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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


    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 (1998-2006)

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    9 October 2009

      Some "recent" 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): 195-203.

    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.

    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.

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    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

    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. 2009 Jul 30. [Epub ahead of print]

    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.

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

    10 June 2008

    alpha-Conotoxin Lp1.1 from Conus leopardus

    Peng C, Han Y, Sanders T, Chew G, Liu J, Hawrot E, Chi C, Wang C. (2008) alpha4/7-conotoxin Lp1.1 is a novel antagonist of neuronal nicotinic acetylcholine receptors. Peptides. 29:1700-1707.

    Institute of Protein Research, Tongji University, Shanghai 200092, China.

    Abstract: Cone snails comprise approximately 700 species of venomous molluscs which have evolved the ability to generate multiple toxins with varied and exquisite selectivity. alpha-Conotoxin is a powerful tool for defining the composition and function of nicotinic acetylcholine receptors which play a crucial role in excitatory neurotransmission and are important targets for drugs and insecticides. An alpha4/7 conotoxin, Lp1.1, originally identified by cDNA and genomic DNA cloning from iConus leopardus, was found devoid of the highly conserved Pro residue in the first intercysteine loop. To further study this toxin, alpha-Lp1.1 was chemically synthesized and refolded into its globular disulfide isomer. The synthetic Lp1.1 induced seizure and paralysis on freshwater goldfish and selectively reversibly inhibited ACh-evoked currents in Xenopus oocytes expressing rat alpha3beta2 and alpha6alpha3beta2 nAChRs. Comparing the distinct primary structure with other functionally related alpha-conotoxins could indicate structural features in Lp1.1 that may be associated with its unique receptor recognition profile.

    Conantokin-P and -E: Novel conopeptides from Conus purpurascens and Conus ermineus

    Gowd KH, Twede V, Watkins M, Krishnan KS, Teichert RW, Bulaj G, Olivera BM.(2008) Conantokin-P, an unusual conantokin with a long disulfide loop. Toxicon 52: 203-213.

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

    Abstract: The conantokins are a family of Conus venom peptides (17-27AA) that are N-methyl-d-aspartate (NMDA) receptor antagonists. Conantokins lack disulfide bridges (six out of seven previously characterized peptides are linear), but contain multiple residues of gamma-carboxyglutamate. These post-translationally modified amino acids confer the largely helical structure of conantokins by coordinating divalent metal ions. Here, we report that a group of fish-hunting cone snails, Conus purpurascens and Conus ermineus, express a distinctive branch of the conantokin family in their venom ducts. Two novel conantokins, conantokin-P (Con-P) and conantokin-E (Con-E) are 24AA long and contain five gamma-carboxyglutamate residues. These two peptides are characterized by a long disulfide loop (12 amino acids including two Gla residues between the Cys residues). The oxidative folding studies of Con-P revealed that the formation of the disulfide bond proceeded significantly faster in the presence of Ca(++) ions. Circular dichroism suggested that Con-P is less helical than other previously characterized conantokins. Con-P blocks NMDA receptors containing NR2B subunit with submicromolar potency. Furthermore, the subtype-selectivity for different NR2 subunits differs from that of the previously characterized conantokins. Our results suggest that different branches of the phylogenetic tree of cone snails have evolved distinct groups of conantokins, each with its own unique biochemical features.

    (abstracts to follow)

    3: Chen P, Garrett JE, Watkins M, Olivera BM.(2008) Purification and characterization of a novel excitatory peptide from Conus distans venom that defines a novel gene superfamily of conotoxins. Toxicon. 2008 Jun 5. [Epub ahead of print]

    4: Yuan DD, Liu L, Shao XX, Peng C, Chi CW, Guo ZY.(2008) Isolation and cloning of a conotoxin with a novel cysteine pattern from Conus caracteristicus. Peptides. 2008 May 25. [Epub ahead of print]

    5: Teichert RW, Garcia CC, Potian JG, Schmidt JJ, Witzemann V, Olivera BM, McArdle JJ.(2008) Peptide-toxin tools for probing the expression and function of fetal and adult subtypes of the nicotinic acetylcholine receptor. Ann N Y Acad Sci. 2008 Jun;1132:61-70.

    6: Wang L, Pi C, Liu J, Chen S, Peng C, Sun D, Zhou M, Xiang H, Ren Z, Xu A.(2008) Identification and characterization of a novel O-superfamily conotoxin from Conus litteratus. J Pept Sci. 2008 Jun 3. [Epub ahead of print]

    7: Schroeder CI, Ekberg J, Nielsen KJ, Adams D, Loughnan ML, Thomas L, Adams DJ, Alewood PF, Lewis RJ.(2008) Neuronally selective mu -conotoxins from conus striatus utlise an alpha -helical motif to target mammalian sodium channels. J Biol Chem. 2008 Jun 3. [Epub ahead of print]

    8: Williams JA, Day M, Heavner JE.(2008) Ziconotide: an update and review. Expert Opin Pharmacother. 2008 Jun;9(9):1575-83.

    9: Imperial JS, Chen P, Sporning A, Terlau H, Daly NL, Craik DJ, Alewood PF, Olivera BM.(2008) Tyrosine-rich conopeptides affect voltage-gated K+ channels. J Biol Chem. 2008 May 27. [Epub ahead of print]

    10: Duda TF Jr, Remigio EA.(2008) Variation and evolution of toxin gene expression patterns of six closely related venomous marine snails. Mol Ecol. 2008 Jun;17(12):3018-32. Epub 2008 May 16.

    11: Fiedler B, Zhang MM, Buczek O, Azam L, Bulaj G, Norton RS, Olivera BM, Yoshikami D.(2008) Specificity, affinity and efficacy of iota-conotoxin RXIA, an agonist of voltage-gated sodium channels Na(V)1.2, 1.6 and 1.7. Biochem Pharmacol. 2008 Jun 15;75(12):2334-44. Epub 2008 Apr 6.

    12: Doura MB, Gold AB, Keller AB, Perry DC.(2008) Adult and periadolescent rats differ in expression of nicotinic cholinergic receptor subtypes and in the response of these subtypes to chronic nicotine exposure. Brain Res. 2008 Jun 18;1215:40-52. Epub 2008 Apr 4.

    13: Wang Y, Shao X, Li M, Wang S, Chi C, Wang C.(2008) mr1e, a conotoxin from Conus marmoreus with a novel disulfide pattern. Acta Biochim Biophys Sin (Shanghai). 2008 May;40(5):391-6.

    14: Dwoskin LP, Wooters TE, Sumithran SP, Siripurapu KB, Joyce BM, Lockman PR, Manda VK, Ayers JT, Zhang Z, Deaciuc AG, McIntosh JM, Crooks PA, Bardo MT.(2008) N,N'-Alkane-diyl-bis-3-picoliniums as Nicotinic Receptor Antagonists: Inhibition of Nicotine-induced Dopamine Release and Hyperactivity. J Pharmacol Exp Ther. 2008 May 6. [Epub ahead of print]

    15: Arredondo J, Chernyavsky AI, Jolkovsky DL, Pinkerton KE, Grando SA.(2008) Receptor-mediated tobacco toxicity: acceleration of sequential expression of alpha5 and alpha7 nicotinic receptor subunits in oral keratinocytes exposed to cigarette smoke. FASEB J. 2008 May;22(5):1356-68.

    16: Papke RL, Dwoskin LP, Crooks PA, Zheng G, Zhang Z, McIntosh JM, Stokes C.(2008) Extending the analysis of nicotinic receptor antagonists with the study of alpha6 nicotinic receptor subunit chimeras. Neuropharmacology. 2008 Jun;54(8):1189-200. Epub 2008 Mar 28.

    6 April 2008

      Conotoxins and receptors

      Olivera, BM (2008) Using Conus venom peptides to understand nervous systems and discover drugs. FASEB J. 22:252.1 [Meeting Abstract]

      Miller MT, Hansen,SB, McIntosh JM, Olivera BM and Taylor P (2006) Structural insights into competitive and non-competitive nicotinic antagonists. FASEB J. 20:A244 [Meeting Abstract]

    25 March 2008

      alpha conotoxin Vc1.1 receives US patent

      US Patent NumberUS Patent Number 7,348,400 B2 “a-Conotoxin Peptides with Analgesic Properties”, Bruce Livett, Zeinab Khalil, Ken Gayler, John Down, David Sandall, David Keays.  Date of Patent : March 25, 2008

    27 February, 2008

      US patent win for unlisted Xenome

      Nick Evans BioTechnologyNews.Net Tuesday, 25 March 2008

      UNLISTED Brisbane peptide company Xenome has won a US patent battle over the company’s novel class of norepinephrine transporter (NET) inhibitors. The patent was reissued this week following a ruling on a patent interference process between Xenome and an unnamed US company.

      Abstract: Xen2174 is derived from the venom of the cone snail. The ruling sees Xenome granted the patent for the class of drugs, originally assigned to the company by the University of Queensland, and means the company’s lead pain compound, Xen2174, is cleared for development without the risk of patent infringement claims in the US.
      Xen2174 is already covered by broadly granted patents in other major markets, including Europe.
      New Xenome chief executive Ian Nisbet told BTN that the ruling was significant for the company. “There was an interference around that patent application, and our position was deemed to be the predominant one, which meant that our patent was ultimately issued. That’s clearly a major win for the company,” he said. Nisbet said that Xenome has recently completed enrolment of a Phase 1/2 trial of the drug in cancer pain patients.

      “We recruited 37 patients into that study and we’re in the process of analysing data, and those results will be available in about the middle of the year,” he said.
      Nisbet said the company hoped that positive results from the Xen2174 clinical trial would generate interest in the company as it considered an initial public offering later this year.
      Xenome is one of a number of Australian biotechs waiting on a recovery in the broader market before considering a stock market listing. Nisbet said the company planned to list within the next 12 months, depending on the state of the market.

        Background notes

      Under the US patent system, “interference” is declared if two patent applications are filed which set forth claims covering the same subject matter. Under those circumstances the patent office can require that each of the parties appear to determine who was the earliest to discover the claimed invention.

      Xen2174 is a synthetic drug modelled on a peptide isolated from the venom of a cone shell found on Australia's Great Barrier Reef.
      Xen2174 selectively targets the norepinephrine transporter (NET), a well-established pharmaceutical target for a number of conditions. Inhibition of this transporter elevates the levels of norepinephrine in the spinal cord, preventing pain signals from reaching the brain.
      Xen2174 successfully completed a Phase 1 human safety trial in healthy volunteers in 2005, where it was tested in systemic circulation, via intravenous administration.

      Xen2174 is currently being tested in a Phase 1/2 clinical trial targeting chronic intractable pain in cancer patients via single dose intrathecal injection. This study again focuses on safety under this type of administration as well as pharmacokinetics and signs of efficacy at various doses.

    18 February, 2008

      Disulfide-rich peptide toxins from Gemmula species

      Heralde FM 3rd, Imperial J, Bandyopadhyay PK, Olivera BM, Concepcion GP, Santos AD (2007) A rapidly diverging superfamily of peptide toxins in venomous Gemmula species.Toxicon. 2007 Dec 28; [Epub ahead of print] doi:10.1016/j.toxicon.2007.12.022
      National Institute of Molecular Biology and Biotechnology, University of the Philippines, Diliman, Quezon City 1001, Philippines; Marine Science Institute, University of the Philippines, Diliman, Quezon City 1001, Philippines.

      Abstract: The gem turrids (genus Gemmula Weinkauff, 1875) are venomous snails in the family Turridae. A gene superfamily of disulfide-rich peptides expressed in Gemmula venom ducts was characterized. Gemmula speciosa (Reeve, 1843) venom duct cDNA clones revealed two different conotoxin-like prepropeptide precursors, with identical signal sequences, a largely conserved pro region, and a cysteine-rich C-terminal mature peptide region. The conserved signal sequence was used to successfully amplify homologous genes from three other Gemmula species; all had the same pattern of Cys residues in the predicted mature venom peptide. Although the signal sequence and propeptide regions were highly conserved, the mature toxin regions diverged greatly in sequence, except that the Cys residues were conserved. We designate this as the Pg-gene superfamily (Pg-superfamily) of Gemmula venom peptides. Purification of two members of the family directly from G. speciosa venom was achieved; amino acid sequence analysis revealed that these peptides are highly posttranslationally modified. With at least 10-fold as many species of turrids as cone snails, identification of rapidly diversifying gene superfamilies such as the Pg-superfamily of Gemmula is essential before the facile and systematic discovery and characterization of peptide toxins from turrid venoms can be achieved.

    15 February, 2008

      Venom peptides from Conus ventricosus

      Romeo C, Di Francesco L, Oliverio M, Palazzo P, Massilia GR, Ascenzi P, Polticelli F and Schininà ME (2008) Conus ventricosus venom peptides profiling by HPLC-MS: A new insight in the intraspecific variation. J Sep Sci. 31:488-498 [Epub ahead of print]
      Consorzio Interuniversitario “Istituto Nazionale Biostrutture e Biosistemi”, Roma, Italy.

      Abstract: Conus is a genus of predatory marine gastropods that poison the prey with a complex mixture of compounds active on muscle and nerve cells. An individual cone snail's venom contains a mixture of pharmacological agents, mostly short, structurally constrained peptides. This study is focused on the composition of the venom employed by Conus ventricosus Gmelin, 1791, a worm-hunting cone snail living in the Mediterranean Sea. For this purpose, LC coupled to MS techniques has been successfully used to establish qualitative and quantitative differences in conopeptides from minute amounts of venom ducts. We were able to prove variability in the venom conopeptide complement, possibly related to different trophic habits of the species in the Mediterranean Sea. Moreover, the information-rich MS techniques enabled us to identify two novel C. ventricosus peptides, here named Conotoxin-Vn and alpha-Conotoxin-Vn. On the basis of the structural data collected so far, we suggest that Conotoxin-Vn is a conopeptide belonging to the omega-family that recognizes calcium channels through a specific pharmacophore. Similarly, molecular modeling data suggest that alpha-Conotoxin-Vn should represent a competitive antagonist of neuronal nicotinic acetylcholine receptors (nAChRs).

    7 February, 2008

      Biogeographical patterns in Cape Verde Conus

      Cunha RL*†, Tenorio MJ, Afonso C, Castilho R and Zardoya R*. (2008) Replaying the tape: recurring biogeographical patterns in Cape Verde Conus after 12 million years. Molecular Ecology 17: 885–901
      *Departamento de Biodiversidad y Biología Evolutiva, Museo Nacional de Ciencias Naturales-CSIC, José Gutiérrez Abascal, 2, 28006 Madrid, Spain, †CCMAR, Campus de Gambelas-Universidade do Algarve, 8005-139 Faro, Portugal, ‡Facultad de Ciencias, Universidad de Cadiz, 11510 Puerto Real, Cádiz, Spain

      Abstract: Isolated oceanic islands are excellent natural laboratories to test the relative role of historical contingency and determinism in evolutionary diversification. Endemics of the marine venomous snail Conus in the Cape Verde archipelago were originated from at least two independent colonizations of ‘small’ and ‘large’ shelled species separated by 12 million years. In this study, we have reconstructed phylogenetic relationships within large-shelled Conus (C. ateralbus, C. pseudonivifer, C. trochulus, and C. venulatus) based on mitochondrial cox1 and nad4 haplotype sequences. The reconstructed molecular phylogeny revealed three well-supported and relatively divergent clades (A, B, and C) that do not correspond to current species classification based on shell colour and banding patterns. Clade A grouped specimens assigned either to C. pseudonivifer or C. trochulus, clade B is composed of specimens assigned to C. venulatus, and clade C comprises specimens assigned either to C. venulatus or C. ateralbus. Geometric morphometric analyses found significant differences between the radular teeth shape of C. pseudonivifer/C. trochulus and C. venulatus/C. ateralbus. In clades A and B, northwestern Boavista and Maio specimens cluster together to the exclusion of eastern Boavista samples. In Sal, populations form a monophyletic island assemblage(clade C). The large-shelled Conus have remarkably replicated biogeographical patterns of diversification of small-shelled Conus. Similar selective forces (i.e. nonplanktonic lecithotrophy with limited larval dispersal and allopatric diversification) together with repeated instances of low sea level stands during glacial maxima that allowed connection between islands, have overcome the effect of historical contingency, and explain the observed recurring biogeographical patterns.

    6 February, 2008

      Structure and function of α-conotoxin RgIA and analogues

      Ellison M1, Feng Z-P2, Park AJ1, Zhang X2, Olivera BM1, McIntosh JM1 and Norton RS2 (2008) α-RgIA, a novel α-conotoxin that blocks the α9α10 nAChR: Structure and identification of key receptor binding residues. Journal of Molecular Biology, In Press, Accepted Manuscript, Available online 4 February 2008
      1Department of Biology, University of Utah, Salt Lake City, Utah, 84112, USA. 2The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville 3050 Australia

      Abstract: α-Conotoxins are small disulfide-constrained peptides from cone snails which act as antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). The 13-residue peptide α-RgIA is a member of the α-4,3 family of α-conotoxins and selectively blocks the α9α10 nAChR subtype, in contrast to another well characterized member of this family, α-ImI, which is a potent inhibitor of the α7 and α3α2 nAChR subtypes. In this study, we have altered side chains in both the 4-residue and 3-residue loops of α-RgIA, and have modified its C-terminus. The effects of these changes on activity against α9α10 and α7 nAChRs were measured, the solution structures of α-RgIA and its Y10W, D5E and P6V analogues were determined from NMR data, and resonance assignments made for α-RgIA[R9A]. The structures for α-RgIA and its three analogues were well-defined except at the chain termini. Comparison of these structures with reported structures of α-ImI reveals a common two-loop backbone architecture within the α-4,3 family, but with variations in side chain solvent accessibility and orientation. Asp5, Pro6 and Arg7 in loop 1 are critical for blockade of both the α9α10 and α7 subtypes. In loop 2, α-RgIA[Y10W] had activity near that of wild-type α-RgIA, with high potency for α9α10 and low potency for α7, and had a similar structure to wild-type. By contrast, Arg9, in loop 2, is critical for specific binding to the α9α10 subtype, probably because it is larger and more solvent accessible than Ala9 in α-ImI. Our findings contribute to a better understanding of the molecular basis for antagonism of the α9α10 nAChR subtype, which is a target for the development of analgesics for treatment of chronic neuropathic pain.
      Keywords: conotoxin; structure; peptide; NMR; nicotinic acetylcholine receptor; pain

      Conotoxin presentations at Society for Neuroscience meeting November 2007

      At the Society for Neuroscience meeting in SanDiego, CA, November 2007 there were a number of presentations on contoxins. A selection of these Abstracts is available here.

    4 February, 2008

      Structure of alpha-conotoxin RgIA: Comparison with ImI and Vc1.1

      Clark RJ1, Daly NL1, Halai R1, Nevin ST2, Adams DJ2, Craik DJ1 (2008). The three-dimensional structure of the analgesic α-conotoxin, RgIA. FEBS Letters (DOI: 10.1016/j.febslet.2008.01.027):Received 10 January 2008; accepted 17 January 2008. published online 31 January 2008
      1Institute for Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia; 2School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia

      Abstract: The α-conotoxin RgIA is a selective antagonist of the α9α10 nicotinic acetylcholine receptor and has been shown to be a potent analgesic and reduces nerve injury associated inflammation. RgIA was chemically synthesized and found to fold into two disulfide isomers, globular and ribbon. The native globular isomer inhibited ACh-evoked currents reversibly in oocytes expressing rat α9α10 nAChRs but the ribbon isomer was inactive. We determined the three-dimensional structure of RgIA using NMR methods to assist in elucidating the molecular role of RgIA in analgesia and inflammation.
      Keywords: Conotoxin, Nuclear magnetic resonance, Analgesic, Oxidative folding, Disulfide isomers

    31 January, 2008

      From Cone Snail to Drugs: Olivera's UP Centenial Address

      On January 16, 2008, Prof. Baldomero M. Olivera PhD received an honorary degree and presented this Centenial address to assembled staff and graduates at the University of the Philippines: From Cone Snails to Drugs: The Scientific Odyssey of a UP Graduate. In 2007, Prof Olivera was named by the Harvard Foundation as "2007 Scientist of the Year". You can read more about his career and award here.
      The Centennial lecture was beamed live via video-conferencing from the UP Film Center to the UP Mindanao , Mintal Campus College of Humanities and Social Sciences Audio-Visual Room and the adjacent Theater Hall.

      Ziconotide (omega-conotoxin MVIIA): non-clinical safety

      Skov MJ, Beck JC, de Kater AW, Shopp GM. (2007) Nonclinical safety of ziconotide: an intrathecal analgesic of a new pharmaceutical class. Int J Toxicol. 26: 411-421.
      Elan Pharmaceuticals, Inc., South San Francisco, California 94080, USA. michael.skov@elan.com

      Abstract: Ziconotide, a potent, selective, reversible blocker of neuronal N-type voltage-sensitive calcium channels, is approved in the United States for the management of severe chronic pain in patients for whom intrathecal therapy is warranted, and who are intolerant or refractory to other treatment, such as systemic analgesics, adjunctive therapies, or intrathecal morphine. In the European Union, ziconotide is indicated for the treatment of severe chronic pain in patients who require intrathecal analgesia. Nonclinical investigations of ziconotide included a comprehensive characterization of its toxicology, incorporating acute and subchronic toxicity studies in rats, dogs, and monkeys; reproductive toxicity assessments in rats and rabbits; and mutagenic, carcinogenic evaluations performed in vivo and in vitro. Additional investigations assessed the potential for cardiotoxicity (rats) and immunogenicity (mice, rats, and guinea pigs), and the presence or absence of intraspinal granuloma formation and local cell proliferation and apoptosis (dogs). The resulting nonclinical toxicology profile was predictive of human adverse events reported in clinical trials and consistent with ziconotide's pharmacological activity. Frequently observed nonclinical behavioral effects included tremoring, shaking, ataxia, and hyperreactivity. Occurrences were generally transient and reversible upon cessation of treatment, and intolerable effects occurred at doses more than 45 times the maximum recommended clinical dose. Ziconotide was not associated with target organ toxicity, teratogenicity, or treatment-related gross or histopathological changes; it displayed no mutagenic or carcinogenic potential and no propensity to induce local cell proliferation or apoptosis. Although guinea pigs developed systemic anaphylaxis, antibodies to ziconotide were not detected in mice, rats, or guinea pigs, indicating low immunogenic potential. No evidence of granuloma formation was observed with intrathecal ziconotide treatment. In summary, the results from these nonclinical safety assessments revealed no significant toxicological risk to humans treated with ziconotide as recommended.

    29 January, 2008

      Conotoxin transcripts in Conus leopardus

      Remigio, EA and Duda TF Jr (2008) Evolution of ecological specialization and venom of a predatory marine gastropod Molecular Ecology, OnlineEarly ArticlesPublished article online: 23-Jan-2008 doi: 10.1111/j.1365-294X.2007.03627.x
      Department of Ecology and Evolutionary Biology/Museum of Zoology, University of Michigan, 1109 Geddes Avenue, Ann Arbor, MI 48109, USA, †Smithsonian Tropical Research Institute, Apartado 0843-03092, Balboa, Ancón, Panama. Correspondence: T. F. Duda Jr, Fax: 734-763-4080;
      E-mail: tfduda@umich.edu
      Keywords: conotoxins, Conus, ecological specialization, gene expression

      Abstract: Understanding the evolution of ecological specialization is important for making inferences about the origins of biodiversity. Members of the predatory, marine gastropod genus Conus exhibit a variety of diets and the ability to capture prey is linked to a venom comprised of peptide neurotoxins, termed conotoxins. We identified conotoxin transcripts from Conus leopardus, a species of Conus that uniquely preys exclusively on hemichordates, and compared its venom duct transcriptome to that of four other Conus species to determine whether a shift to a specialized diet is associated with changes in the venom composition of this species. We also examined the secondary structure of predicted amino acid sequences of conotoxin transcripts of C. leopardus to identify substitutions that may be linked to specialization on hemichordates. We identified seven distinct conotoxin sequences from C. leopardus that appear to represent transcripts of seven distinct loci. Expression levels and the diversity of conotoxins expressed by C. leopardus are considerably less than those of other Conus. Moreover, gene products of two transcripts exhibited unique secondary structures that have not been previously observed from other Conus. These results suggest that transition to a specialist diet is associated with reduction in the number of components expressed in venoms of Conus and that diverse venoms of Conus are maintained in species with a broad dietary width.

    25 January, 2008

      Conotoxins from Conus austini

      Zugasti-Cruz A, Aguilar MB, Falcón A, Olivera BM, Heimer de la Cotera EP (2008). Two new 4-Cys conotoxins (framework 14) of the vermivorous snail Conus austini from the Gulf of Mexico with activity in the central nervous system of mice. Peptides. 2007 Dec 5; [Epub ahead of print]
      Posgrado en Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Ciudad Universitaria, México 04510, D.F., Mexico; Laboratorio de Neurofarmacología Marina, Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro 76230, Mexico.

      Abstract: As part of continuing studies of the venom components present in Conus austini (syn.: Conus cancellatus), a vermivorous cone snail collected in the western Gulf of Mexico, Mexico, two major peptides, as14a and as14b, were purified and characterized. Their amino acid sequences were determined by automatic Edman sequencing after reduction and alkylation. Their molecular masses, established by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, confirmed the chemical analyses and indicated that as14a and as14b have free C-termini. Each peptide contains 4-Cys residues arranged in a pattern (C-C-C-C, framework 14). The primary structure of as14a is GGVGRCIYNCMNSGGGLNFIQCKTMCY (experimental monoisotopic mass 2883.92Da; calculated monoisotopic mass 2884.20Da), whereas that of as14b is RWDVDQCIYYCLNGVVGYSYTECQTMCT (experimental monoisotopic mass 3308.63Da; calculated monoisotopic mass 3308.34Da). Both purified peptides elicited scratching and grooming activity in mice, and as14b also caused body and rear limb extension and tail curling immediately upon injection. The high sequence similarity of peptide as14a with peptide vil14a from the vermivorous C. villepinii suggests that the former might block K(+) channels.

      3-D structure of conotoxin tx3a from Conus textile

      McDougal OM, Turner MW, Ormond AJ, Poulter CD (2008). Three-Dimensional Structure of Conotoxin tx3a: An m-1 Branch Peptide of the M-Superfamily. Biochemistry. 2008 Jan 19; [Epub ahead of print]
      Department of Chemistry and Biochemistry, Boise State University, Boise, Idaho 83725, and Department of Chemistry, University of Utah, Salt Lake City, Utah 84112 .

      Abstract: The M-superfamily, one of eight major conotoxin superfamilies found in the venom of the cone snail, contains a Cys framework with disulfide-linked loops labeled 1, 2, and 3 (-CC (1) C (2) C (3) CC-). M-Superfamily conotoxins can be divided into the m-1, -2, -3, and -4 branches, based upon the number of residues located in the third Cys loop between the fourth and fifth Cys residues. Here we provide a three-dimensional solution structure for the m-1 conotoxin tx3a found in the venom of Conus textile. The 15-amino acid peptide, CCSWDVCDHPSCTCC, has disulfide bonds between Cys (1) and Cys (14), Cys (2) and Cys (12), and Cys (7) and Cys (15) typical of the C1-C5, C2-C4, and C3-C6 connectivity pattern seen in m-1 branch peptides. The tertiary structure of tx3a was determined by two-dimensional (1)H NMR in combination with the combined assignment and dynamics algorithm for nuclear magnetic resonance (NMR) applications CYANA program. Input for structure calculations consisted of 62 inter- and intraproton, five phi angle, and four hydrogen bond constraints. The root-mean-square deviation values for the 20 final structures are 0.32 +/- 0.07 and 0.84 +/- 0.11 A for the backbone and heavy atoms, respectively. Surprisingly, the structure of tx3a has a "triple-turn" motif seen in the m-2 branch conotoxin mr3a, which is absent in mr3e, the only other member of the m-1 branch of the M-superfamily whose structure is known. Interestingly, injection of tx3a into mice elicits an excitatory response similar to that of the m-2 branch peptide mr3a, even though the conotoxins have different disulfide connectivity patterns.

    20 January, 2008

      Conorfamide-Sr2 from Conus spurius

      Aguilar MB, Luna-Ramírez KS, Echeverría D, Falcón A, Olivera BM, Heimer de la Cotera EP, Maillo M. (2008) Conorfamide-Sr2, a gamma-carboxyglutamate-containing FMRFamide-related peptide from the venom of Conus spurius with activity in mice and mollusks. Peptides. 2007 Dec 5; [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, Mexico.

      Abstract: A novel peptide, conorfamide-Sr2 (CNF-Sr2), was purified from the venom extract of Conus spurius, collected in the Caribbean Sea off the Yucatan Peninsula. Its primary structure was determined by automated Edman degradation and amino acid analysis, and confirmed by electrospray ionization mass spectrometry. Conorfamide-Sr2 contains 12 amino acids and no Cys residues, and it is only the second FMRFamide-related peptide isolated from a venom. Its primary structure PMgammaDPLgammaIIRI-nh(2), (gamma, gamma-carboxyglutamate; -nh(2), amidated C-terminus; calculated monoisotopic mass, 1468.72Da; experimental monoisotopic mass, 1468.70Da) shows two features that are unusual among FMRFamide-related peptides (FaRPs, also known as RFamide peptides), namely the novel presence of gamma-carboxyglutamate, and a rather uncommon C-terminal residue, Ile. CNF-Sr2 exhibits paralytic activity in the limpet Patella opea and causes hyperactivity in the freshwater snail Pomacea paludosa and in the mouse. The sequence similarities of CNF-Sr2 with FaRPs from marine and freshwater mollusks and mice might explain its biological effects in these organisms. It also resembles FaRPs from polychaetes (the prey of C. spurius), which suggests a natural biological role. Based on these similarities, CNF-Sr2 might interact with receptors of these three distinct types of FaRPs, G-protein-coupled receptors, Na(+) channels activated by FMRFamide (FaNaCs), and acid-sensing ion channels (ASICs). The biological activities of CNF-Sr2 in mollusks and mice make it a potential tool to study molecular targets in these and other organisms.

    17 January, 2008

      Do hydroxyprolines determine the folding and activity of conotoxins ?



      Lopez-Vera E, Walewska A, Skalicky JJ, Olivera BM, Bulaj G. (2008) Role of Hydroxyprolines in the in Vitro Oxidative Folding and Biological Activity of Conotoxins. Biochemistry. 2008 Jan 12; [Epub ahead of print]
      Department of Biology, Department of Medicinal Chemistry, and Department of Biochemistry, University of Utah, Salt Lake City, Utah 84108, and Faculty of Chemistry, University of Gdansk, 80-952 Gdansk, Poland.

      Abstract: Hydroxylation of proline residue occurs in specific peptides and proteins derived from plants and animals, but the functional role of this modification has been characterized primarily in collagen. Marine cone snails produce disulfide-rich peptides that have undergone a plethora of posttranslational modifications, including proline hydroxylation. Although Conus snails extensively utilize proline hydroxylation, the consequences of this modification remain largely unexplored. In this work, we investigated the function of 4-hydroxyproline (Hyp) in conotoxins from three distinct gene families: mu-, omega-, and alpha-conotoxins. Analogues of mu-GIIIA, omega-MVIIC, alpha-GI, and alpha-ImI were synthesized with either Pro or Hyp, and their in vitro oxidative folding and biological activity were characterized. For GIIIA, which naturally contains three Hyp residues, the modifications improved the ability to block NaV1.4 sodium channels but did not affect folding. In contrast, the presence of Hyp in MVIIC had a significant impact on the oxidative folding but not on the biological activity. The folding yields for the MVIIC[Pro7Hyp] analogue were approximately 2-fold higher than for MVIIC under a variety of optimized oxidation conditions. For alpha-conotoxins ImI and GI, the hydroxylation of the conserved Pro residue improved their folding but impaired their activities against target receptors. Since prolyl-4-hydroxylase and protein disulfide isomerase coexist as a heterotetramer in the ER, we discuss the effects of Hyp on the folding of conotoxins in the context of cis-trans isomerization of Pro and Hyp. Taken together, our data suggest that proline hydroxylation is important for both in vitro oxidative folding and the bioactivity of conotoxins.

      [The following hydroxyproline-containing conotoxins are included: mu-conotoxins GIIA, GIIIB, GIIIC, PIIIA; kM-conotoxin RIIK; psi-conotoxin PIIIE; alpha-conotoxin (4/7) EI; alpha-conotoxin (4/3) Reg1b; delta-conotoxin PVIA, SVIE; alphaA-conotoxins PIVA, EIVA; omega-conotoxin GVIA; chi-conotoxin MrIA, MrIB; and contryphans R, and Tx]

      Two Cone Shell Articles
      (from "Man and Mollusc" by Ross Mayhew of Schooner Specimen Shells)

    • The "Glory of" Gang of Cones
      Conus gloriamaris Chemnitz 1777 (Glory of the Seas Cone); Conus bengalensis Okutani 1968 (Glory of Bengal Cone); Conus milneedwardsi Jousseaume 1894 (Glory of India Cone) and Conus granulatus Linneus 1758 (Glory of the Atlantic Cone)
    • Killer Conesnails
      Conus geographus Linne 1758 (Geography cone) and Conus textile Linne 1758 (Textile cone)
    16 January, 2008

      New Species of Cone Shells

      One new species: Conus beatrix Tenorio, Poppe & Tagaro, 2007, collected on Aliguay Is., P.I. by Manuel J. Tenorio and Paul Kersten, and one new subspecies Conus recluzianus simanoki Tenorio, Poppe & Tagaro, 2007 collected at the Burma / Thailand border by Philippe Quisquandon, were published in Visaya, vol. 2, No. 2 (November, 2007).

      The Cone Collector Volume 5 January 2008



      Click here to download The Cone Collector #5 (PDF - 1.9 MB).
      [Earlier issues, #0 - #4, are available for download at www.seashell-collector.com]

      Edited by António Monteiro, with layout by André Poremski.
      Contributions include:

    • Who's Who in Cones : by Gabriella Raybaudi Massilia
    • Live Taken Specimen of Rare Species (Conus darkini Röckel, Korn & Richard) , 1992), by Richard Goldberg
    • Special Population of Conus fuvus Reeve, 1843, by António Monteiro
      Reference: BEN-SAÂD, AÏCHA, JANSSENS, ADRIAAN & NOLF, FRANK 2007. Another population variant of Conus furvus Reeve, 1843 (Mollusca: Gastropoda: Conoidea: Conidae) from the Cuyo Islands (Palawan, Philippines).Neptunea, Vol. 6 No. 1
    • Two Exceptional Specimens (Conus vexillum Gmelin, 1791 and Conus ammiralis Linnaeus, 1758) by Philippe Quiquandon
    • Distribution of Conus kohni Mclean & Nybakken, 1979 by John K. Tucker
    • The Most Variable Cone? by António Monteiro and by Paul Kersten
      Is it Conus mercator Linnaeus, 1758, C. venulatus Hwass, 1792, or C. generalis Linnaeus, 1767, or C. magus Linnaeus, 1758 and its different forms such as assimilis Adams, 1854, borneensis Sowerby, 1866, carinatus Swainson, 1822, cernohorskyi da Motta, 1983, circae Sowerby, 1858, consul Boivin, 1864, fr auenfeldi Crosse, 1865, fulvobullatus da Motta, 1982, metcalfi i Reeve, 1843, raphanus Hwass, 1792, signifer Crosse, 1865 and ustulatus Reeve, 1843.
    • Australis or Anonymous? by Jon Singleton
      C. australis vs. C. cebuganus Reference: DA MOTTA, A. J. 1982. C. cebuganus sp. nov. Carfel Shell News 4(3) 2007; For images of these species SEARCH on Eurasiashells. de Suduiraut, E. G. www.eurasiashells.net.
    • About Conus aurantius, Hwass 1792 by Frits Fontein
    • Caribbean Corner: Notes on Conus stearnsii Conrad, 1869 by André Poremski
    • Scuba Diving For Shells Part 1: Night Snorkeling In The Grenadines - A Blessing by David Touitou
      Conus dominicanus
    • Conus queketti E.A. Smith, 1906. What is it? by R. M. (Mike) Filmer
      Mike concludes that C. queketti is only a form of C. imperialis. Other species discussed are C. pennaceus Born, 1778 and C. biliosus Röding, 1798, C. lohri Kilburn, 1972 and C. meyeri Walls, 1979).
    • Why Are Some Cone Shells So Often Eroded? by António Monteiro and Carlos Afonso
      Conus miruchae
    • Australian Corner: Cone News from Australia
      - 12 Any Votes for blainvillii ?
      by John Singleton
      Included in the discussion are Conus pseudocedonulli Blainville, 1818, C. ammiralis Linneaus, 1758, C. archithalassus Hwass, 1792, C. da Motta 1987
    • Cone News from Australia
      - 13. Just White or Albino?
      by Jon Singleton
      Included in the discussion are C. cloveri and ateralbus, C. trigonus, C. rufi maculosus and C. pertusus, C. victoriae and C. anemone, C. amadis, magus, furvus and spectrum; C. araneosus, lynceus, malaccanus and nobilis. The Atlantic cones, C. mindanus and spurius. C. puncticulatus form columba, C. mappa granarius , C. cloveri and ateralbus
    • Conus cordigera vs C. nobilis by António Monteiro
      Conus cordigera Sowerby II in 1866, C. nobilis Linnaeus, 1758
    • The Golden Nocturnus by Jon Singleton
      Conus nocturnus Hwass, 1792; C. marmoreus Linneaus, 1758
    • Fossil Conus from Castell’Arquato (Piacenza) by Giancarlo Paganelli
      Fossil cones discussed include Conus antidiluvianus Bruguière, 1792, Conus brocchii Bronn, 1828, Conus canaliculatus Brocchi, 1814, Conus pelagicus Brocchi, 1814, Conus ponderosus Brocchi, 1814, Conus pyrula Brocchi, 1814, Conus striatulus Brocchi, 1814, and Conus virginalis Brocchi, 1814
    • Conus kawamurai Habe, 1962 An Extinct Species? by António Monteiro
      Conus kawamurai Habe, 1962; Conus granulatus , Conus ochroleucus tmetus Tomlin, 1937 and Conus ochroleucus tmetus Tomlin, 1937

    5 January, 2008

      Folding of conotoxins

      Bulaj G, Olivera BM. (2008) Folding of conotoxins: formation of the native disulfide bridges during chemical synthesis and biosynthesis of Conus peptides. Antioxid Redox Signal. 10:141-56.
      Department of Medicinal Chemistry, College of Pharmacy, Salt Lake City, Utah., Department of Biology University of Utah, Salt Lake City, Utah.

      Abstract: Conopeptides from >700 species of predatory marine Conus snails provide an impressive molecular diversity of cysteine-rich peptides. Most of the estimated 50,000-100,000 distinct conopeptides range in size from 10 to 50 amino acid residues, often with multiple posttranslational modifications. The great majority contain from two to four disulfide bridges. As the biosynthetic and chemical production of this impressive repertoire of disulfide-rich peptides has been investigated, particularly the formation of native disulfide bridges, differences between in vivo and in vitro oxidative folding have become increasingly evident. In this article, we provide an overview of the molecular diversity of conotoxins with an emphasis on the cysteine patterns and disulfide frameworks. The conotoxin folding studies reviewed include regioselective and direct oxidation strategies, recombinant expression, optimization of folding methods, mechanisms of in vitro folding, and preliminary data on the biosynthesis of conotoxins in venom ducts. Despite these studies, how the cone snails efficiently produce properly folded conotoxins remains unanswered. As chemists continue to master oxidative folding techniques, insights gleaned from how conotoxins are folded in vivo will likely lead to the development of the new folding methods, as well as shed some light on fundamental mechanisms relevant to the protein folding problem.

    1 January, 2008

      Welcome to 2008




    Continued in What's new in 2007
    See also : 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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