in genomic alleles at Okinawa, Guam and Hawaii

    Duda TF Jr, Chang D, Lewis BD, Lee T Geographic variation in venom allelic composition and diets of the widespread predatory marine gastropod Conus ebraeus PLoS One. 2009 4:e6245.

    Department of Ecology and Evolutionary Biology and Museum of Zoology, University of Michigan, Ann Arbor, MI, USA. tfduda@umich.edu

    Abstract: BACKGROUND: Members of the predatory gastropod genus Conus use a venom comprised of a cocktail of peptide neurotoxins, termed conotoxins or conopeptides, to paralyze prey and conotoxin gene family members diversify via strong positive selection. Because Conus venoms are used primarily to subdue prey, the evolution of venoms is likely affected by predator-prey interactions. METHODOLOGY/PRINCIPAL FINDINGS: To identify the selective forces that drive the differentiation of venoms within species of Conus, we examined the distribution of alleles of a polymorphic O-superfamily conotoxin locus of Conus ebraeus at Okinawa, Guam and Hawaii. Previous analyses of mitochondrial cytochrome oxidase I gene sequences suggest that populations of C. ebraeus, a worm-eating Conus, are not structured genetically in the western and central Pacific. Nonetheless, because the sample size from Guam was relatively low, we obtained additional data from this location and reexamined patterns of genetic variation at the mitochondrial gene at Okinawa, Guam and Hawaii. We also utilized a DNA-based approach to identify prey items of individuals of C. ebraeus from Guam and compared this information to published data on diets at Okinawa and Hawaii. Our results show that conotoxin allelic frequencies differ significantly among all three locations, with strongest differentiation at Hawaii. We also confirm previous inferences that C. ebraeus exhibits no genetic differentiation between Okinawa, Guam and Hawaii at the mitochondrial locus. Finally, DNA-based analyses show that eunicid polychaetes comprise the majority of the prey items of C. ebraeus at Guam; while this results compares well with observed diet of this species at Okinawa, C. ebraeus preys predominantly on nereid polychaetes at Hawaii. CONCLUSIONS/SIGNIFICANCE: These results imply that strong selection pressures affect conotoxin allelic frequencies. Based on the dietary information, the selection may derive from geographic variation in dietary specialization and local coevolutionary arms races between Conus and their prey.

    Peptide interactions with the nicotinic receptor

    Kasheverov IE, Utkin YN, Tsetlin VI (2009)Naturally occurring and synthetic peptides acting on nicotinic acetylcholine receptors. Curr Pharm Des. 15:2430-2452.

    Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation.

    Abstract: Nicotinic acetylcholine receptors (nAChRs) are pentameric membrane-bound proteins belonging to the large family of ligand-gated ion channels. nAChRs possess various binding sites which interact with compounds of different chemical nature, including peptides. Historically first peptides found to act on nAChR were synthetic fragments of snake alpha-neurotoxins, competitive receptor antagonists. Later it was shown that fragments of glycoprotein from rabies virus, having homology to alpha-neurotoxins, and polypeptide neurotoxins waglerins from the venom of Wagler's pit viper Trimeresurus (Tropidolaemus) wagleri bind in a similar way, waglerins being efficient blockers of muscle-type nAChRs. Neuropeptide substance P appears to interact with the channel moiety of nAChR. beta-Amyloid, a peptide forming senile plaques in Alzheimer's disease, also can bind to nAChR, although the mode of binding is still unclear. However, the most well-studied peptides interacting with the ligand-binding sites of nAChRs are so-called alpha-conotoxins, peptide neurotoxins from marine snails of Conus genus. First alpha-conotoxins were discovered in the late 1970s, and now it is a rapidly growing family due to isolation of peptides from multiple Conus species, as well as to cloning, and chemical synthesis of new analogues. Because of their unique selectivity towards distinct nAChR subtypes, alpha-conotoxins became valuable tools in nAChR research. Recent X-ray structures of alpha-conotoxin complexes with acetylcholine-binding protein, a model of nAChR ligand-binding domains, revealed the details of the nAChR ligand-binding sites and provided the basis for design of novel ligands.

    Novel I-superfamily conotoxins from Conus in the South China Sea

    Liu Z, Xu N, Hu J, Zhao C, Yu Z, Dai Q (2009) Identification of novel I-superfamily conopeptides from several clades of Conus species found in the South China Sea. Peptides 30:1782-1787

    Beijing Institute of Biotechnology, Beijing 100071, People's Republic of China.

    Abstract: The I-superfamily of Conus peptides represents a new class of peptides with four disulfide bridges (-C-C-CC-CC-C-C-) that falls into three (I1, I2 and I3) categories according to the different signal peptide sequences. The I-superfamily has received increasing attention because it targets K+ ion channels, a function that is relatively rare in conotoxins. Herein we report 11 novel I-superfamily conotoxins from the venom ducts of five Cone snails (Conus eburneus, Conus imperialis, Conus vitulinus, Conus emaciatus and Conus litteratus) native to the South China Sea using a primer designed according to the N-terminus of the signal sequence of I2-superfamily conotoxins. The alignment of sequences revealed that signal regions exhibited moderate conservation with the exception of Eb11.3 from C. eburneus with homologies of 21.1%, 38.5% and 30.0% to the signal peptides of I1, I2 and I3 superfamily conotoxins, respectively. The mature peptides ranged from almost identical to highly divergent between species. Analyses of the evolutionary trees of these peptides with those of reported I-superfamily conotoxins showed that nine of them fall in I2 superfamily clades, but two of them were neither I1- and I2- nor I3-superfamily clades. Notably, some peptides exhibited significantly different amino acid residues in the intercysteine loops compared with group A, B and C of I-superfamily conopeptides, suggesting that they may have different bioactivities and functions.

    Novel M-superfamily conotoxin from Conus litteratus

    Wang L, Liu J, Pi C, Zeng X, Zhou M, Jiang X, Chen S, Ren Z, Xu A. (2009) Identification of a novel M-superfamily conotoxin with the ability to enhance tetrodotoxin sensitive sodium currents. Arch Toxicol. 83:925-932

    State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, Department of Biochemistry, College of Life Sciences, Sun Yat-sen (Zhongshan) University, 135 Xingangxi Road, 510275 Guangzhou, People's Republic of China.

    Abstract: In this work, a novel M-superfamily conotoxin, designated lt3a, was purified from the crude venom of Conus litteratus. Combined with peptide sequencing, MALDI-TOF mass spectrometry and cDNA cloning techniques, the amino acid sequence of lt3a was supposed to be DgammaCCgamma OQWCDGACDCCS, where O is hydroxyproline and gamma is carboxyglutamate. The Cys framework of lt3a (-CC-C-C-CC-) is similar to that of psi-, mu-, kappaM-conotoxins, which are representatives of M-conotoxins. Peptide lt3a is categorized into M1 branch based on the number of residues in the last Cys loop. Whole cell patch-clamp study on adult rat dorsal root ganglion neurons indicated that lt3a could enhance tetrodotoxin-sensitive sodium currents. This is a previously unknown function of M-superfamily conotoxins.

    Use of selective conotoxins to block nicotinic receptors

    Anderson DJ, Malysz J, Grønlien JH, El Kouhen R, Håkerud M, Wetterstrand C, Briggs CA, Gopalakrishnan M.(2009) Stimulation of dopamine release by nicotinic acetylcholine receptor ligands in rat brain slices correlates with the profile of high, but not low, sensitivity alpha4beta2 subunit combination. Biochem Pharmacol.78:844-851.

    Abstract: Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA. david.j.anderson@abbott.com alpha4beta2 neuronal nicotinic receptors (nAChRs) can exist in high and low sensitivity states possibly due to distinct stoichiometries during subunit assembly: (alpha4)(2)(beta2)(3) pentamer (high sensitivity, HS) and (alpha4)(3)(beta2)(2) pentamer (low sensitivity, LS). To determine if there is a linkage between HS or LS states and receptor-mediated responses in brain, we profiled several clinically studied alpha4beta2* nAChR agonists for the displacement of radioligand binding to alpha4beta2 [(3)H]-cytisine sites in rat brain membranes, effects on stimulation of [(3)H]-dopamine release from slices of rat prefrontal cortex and striatum, and activation of HS and LS human alpha4beta2 nAChRs expressed in Xenopus laevis oocytes. Binding affinities (pK(i)) and potency (pEC(50)) values for [(3)H]-dopamine release closely correlated with a rank order: varenicline>(-)-nicotine>AZD3480>dianicline congruent with ABT-089. Further, a good correlation was observed between [(3)H]-dopamine release and HS alpha4beta2 pEC(50) values, but not between [(3)H]-dopamine release and LS alpha4beta2. The relative efficacies of the agonists ranged from full to partial agonists. Varenicline behaved as a partial agonist in stimulating [(3)H]-dopamine release and activating both HS and LS alpha4beta2 nAChRs expressed in oocytes. Conversely, ABT-089, AZD3480 and dianicline exhibited little efficacy at LS alpha4beta2 (<5%), were more effective at HS alpha4beta2 nAChRs, and in stimulating cortical and striatal [(3)H]-dopamine release >or=30%. In the presence of alpha-conotoxin MII to block alpha6beta2* nAChRs, the alpha4beta2* alpha-conotoxin-insensitive [(3)H]-dopamine release stimulated by these ligands correlates well with their interactions at HS, but not LS. In summary, this study provides support for HS alpha4beta2* nAChR involvement in neurotransmitter release.

    Complexity of conotoxins revealed by MS

    Davis J, Jones A, Lewis RJ. (2009) Remarkable inter- and intra-species complexity of conotoxins revealed by LC/MS. Peptides 30:1222-1227.

    The University of Queensland, Institute for Molecular Biosciences, Brisbane Q4072, Australia.

    Abstract: Cone snails have evolved an assortment of venom peptides as an evolutionary strategy for rapid prey immobilization and defence. Earlier studies estimated approximately 100 conopeptides per species. In this study we optimized liquid chromatography and electrospray ionization mass spectrometry for the detection of conopeptides in crude venom to characterize conopeptides present in the venom of individual specimens of Conus textile, C. imperialis and C. marmoreus. Using this approach, we have expanded the predicted number of venom peptides 10-fold to an estimate of 1000-1900 conopeptides per species. Our investigation has also revealed a surprisingly high level of intra-species variation that distinguishes cone snails from other venomous species including spiders and scorpions. Given this inherent diversity and variability, more sensitive bioassays and sequencing techniques will be required to fully explore conotoxin bioactivity.

    Complications with Ziconotide

    [No Authors listed] (2008). Ziconotide: new drug. Limited analgesic efficacy, too many adverse effects. Prescrire Int. 17:179-182. [No authors listed]

    Abstract: (1) When oral morphine does not relieve severe pain and when there is no specific treatment for the underlying cause, the first option is to try subcutaneous or intravenous administration. If this standard treatment fails or is poorly tolerated, intrathecal injection is usually preferred as the direct route to the central nervous system. However, one-quarter to one-half of patients still do not achieve adequate pain relief, and adverse effects are relatively frequent; (2) Ziconotide is not an opiate and is not related to the usual classes of drugs that interfere with nervous transmission in the posterior horn of the spinal cord. Marketing authorization has been granted for "severe, chronic pain in patients who require intrathecal analgesia". The Summary of Product Characteristics (SPC) recommends continuous infusion via an intrathecal catheter connected to a pump; (3) Clinical evaluation of ziconotide does not include any trials versus morphine in patients with nociceptive pain, or any trials versus tricyclic or antiepileptic drugs in patients with neurogenic pain; (4) In a trial in 220 patients in whom systemic morphine had failed, the mean pain score on a 100-mm visual analogue scale was 69.8 mm after three weeks on ziconotide, compared to 75.8 mm with placebo. This difference, although statistically significant, is clinically irrelevant. The proportion of "responders" (reduction of at least 30% in the initial pain score) was respectively 16.1% and 12.0% (no statistically significant difference); (5) The two other placebo-controlled trials included 112 patients with pain linked to cancer or HIV infection, and 257 patients with non-cancer pain. After a titration phase lasting 5 to 6 days, a combined analysis of the two trials showed that the mean pain score was 48.8 mm with ziconotide and 68.4 mm with placebo (statistically significant difference). However, many patients did not complete the titration phase. Efficacy also appeared to differ according to the type of pain; ziconotide was more effective on cancer pain than on neurogenic pain; (6) The main adverse effects of ziconotide in clinical trials were cerebellovestibular disorders such as ataxia, dizziness, and gait disorders, as well as confusion, hallucinations (increased in cases of overdose), nausea, vomiting, postural hypotension, and urine retention. About 40% of patients had an elevation in muscle creatine kinase activity, through an unknown mechanism; (7) Intrathecal administration carries a risk of infection (especially meningitis). Some patients might experience a paradoxical increase in pain with ziconotide; (8) In practice, the efficacy of ziconotide in relieving neurogenic pain remains to be established. In cancer pain, the available evidence showing that ziconotide is effective after opiate failure is too weak in view of the potential risks. It is better to re-examine and, if possible, correct the reasons for opiate treatment failure rather than prescribe ziconotide.

    Peptidergic toxins as tools for nicotinic receptors

    Tsetlin V, Utkin Y, Kasheverov I. (2009) Polypeptide and peptide toxins, magnifying lenses for binding sites in nicotinic acetylcholine receptors. Biochem Pharmacol. 78:720-731

    Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russian Federation. vits@mx.ibch.ru

    Abstract: At present the cryo-electron microscopy structure at 4A resolution is known for the Torpedo marmorata nicotinic acetylcholine receptor (nAChR), and high-resolution X-ray structures have been recently determined for bacterial ligand-gated ion channels which have the same type of spatial organization. Together all these structures provide the basis for better understanding functioning of muscle-type and neuronal nAChRs, as well as of other Cys-loop receptors: 5HT3-, glycine-, GABA-A and some other. Detailed information about the ligand-binding sites in nAChRs, necessary both for understanding the receptor functioning and for rational drug design, became available when the X-ray structures were solved for the acetylcholine-binding proteins (AChBP), excellent models for the ligand-binding domains of all Cys-loop receptors. Of special value in this respect are the X-ray structures of AChBP complexes with agonists and antagonists. Among the latter are the complexes with polypeptide and peptide antagonists, that is with protein neurotoxins from snake venoms and peptide neurotoxins (alpha-conotoxins) from poisonous marine snails of Conus genus. The role of a bridge between the AChBP and nAChRs is played by the X-ray structure of the ligand-binding domain of alpha1 subunit of nAChR in the complex with alpha-bungarotoxin. The purpose of this review is to show the role of well-known and new polypeptide and peptide neurotoxins, from the earlier days of nAChRs research until present time, in identification of different nAChR subtypes and mapping their binding sites.

    Intrathecal Ziconotide

    Kapural L, Lokey K, Leong MS, Fiekowsky S, Stanton-Hicks M, Sapienza-Crawford AJ, Webster LR.(2009) Intrathecal ziconotide for complex regional pain syndrome: seven case reports. Pain Pract 9:296-303

    The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA. KAPURAL@ccf.org

    Abstract: Ziconotide is a nonopioid analgesic currently indicated as monotherapy, but frequently used in combination with opioids, for the management of severe chronic pain in patients for whom intrathecal (IT) therapy is warranted and who are intolerant of, or whose pain is, refractory to other treatments. There is a paucity of information regarding ziconotide use in patients with complex regional pain syndrome (CRPS). Seven cases in which IT ziconotide was used in patients with CRPS were analyzed. All patients (4 male, 3 female; age range, 14 to 52 years) had experienced inadequate pain relief with multiple conventional and interventional treatments. Three patients received ziconotide monotherapy exclusively; 4 patients received ziconotide monotherapy initially, then combination IT therapy. The mean ziconotide dose was 5.2 mcg/d (range, 0.5 to 13 mcg/d) at initiation and 24.7 mcg/d (range, 0.06 to 146 mcg/d) at the last available assessment. The mean duration of ziconotide therapy was 3.1 years (range, 26 days to 8 years). At ziconotide initiation, the mean visual analog scale (VAS) score was 89.3 mm (range, 75 to 100 mm); VAS scores decreased by a mean of 47.5% (range, 5% to 100%) at last assessment. Of the 5 patients who experienced substantial improvement in pain, edema, skin abnormalities, and/or mobility with ziconotide therapy, 2 have discontinued ziconotide and are pain free. Another patient experienced marked reversal of both edema and advanced skin trophic changes. Adverse events included urinary retention, depression, anxiety, and hallucinations. Adverse events generally resolved spontaneously, with treatment, or with ziconotide discontinuation/dose reduction. Although further studies are required, ziconotide holds promise as an effective treatment for CRPS.


    Yang KC, Jin GZ, Wu J.(2009) Mysterious alpha6-containing nAChRs: function, pharmacology, and pathophysiology. Acta Pharmacol Sin. 30:740-751

    Divisions of Neurology, Barrow Neurological Institute, St Joseph's Hospital and Medical Center, Phoenix, AZ 85013, USA.

    Abstract: Neuronal nicotinic acetylcholine receptors (nAChRs) are the superfamily of ligand-gated ion channels and widely expressed throughout the central and peripheral nervous systems. nAChRs play crucial roles in modulating a wide range of higher cognitive functions by mediating presynaptic, postsynaptic, and extrasynaptic signaling. Thus far, nine alpha (alpha2-alpha10) and three beta (beta2, beta3, and beta4) subunits have been identified in the CNS, and these subunits assemble to form a diversity of functional nAChRs. Although alpha4beta2- and alpha7-nAChRs are the two major functional nAChR types in the CNS, alpha6*-nAChRs are abundantly expressed in the midbrain dopaminergic (DAergic) system, including mesocorticolimbic and nigrostriatal pathways, and particularly present in presynaptic nerve terminals. Recently, functional and pharmacological profiles of alpha6*-nAChRs have been assessed with the use of alpha6 subunit blockers such as alpha-conotoxin MII and PIA, and also by using alpha6 subunit knockout mice. By modulating DA release in the nucleus accumbens (NAc) and modulating GABA release onto DAergic neurons in the ventral tegmental area (VTA), alpha6*-nAChRs may play important roles in the mediation of nicotine reward and addiction. Furthermore, alpha6*-nAChRs in the nigrostriatal DAergic system may be promising targets for selective preventative treatment of Parkinson's disease (PD). Thus, alpha6*-nAChRs may hold promise for future clinical treatment of human disorders, such as nicotine addiction and PD. In this review, we mainly focus on the recent advances in the understanding of alpha6*-nAChR function, pharmacology and pathophysiology.

    Intrathecal Ziconotide - a REVIEW

    Rauck RL, Wallace MS, Burton AW, Kapural L, North JM.(2009) Intrathecal ziconotide for neuropathic pain: a review. Pain Pract.9:327-337

    Wake Forest University Health Sciences, Winston-Salem, North Carolina 27103, USA. rrauck@ccrpain.com

    Abstract: Neuropathic pain is a considerable burden that affects activities of daily living. The management of neuropathic pain can be challenging because of multiple etiologies and complex manifestations. Ziconotide is a nonopioid intrathecal (IT) analgesic option for patients with neuropathic pain refractory to conventional treatments. The objective of this article is to review the published literature on ziconotide for the treatment of neuropathic pain. Relevant publications were identified through searches of all years of 6 databases, which included PubMed, EMBASE, and CINAHL. Search terms used were ziconotide, SNX-111, MVIIA, Prialt, and neuropathic pain. Publications were included if ziconotide was intrathecally administered (either alone or in combination with other IT agents) to treat neuropathic pain of any etiology and if pain assessment was an outcome measure. Data extracted included study design, IT drug doses, pain outcome measures, and adverse events (AEs). Twenty-eight articles met the inclusion criteria: 5 were preclinical studies and 23 were clinical studies. In the preclinical studies, ziconotide demonstrated antiallodynic effects on neuropathic pain. Data from double-blind, placebo-controlled (DBPC) trials indicated that patients with neuropathic pain reported a mean percent improvement in pain score with ziconotide monotherapy that ranged from 15.7% to 31.6%. A low starting dose and slow titration of ziconotide resulted in an improved safety profile in the aforementioned trials. Common AEs associated with ziconotide include nausea and/or vomiting, dizziness, confusion, urinary retention, and somnolence. Evidence from DBPC trials, open-label studies, case series, and case studies suggests that ziconotide, as either monotherapy or in combination with other IT drugs, is a potential therapeutic option for patients with refractory neuropathic pain. Additional studies are needed to establish the long-term efficacy and safety of ziconotide for neuropathic pain.

    Intrathecal Ziconotide in Europe

    Kress HG, Simpson KH, Marchettini P, Ver Donck A, Varrassi G. (2009) Intrathecal therapy: what has changed with the introduction of ziconotide. Pain Pract. 9:338-347

    Medical University of Vienna, A-1090 Vienna, Austria. hans-georg.kress@meduniwien.ac.at

    Abstract: Administering drugs into the intrathecal space is becoming more popular in the treatment of patients with intractable pain or intolerable side effects of systemic analgesic treatments. Although morphine and ziconotide are the only intrathecal analgesics currently approved by regulatory authorities in the U.S. (Food and Drug Administration) and Europe (national-level approval by individual countries for morphine and European Agency for the Evaluation of Medicinal Products approval for ziconotide), a wide variety of opioid and non-opioid drugs are being used in this way. There is no official guidance concerning the selection of these drugs or their use in combinations and a paucity of efficacy and safety data from randomized controlled trials. The polyanalgesic initiative aims to summarize the current knowledge and to facilitate rational choices of intrathecal drug and drug combinations for the management of chronic pain. The most recent polyanalgesic consensus recommendations were published in 2007. In this review, we shall examine these recommendations, which are tailored toward those practicing intrathecal analgesia in the U.S., and discuss how they should be implemented in Europe, where the healthcare systems and regulations of the medical authorities are different.

    Cost effectiveness of Ziconotide treatment in the UK

    Dewilde S, Veridian L, Maclaine GDH (2009) Cost-effectiveness of ziconotide in intrathecal pain management for severe chronic pain patients in the UK. Curr Med Res Opin 25:2007-2019

    Services in Health Economics, 1000 Brussels, Belgium. sd@SHE-consulting.be

    Abstract: OBJECTIVE: To examine the cost-effectiveness of using intrathecal ziconotide in the treatment of severe chronic pain compared to best supportive care for patients with intractable chronic pain in the United Kingdom. METHODS: Using a simulation model, the analysis evaluated the cost and health economic consequences of using ziconotide as a treatment for severe chronic pain. The modelled population and clinical data were based on a randomised controlled trial in which the main outcome was reduction in pain as measured by the visual analogue scale of pain intensity (VASPI). Resource use data were elicited using a modified Delphi panel and costed using published sources. Utility values were derived from a separate research study. The main outcome measure was the cost per quality-adjusted life-year (QALY). Extensive scenario analysis was conducted to evaluate parameter uncertainty. RESULTS: Overall, findings were robust to most assumptions. The cost-effectiveness of ziconotide compared to best supportive care (BSC) was pound 27,443 per QALY (95% CI pound 18,304-38,504). Scenarios were investigated in which discount rates, the time horizon, the threshold for qualifying as a responder, pump-related assumptions, utilities, ziconotide drug dose, and the patient discontinuation rate with ziconotide were varied. The most sensitive parameter was the dosage of ziconotide: using the lower and upper bounds of the average ziconotide dosage observed in the long-term open-label study changed the incremental cost-effectiveness ratio (ICER) to pound 15,500 [pound 8206-25,405] and pound 44,700 [pound 30,541-62, 670]. CONCLUSIONS: Ziconotide may offer an economically feasible alternative solution for patients for whom current treatment is inappropriate or ineffective. The main study limitation is that some model inputs, mainly related to resource use, are based on assumptions or expert interviews.

    28 April 2009

    Proteomics on a single Conus textile venom duct

    Ueberheide BM, Fenyö D, Alewood PF, Chait BT. (2009) Rapid sensitive analysis of cysteine rich peptide venom components. Proc Natl Acad Sci U S A. 106: 6910-6915.

    Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, 1230 York Avenue, New York, NY, 10065, USA.

    Abstract: Disulfide-rich peptide venoms from animals such as snakes, spiders, scorpions, and certain marine snails represent one of nature's great diversity libraries of bioactive molecules. The various species of marine cone shells have alone been estimated to produce >50,000 distinct peptide venoms. These peptides have stimulated considerable interest because of their ability to potently alter the function of specific ion channels. To date, only a small fraction of this immense resource has been characterized because of the difficulty in elucidating their primary structures, which range in size between 10 and 80 aa, include up to 5 disulfide bonds, and can contain extensive posttranslational modifications. The extraordinary complexity of crude venoms and the lack of DNA databases for many of the organisms of interest present major analytical challenges. Here, we describe a strategy that uses mass spectrometry for the elucidation of the mature peptide toxin components of crude venom samples. Key to this strategy is our use of electron transfer dissociation (ETD), a mass spectrometric fragmentation technique that can produce sequence information across the entire peptide backbone. However, because ETD only yields comprehensive sequence coverage when the charge state of the precursor peptide ion is sufficiently high and the m/z ratio is low, we combined ETD with a targeted chemical derivatization strategy to increase the charge state of cysteine-containing peptide toxins. Using this strategy, we obtained full sequences for 31 peptide toxins, using just 7% of the crude venom from the venom gland of a single cone snail (Conus textile).

    Continued in What's new in 2008
    See also : What's new in 2007, What's new in 2006, What's new in 2005, What's new in 2004, What's new in 2003, What's new in 2002, What's new in 2001, What's new in 2000, What's new in 1999, What's new in 1998, What's New in 1997 and What's New in 1996

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