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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

Bruce Livett's more recent publications (1998-2005)

Site Map of Cone Shells and Conotoxins HomePage


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    31 December, 2005

      Comment on use of Ziconotide for palliative care

      Narayana, A.K. (2005) Elan: ziconotide review focused on off-label uses. Am J Hosp Palliat Care. 22 (6): 408.

      [No abstract available. Comment on Prommer, E.E. (2005) Ziconotide: can we use it in palliative care? Am J Hosp Palliat Care. 22(5):369-374. ]

    25 December, 2005. HAPPY CHRISTMAS ALL !!

      VISAYA Vol. 5 Published. New Conus species described.

      VISAYA, 5 [2005], 95pp., 54col.pls. Euro 50.00-55.00 [indication]

      Abstract: The 5th issue of Visaya contains 16 articles, which were written by M.Chiapponi, F. Déniz, W. Engl, K. Fraussen, J.M. Hernandez, F. Lorenz, D. Massiglia, S. Patamakanthin, G.T. Poppe, C. Redfern, E. Rolán, R. Salisbury, D. Stratmann, F. Swinnen, S. Tagaro, Y. Terryn, R. Vanwalleghem, Y. Verhaeghe, C. Vos, J. Wolff and G. Raybaudi Massilia. The following new Conus species was described:

      Conus guidopoppe

      Some references to past papers on Conus available from :A.N. van der Bijl, Burgemeester van Bruggenstraat 41, 1165 NV Halfweg, Nederland +31-20-4977772 (phone); anvdbijl@xs4all.nl (e-mail)
      RICHARD, 1983. 2 n.sp. of Conus from New Caledonia: C. boucheti & kanakinus. 6pp., 9figs. Euro 1.55
      LE GALL & al., 1999. Biodiversity of the genus Conus: a rich source of bioactive peptides. 26pp., 7tabs., 2figs.
      (1col.fig.). Euro 4.45 LE GALL & al., 1999. Les venins de cônes: source de toxines qui interagissent avec les canaux sodium dépendant du potentiel de membrane. 13pp., 8figs. (3col.figs.), 1tab., English summ., xerox-copy. Euro 0.80
      SEVERAL AUTHORS,2003. La Rochelle 24-7 Juin 2003 Pôle sciences & technologies de l'Université 3e Congrès International des Sociétés Européennes de Malacologie Les mollusques dans la recherche actuelle. 112pp., 2figs., 84abstracts. Euro 13.50
      RICHARD & al., 2005. Status de Conus nigropunctatus de Mer Rouge par rapport a C. catus a partir de la biometrie des coquilles. 7pp.,
      4col.figs., English summ. Euro 1.30
      SÉGUIGNES & al., 2005. Nouvelle approche morphometrique des coquilles du genre Conus. 6pp., 4col.figs., English summ. Euro 1.10

    23 December, 2005

      Conantokin-G from Conus geographus: Interaction with receptors

      L, Yasuda T, Lewis RJ, Dodd PR, Adams DJ. (2005) )NMDA receptor subunit-dependent modulation by conantokin-G and Ala(7)-conantokin-G. Ragnarsson. J Neurochem. 2005 Nov 29; [Epub ahead of print]

      School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland, Australia.

      Abstract: The modulation of recombinant NMDA receptors by conantokin-G (con-G) and Ala(7)-conantokin-G (Ala(7)-Con-G) was investigated in Xenopus oocytes injected with capped RNA coding for NR1 splice variants and NR2 subunits using the two-electrode voltage clamp technique. Glutamate exhibited a marginally higher apparent affinity for NR2A-containing receptors than NR2B-containing receptors, regardless of the NR1 subunit present. Conantokins were bath applied to give cumulative concentration responses in the presence of 3 and 30 mum glutamate. Both contantokins exhibited biphasic concentration-response relationships at NR2A-containing NMDA receptors, producing potentiation at low conantokin concentrations and inhibition at high concentrations. These effects were stronger with glutamate concentrations near its EC(50), and less marked at saturating concentrations. In contrast, the conantokin concentration-response relation was monophasic and inhibitory at NR2B-containing receptors. We conclude that the combinations of subunits that comprise the NMDA receptor complex influence conantokin and glutamate affinities and the nature of the responses to conantokins.

    2 December, 2005

      Use of Conus and other peptide toxins in the analysis of nicotinic receptors

      Fruchart-Gaillard C, Menez A, Servent D. (2005) [Critical role of peptidic toxins in the functional and structural analysis of nicotinic acetylcholine receptors] . J Soc Biol. 199: 45-53. Review. French.

      Departement d'lngenierie et d'Etudes des Proteines, CE Saclay, 91191 Gif, Yvette.

      Abstract: Animal toxins which interact on various receptors and channels have been often used in the studies of the functional roles of these targets. Nicotinic toxins have been purified from snake and cone venoms and are characterized by high affinity and various selectivity of interactions on the different nicotinic receptors subtypes. Since 30 years they have been used as molecular probes to identify, localize and purify these receptors. Furthermore, they have played a crucial role in the better understanding of their functional properties and have been useful in their structural studies. These peptidic toxins could be chemically synthesized or recombinantly expressed and nonnatural residues could be introduced in their sequences in order to delineate their functional interaction sites. The structural modelisation of toxin-nAChR interaction allows us to understand the antagonistic property of these toxins and open the way to the design of engineered ligands with predetermined specificity, useful as pharmacological tools or therapeutic agents in the numerous diseases involving this receptor family.

      Conantokin-G from Conus geographus lessens cerebral ischemia

      Lu, X.C., Williams, A.J., Wagstaff, J.D., Tortella, F.C. and Hartings, J.A. (2005). Effects of delayed intrathecal infusion of an NMDA receptor antagonist on ischemic injury and peri-infarct depolarizations. Brain Res. 1056: 200-208.

      Division of Psychiatry and Neurosciences, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD 20910, USA.

      Abstract: The potent NMDA receptor antagonist, Conantokin-G (CGX-1007), a snail peptide, has an 8-h therapeutic window in rat focal cerebral ischemia. We hypothesized that the mechanism of neuroprotection is the inhibition of 'secondary phase' peri-infarct depolarizations (PIDs), recently shown to recur 6--24 h post-reperfusion. Rats were implanted with intrathecal (i.t.) catheters for drug delivery and DC-compatible electrodes for continuous PID monitoring and subjected to transient (2 h) middle cerebral artery occlusion. Four groups were studied. In two groups (C(40)C and C(20)C), continuous infusion of CGX-1007 was administered over 8--24 h post-occlusion at 0.1 microg/h (0.04 nmol/h) following either a 40- or 20-nmol bolus dose at 8 h. Another group (C(40)S) received the 40-nmol bolus followed by saline infusion, and a control group received saline. Intrathecal drug treatment reduced infarct volumes relative to controls by 61%, 31%, and 10% in C(40)C, C(40)S, and C(20)C groups, respectively, but also induced dose-dependent paralysis and elevated mortality. All rats had PID rates similar to the control group prior to treatment, but following treatment secondary phase PIDs were reduced by 47--57% in each drug group compared to controls. Because several animals exhibited PID inhibition but no neuroprotection, there was no significant correlation between these endpoints across groups. However, drug-treated animals that did not exhibit secondary phase PIDs prior to treatment had significantly smaller infarcts and reduced subsequent PID activity than corresponding control rats. Results suggest that post-reperfusion PIDs play a substantial, though still undefined pathogenic role in delayed maturation of cerebral infarction and NMDA receptor-targeted neuroprotection.

      Conantokin G, an antagonist of NMDA (NR1/2A/2B and NR1/2B) receptors, inhibits p38 phosphorylation.

      Waxman, E.A. and Lynch, D.R. (2005). N-methyl-D-aspartate receptor subtype mediated bidirectional control of p38 mitogen-activated protein kinase. J Biol Chem. 280: 29322-29333..

      Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

      Abstract: N-methyl-d-aspartate receptor (NMDAR) stimulation activates many downstream mechanisms involved in both cell survival and cell death. The manner in which the NMDAR regulates one of these pathways, the p38 mitogen-activated protein kinase (p38) pathway, is currently unknown. In the present study, we have defined a developmental-, concentration-, and time-dependent phosphorylation and subsequent dephosphorylation of p38. In cultured hippocampal neurons 7-8 days in vitro (DIV7-8), NMDAR stimulation leads to a concentration-dependent increase in p38 phosphorylation (phospho-p38). However, in more mature neurons (>DIV17) application of NMDA produces concentration-dependent effects, such that low concentrations result in sustained increases in phospho-p38 levels, and high concentrations dephosphorylate p38 within 5 min. Conantokin G, an antagonist of NR1/2A/2B and NR1/2B receptors, inhibits p38 phosphorylation, while NR1/2B-specific antagonists prevent the rapid dephosphorylation of p38 without affecting p38 activation. Furthermore, inhibition of calcineurin prevents the activation of p38, whereas inhibition of phosphoinositide 3-kinase (PI3K) prevents the rapid dephosphorylation of p38. Our results support the presence of subtype-dependent pathways regulating p38 activation and deactivation: one involves NR1/2A/2B receptors activating calcineurin and resulting in p38 phosphorylation, and the other utilizes NR1/2B receptors binding to and activating PI3K and leading to the dephosphorylation of p38 in a manner involving both NR1/2A/2B receptor activation and tyrosine phosphorylation of NR2B. The ability of NMDAR subtype-specific mechanisms to regulate p38 has implications for NMDAR-mediated synaptic plasticity, gene regulation, and excitotoxicity.

      Acrorhagin II, a novel peptide toxin in the sea anemone, Actinia equina, resembles a cone snail neurotoxin

      Honma, T., Minagawa, S., Nagai, H., Ishida, M., Nagashima, Y. and Shiomi K. (2005). Novel peptide toxins from acrorhagi, aggressive organs of the sea anemone Actinia equina. Toxicon. 46: 768-774..

      Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Konan-4, Minato-ku, Tokyo 108-8477, Japan.

      Abstract: Two peptide toxins, acrorhagin I (50 residues) and II (44 residues), were isolated from special aggressive organs (acrorhagi) of the sea anemone Actinia equina by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T. The LD50 against crabs of acrorhagin I and II were estimated to be 520 and 80 microg/kg, respectively. 3'- and 5'-RACE established the amino acid sequences of the acrorhagin precursors. The precursor of acrorhagin I is composed of both signal and mature peptides and that of acrorhagin II has an additional sequence (propart) between signal and mature peptides. Acrorhagin I has no sequence homologies with any toxins, while acrorhagin II is somewhat similar to spider neurotoxins (hainantoxin-I from Selenocosmia hainana and Tx 3-2 from Phoneutria nigriventer) and cone snail neurotoxin (omega-conotoxin MVIIB from Conus magus). In addition, analogous peptides (acrorhagin Ia and IIa) were also cloned during RT-PCR experiments performed to confirm the nucleotide sequences of acrorhagins. This is the first to demonstrate the existence of novel peptide toxins in the sea anemone acrorhagi.

      Marine neurotoxins: recognition and treatments. Review

      Watters, M.R. and Stommel, E.W. (2005). Marine Neurotoxins: Envenomations and Contact Toxins. Curr Treat Options Neurol. 6: 115-123 .

      Department of Medicine, Division of Neurology, University of Hawaii, 1356 Lusitana Street, 7th Floor, Honolulu, HI 96813, USA. mwatters@hawaii.edu

      Abstract: Familiarity with the appearance and habitat of venomous sea creatures, the location of their stinging apparatus, and surveillance of population concentrations within recreational waters are essential in avoiding envenomations. Compared with the thermo-stable low molecular weighted ingestible seafood toxins, venomous toxins are often large molecular weight proteins and many are heat labile, which provides opportunity for therapeutic intervention. Heat therapy may denature the toxins, and provide immediate relief of pain in coelenterate and venomous fish envenomations. Injections of local anesthetic agents may also be used. First aid measures at the seashore may limit the spread of venom, and include immobilization of the affected sites, compression bandaging, and venous-lymphatic occlusive bandages. Measures to limit continued envenomation by attached stinging cells include topical vinegar for jellyfish tentacles and irrigation with debridment for spines of venomous fish. Antivenins are of limited availability and may be used for envenomations with sea snakes, Chironex box jellyfish, and some venomous fish. Sea snakes bites may be treated with antivenin from land snakes or with hemodialysis when antivenin is not available. Neuromuscular paralysis occurs with bites by sea snakes, cone snails, blue octopuses, and some jellyfish. Supportive treatment includes attention to cardiopulmonary resuscitation and intubation. Exposure to Pfeisteria may result in cognitive and behavioral abnormalities. Treatment with cholestyramine may be helpful in binding the toxin and improve recovery.

    1 December, 2005

      View these Real Top Seashells

      Philippe Quiquandon and Olivier Santini of Real Top Seashells have some tempting examples of Conus on display at their 'Shells Passion.com' site.
      Take for example this Conus bengalensis from Burma.
      OR this Conus gauguini from Marquese Is.
      OR this Conus Hirasei from the Philippines.

      On this web site you can see reference shells classified in TOP & World Record Specimens (WRS) sections. With respect to Conus species, there are already 61 WRS and 523 TOP specimens including 7 New Additions of which 3 are WRS (C. bandanus nigrescens; C. betulinus rifoluteus; C. cordigera) and 4 are TOP (C. fischolderi; C. nocturnus deburgliae; C. suratensis and C. terryni). Read more about this site here.

      A regular update (daily or weekly) will keep you informed of additions to both sections. In all, over 4000 shell images in the image bank (currently 3459 TOP pictures). Subscriptions available for 6 months and 12 months access. (Click here for Details of membership).

    30 November, 2005

      REVIEW : Posttranslational modifications of conotoxins

      Buczek, O., Bulaj, G. and Olivera, B.M. (2005) Conotoxins and the posttranslational modification of secreted gene products. Cellular and Molecular Life Sciences (CMLS) [REVIEW] Published online: 28 November 2005.

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

      Abstract: The venoms of predatory cone snails (genus Conus) have yielded a complex library of about 50-100,000 bioactive peptides, each believed to have a specific physiological target (although peptides from different species may overlap in their target specificity). Conus has evolved the equivalent of a drug development strategy that combines the accelerated evolution of toxin sequences with an unprecedented degree of posttranslational modification. Some Conus venom peptide families are the most highly posttranslationally modified classes of gene products known. We review the variety and complexity of posttranslational modifications documented in Conus peptides so far, and explore the potential of Conus venom peptides as a model system for a more general understanding of which secreted gene products may have modified amino acids. Although the database of modified conotoxins is growing rapidly, there are far more questions raised than answers provided about possible mechanisms and functions of posttranslational modifications in Conus.
      Key words. Posttranslational modification - conotoxin - D-amino acids - 6-bromotryptophan - O-glycosylation
      Received 24 June 2005; received after revision 13 August 2005; accepted 19 September 2005

    29 November, 2005

      Conus spurius reveals a hydrophobic T-1-conotoxin

      Aguilar, M.B., Lezama-Monfil, L., Maillo, M., Pedraza-Lara, H., Lopez-Vera, E., Heimer de la Cotera, E.P. (2005) A biologically active hydrophobic T-1-conotoxin from the venom of Conus spurius. Peptides. 2005 Nov 15; [Epub ahead of print]
      Laboratory of Marine Neuropharmacology, Institute of Neurobiology, Universidad Nacional Autonoma de Mexico, Campus UNAM-UAQ Juriquilla, Km 15 Carr. Queretaro-S.L.P., Juriquilla, Qro. 76230, Mexico.

      Abstract: A major, very hydrophobic peptide, sr5a, was purified from the venom duct of Conus spurius specimens collected in the Yucatan Channel, Mexico. Its amino acid sequence (IINWCCLIFYQCC; calculated monoisotopic mass assuming two disulfide bridges 1616.68Da) was determined by automatic Edman degradation after reduction and alkylation, and confirmed by mass spectrometry (ESI monoisotopic mass, 1616.60; MALDI monoisotopic mass 1616.42Da). The primary structure of sr5a showed the pattern that characterizes the family of the T-1-conotoxins, which belong to the T-superfamily of conotoxins. The disulfide bonds were determined by partial reduction and alkylation with N-ethylmaleimide, followed by total reduction and alkylation with 4-vinylpyridine, and automatic Edman sequencing. The connectivity of the Cys residues (I-III, II-IV) is the same as that found in the T-1-conotoxin family. When injected intracranially (2.0nmol) into mice, peptide sr5a caused depressed behavioral activity.

      See also:

      Aguilar, M.B., Lopez-Vera, E., Ortiz, E., Becerril, B., Possani, L.D., Olivera, B.M. and Heimer de la Cotera, E.P. (2005) A novel conotoxin from Conus delessertii with posttranslationally modified lysine residues. Biochemistry 44: 11130-11136.

      Aguilar, M.B., Lopez-Vera, E., Imperial, J.S., Falcon, A., Olivera, B.M. and de la Cotera, E.P. (2005) Putative gamma-conotoxins in vermivorous cone snails: the case of Conus delessertii. Peptides. 26: 23-27.

    20 November, 2005

      Conus magus and other marine creatures : a source of Nature's Drugs

      Underhill, W. (2005) Nature's Drugs. Newsweek International, Nov. 7, 2005 Issue.

      Synopsis: Scientists tend to prefer the lab to the mess and complication of living beings. Now they realize that forests and oceans hold a bounty of useful chemicals. Swimmers in the coral reefs of the Philippines know to stay away from Conus magus. The sea snail may be small—just a few inches long—but it's deadly mean. One dose of its venom can paralyze the passing fish that make up its diet. To drugmakers, though, the potency of its toxin is sheer poetry. Scientists who recently broke down the poison discovered—and copied exactly—a chemical compound that blocked nerve cells from sending signals to the brain. Result: Prialt, a new painkilling drug 1,000 times more powerful than morphine, the most potent analgesic now available to medicine.

      Evolution of a Scientist

      He had planned to enter the ministry, but his discoveries on a fateful voyage 170 years ago shook his faith and changed our conception of the origins of life. See article about Charles Darwin by Jerry Adler in Newsweek Technology and Science

      Chi, S.W., Lee, S.H., Kim, D.H., Kim, J.S., Olivera, B.M., McIntosh, J.M. and Han, K.H. (2005) Solution structure of alpha-conotoxin PIA, a novel antagonist of alpha6 subunit containing nicotinic acetylcholine receptors. Biochem Biophys Res Commun. 338:1990-1997.

      Protein Analysis and Design Laboratory, Division of Drug Discovery, Korea Research Institute of Bioscience and Biotechnology, Yusong P. O. Box 115, Daejon, Republic of Korea.

      Abstract: alpha-Conotoxin PIA is a novel nicotinic acetylcholine receptor (nAChR) antagonist isolated from Conus purpurascens that targets nAChR subtypes containing alpha6 and alpha3 subunits. alpha-conotoxin PIA displays 75-fold higher affinity for rat alpha6/alpha3beta2beta3 nAChRs than for rat alpha3beta2 nAChRs. We have determined the three-dimensional structure of alpha-conotoxin PIA by nuclear magnetic resonance spectroscopy. The alpha-conotoxin PIA has an "omega-shaped" overall topology as other alpha4/7 subfamily conotoxins. Yet, unlike other neuronally targeted alpha4/7-conotoxins, its N-terminal tail Arg(1)-Asp(2)-Pro(3) protrudes out of its main molecular body because Asp(2)-Pro(3)-Cys(4)-Cys(5) forms a stable type I beta-turn. In addition, a kink introduced by Pro(15) in the second loop of this toxin provides a distinct steric and electrostatic environment from those in alpha-conotoxins MII and GIC. By comparing the structure of alpha-conotoxin PIA with other functionally related alpha-conotoxins we suggest structural features in alpha-conotoxin PIA that may be associated with its unique receptor recognition profile.

      Mordvintsev, D.Y., Polyak, Y.L., Levtsova, O.V., Tourleigh, Y.V., Kasheverov, I.E., Shaitan, K.V., Utkin, Y.N., Tsetlin, V.I. (2005) A model for short alpha-neurotoxin bound to nicotinic acetylcholine receptor from Torpedo californica: Comparison with long-chain alpha-neurotoxins and alpha-conotoxins. Comput Biol Chem. 29: 398-411.

      Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, 117997, Miklukho-Maklaya str., 16/10, GSP-7, Moscow, Russia.

      Abstract: Short-chain alpha-neurotoxins from snakes are highly selective antagonists of the muscle-type nicotinic acetylcholine receptors (nAChR). Although their spatial structures are known and abundant information on topology of binding to nAChR is obtained by labeling and mutagenesis studies, the accurate structure of the complex is not yet known. Here, we present a model for a short alpha-neurotoxin, neurotoxin II from Naja oxiana (NTII), bound to Torpedo californica nAChR. It was built by comparative modeling, docking and molecular dynamics using (1)H NMR structure of NTII, cross-linking and mutagenesis data, cryoelectron microscopy structure of Torpedo marmorata nAChR [Unwin, N., 2005. Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J. Mol. Biol. 346, 967-989] and X-ray structures of acetylcholine-binding protein (AChBP) with agonists [Celie, P.H., van Rossum-Fikkert, S.E., van Dijk, W.J., Brejc, K., Smit, A.B., Sixma, T.K., 2004. Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41 (6), 907-914] and antagonists: alpha-cobratoxin, a long-chain alpha-neurotoxin [Bourne, Y., Talley, T.T., Hansen, S.B., Taylor, P., Marchot, P., 2005. Crystal structure of Cbtx-AChBP complex reveals essential interactions between snake alpha-neurotoxins and nicotinic receptors. EMBO J. 24 (8), 1512-1522] and alpha-conotoxin [Celie, P.H., Kasheverov, I.E., Mordvintsev, D.Y., Hogg, R.C., van Nierop, P., van Elk, R., van Rossum-Fikkert, S.E., Zhmak, M.N., Bertrand, D., Tsetlin, V., Sixma, T.K., Smit, A.B., 2005. Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant. Nat. Struct. Mol. Biol. 12 (7), 582-588]. In complex with the receptor, NTII was located at about 30A from the membrane surface, the tip of its loop II plunges into the ligand-binding pocket between the alpha/gamma or alpha/delta nAChR subunits, while the loops I and III contact nAChR by their tips only in a 'surface-touch' manner. The toxin structure undergoes some changes during the final complex formation (for 1.45rmsd in 15-25ps according to AMBER'99 molecular dynamics simulation), which correlates with NMR data. The data on the mobility and accessibility of spin- and fluorescence labels in free and bound NTII were used in MD simulations. The binding process is dependent on spontaneous outward movement of the C-loop earlier found in the AChBP complexes with alpha-cobratoxin and alpha-conotoxin. Among common features in binding of short- and long alpha-neurotoxins is the rearrangement of aromatic residues in the binding pocket not observed for alpha-conotoxin binding. Being in general very similar, the binding modes of short- and long alpha-neurotoxins differ in the ways of loop II entry into nAChR.

    18 November, 2005

      Novel conotoxins from Conus floridanus floridensis and Conus villepinii,

      Möller, C., Rahmankhah, S., Lauer-Fields, J., Bubis, J., Fields, G.B. and Marí, F. (2005). A novel conotoxin framework with a helix-loop-helix (Cs alpha/alpha) fold. Biochemistry (in-press)

      Department of Chemistry and Biochemistry and Center of Excellence in Biomedical and Marine Biotechnology, Florida Atlantic University, Boca Raton, Florida 33431, and Departamento de Biología Celular, Universidad Simón Bolívar, Sartenejas 1080, Venezuela.

      Abstract: Venomous predatory animals, such as snakes, spiders, scorpions, sea anemones, and cone snails, produce a variety of highly stable cystine-constrained peptide scaffolds as part of their neurochemical strategy for capturing prey. Here we report a new family of four-cystine, three-loop conotoxins (designated framework 14). Three peptides of this family (flf14a-c) were isolated from the venom of Conus floridanus floridensis, and one (vil14a) was isolated from the venom of Conus villepinii, two worm-hunting Western Atlantic cone snail species. The primary structure for these peptides was determined using Edman degradation sequencing, and their cystine pairing was assessed by limited hydrolysis with a combination of CNBr and chymotrypsin under nonreducing, nonalkylating conditions in combination with MALDI-TOF MS analysis of the resulting peptidic fragments. CD spectra and nanoNMR spectroscopy of these conotoxins directly isolated from the cone snails revealed a highly helical secondary structure for the four conotoxins. Sequence-specific nanoNMR analysis at room temperature revealed a well-defined helix-loop-helix tertiary structure that resembles that of the Cs alpha/alpha scorpion toxins kappa-hefutoxin, kappa-KTx1.3, and Om-toxins, which adopt a stable three-dimensional fold where the two alpha-helices are linked by the two disulfide bridges. One of these conotoxins (vil14a) has a Lys/Tyr dyad, separated by approximately 6Å, which is a conserved structural feature in K+ channel blockers. The presence of this framework in scorpions and in cone snails indicates a common molecular imprint in the venom of apparently unrelated predatory animals and suggests a common ancestral genetic origin.

      Interactions of fluorescent conotoxin GI with the nicotinic acetylcholine receptor

      Schreiter, C., Gjoni, M., Hovius, R., Martinez, K.L., Segura, J.M., Vogel, H. (2005). Reversible Sequential-Binding Probe Receptor-Ligand Interactions in Single Cells. Chembiochem. 2005 Nov 4; [Epub ahead of print] PMID:

      Ecole Polytechnique Federale de Lausanne (EPFL) Laboratoire de Chimie Physique des Polymeres et Membranes 1015 Lausanne, Switzerland, Fax: (+41) 21-693-6190.

      Abstract: With the reversible sequential (ReSeq) binding assay,we present a novel approach for the ultrasensitive profiling of receptor function in single living cells. This assay is based on the repetitive application of fluorescent ligands that have fast association-dissociation kinetics. We chose the nicotinic-acetylcholine receptor (nAChR) as a prototypical example and performed ReSeq equilibrium, kinetic, and competition-binding assays using fluorescent derivatives of the antagonist alpha-conotoxin GI (alpha-CnTx). Thereby, we determined the binding constants of unlabeled alpha-CnTx and d-tubocurarine. The high selectivity of alpha-CnTx for muscle-type nAChR made it possible to observe specific binding even in the presence of other nAChR subtypes. Imaging of individual nAChRs and ligand-binding cycles to single cells in microfluidic devices demonstrated the ultimate miniaturization and accuracy of ReSeq-binding assays even at low receptor-expression levels. We expect our approach to be of generic importance for functional screening of compounds or membrane receptors, and for the detailed characterization of rare primary cells.

      Molluscan nicotinic acetylcholine receptors

      van Nierop, P., Keramidas, A., Bertrand, S., van Minnen, J., Gouwenberg, Y., Bertrand, D. and Smit, A.B. (2005) Identification of molluscan nicotinic acetylcholine receptor (nAChR) subunits involved in formation of cation- and anion-selective nAChRs. J Neurosci. 25:10617-10626.

      Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Faculty of Earth and Life Sciences, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands, and Department of Neuroscience, University Medical Centre, 1211 Geneva 4, Switzerland.

      Abstract: Acetylcholine (ACh) is a neurotransmitter commonly found in all animal species. It was shown to mediate fast excitatory and inhibitory neurotransmission in the molluscan CNS. Since early intracellular recordings, it was shown that the receptors mediating these currents belong to the family of neuronal nicotinic acetylcholine receptors and that they can be distinguished on the basis of their pharmacology. We previously identified 12 Lymnaea cDNAs that were predicted to encode ion channel subunits of the family of the neuronal nicotinic acetylcholine receptors. These Lymnaea nAChRs can be subdivided in groups according to the residues supposedly contributing to the selectivity of ion conductance. Functional analysis in Xenopus oocytes revealed that two types of subunits with predicted distinct ion selectivities form homopentameric nicotinic ACh receptor (nAChR) subtypes conducting either cations or anions. Phylogenetic analysis of the nAChR gene sequences suggests that molluscan anionic nAChRs probably evolved from cationic ancestors through amino acid substitutions in the ion channel pore, a mechanism different from acetylcholine-gated channels in other invertebrates.

      Effects of alpha-conotoxin GI with

      Barik, J. and Wonnacott, S. (2005) Indirect modulation by {alpha}7 nicotinic acetylcholine receptors of noradrenaline release in rat hippocampal slices: interaction with glutamate and GABA systems and effect of nicotine withdrawal. Mol Pharmacol. 2005 Nov 3; [Epub ahead of print]

      University of BATH, UK.

      Abstract: Nicotinic acetylcholine receptors (nAChR) can modulate transmitter release. Striatal [(3)H]dopamine ([(3)H]DA) release is regulated by presynaptic nAChR on dopaminergic terminals and alpha7 nAChR on neighbouring glutamatergic afferents. Here, we explored the role of alpha7 nAChR in the modulation of [(3)H]noradrenaline ([(3)H]NA) release from rat hippocampal slices. The nicotinic agonist anatoxin-a (AnTx) evoked monophasic [(3)H]NA release (EC50=1.2 microM) that was unaffected by alpha-conotoxin-MII or dihydro-beta-erythroidine, antagonists of alpha3/alpha6beta2* and beta2* nAChR respectively. In contrast AnTx-evoked striatal [(3)H]DA release was biphasic (EC50=138.9 nM; 7.1 microM) and blocked by these antagonists. At a high AnTx concentration (25 microM), alpha7 nAChR antagonists (methyllycaconitine, alpha-conotoxin-ImI) and glutamate receptor (GluR) antagonists (kynurenic acid, DNQX) partially inhibited [(3)H]NA release . The alpha7 nAChR-selective agonist choline evoked [(3)H]NA release (Emax=33% of that of AnTx) that was blocked by GluR antagonists, supporting a model in which alpha7 nAChR trigger glutamate release that subsequently stimulates [(3)H]NA release. A GABAergic component was also revealed: choline-evoked [(3)H]NA release was partially blocked by the GABAA receptor antagonist bicuculline, and co-application of bicuculline and DNQX fully abolished this response. These findings support alpha7 nAChR on GABAergic neurones that can promote GABA release that, in turn, leads to [(3)H]NA release, probably by disinhibition. To investigate the impact of chronic nicotine exposure on this model, rats were exposed for 14 days to nicotine (4 mg/kg/day) with or without 3 or 7 days withdrawal. alpha7 nAChR responses were selectively and transiently up-regulated after 3 days withdrawal. This functional up-regulation could contribute to the withdrawal effects of nicotine.

      Mondal, S., Vijayan, R., Shichina, K., Babu, R.M., Ramakumar, S. (2005). I-superfamily conotoxins: sequence and structure analysis. In Silico Biol. 5 (4):0050 [Epub ahead of print]

      Department of Physics, Indian Institute of Science, Bangalore 560 012, India; Email: ramak@physics.iisc.ernet.in.

      Abstract: I-superfamily conotoxins have four-disulfide bonds with cysteine arrangement C-C-CC-CC-C-C, and they inhibit or modify ion channels of nerve cells. They have been characterized only recently and are relatively less well studied compared to other superfamily conotoxins. We have detected selective and sensitive sequence pattern for I-superfamily conotoxins. The availability of sequence pattern should be useful in protein family classification and functional annotation. We have built by homology modeling, a theoretical structural 3D model of ViTx from Conus virgo, a typical member of I-superfamily conotoxins. The modeling was based on the available 3D structure of Janus-atracotoxin-Hv1c of Janus-atracotoxin family whose members have been suggested as possible biopesticides. A study comparing the theoretically modeled structure of ViTx, with experimentally determined structures of other toxins, which share functional similarity with ViTx, reveals the crucial role of C-terminal region of ViTx in blocking therapeutically important voltage-gated potassium channels.

      Wada, T., Imanishi, T., Kawaguchi, A., Mori, M.X., Mori, Y., Imoto, K. and Ichida, S. (2005). Effects of Calmodulin and Ca(2+) Channel Blockers on omega-conotoxin GVI A Binding to Crude Membranes from alpha(1B) Subunit (Ca(v)2.2) Expressed BHK Cells and Mice Brain Lacking the alpha(1B) Subunits. Neurochem Res. 30:1045-1054.

      Departments of Biological Chemistry, School of Pharmaceutical Sciences, Kinki University , Kowakae 3-4-1, 577-8502, Higashiosaka , Japan, seiji@phar.kindai.ac.jp.

      Abstract:Characteristics for the specific binding of (125)I-omega-CTX GVIA and (125)I-omega-CTX MVIIC to crude membranes from BHKN101 cells expressing the alpha(1B) subunits of Ca(v)2.2 channels and from mice brain lacking the alpha(1B) subunits of Ca(v)2.2 channels, particularly, the effects of CaM and various Ca(2+) channel blockers on these specific bindings were investigated. Specific binding of (125)I-omega-CTX GVIA to the crude membranes from BHKN101 cells was observed, but not from control BHK6 cells. omega-CTX GVIA, omega-CTX MVIIC and omega-CTX SVIB inhibited the specific binding of (125)I-omega-CTX GVIA to crude membranes from BHKN101 cells, and the IC(50) values for omega-CTXGVIA, omega-CTX MVIIC and omega-CTX SVIB were 0.07, 8.5 and 1.7 nM, respectively. However, omega-agatoxin IVA and calciseptine at concentrations of 10(-9)-10(-6) M did not inhibit specific binding. Specific binding was also about 80% inhibited by 20 mug protein/ml CaM. The amount of (125)I-omega-CTX GVIA (30 pM) specifically bound to membranes from brain of knockout mice lacking alpha(1B) subunits of Ca(v)2.2 channels was about 30% of that to the crude membranes from brain of wild-type. On the other hand, specific binding of (125)I-omega-CTX MVIIC (200 pM) was observed on the crude membranes of both BHKN101 and control BHK6 cells. The specific binding of (125)I-omega-CTX MVIIC (200 pM) was not inhibited by omega-CTX GVIA and omega-CTX SVIB, and also omega-Aga IVA and calciseptine at concentrations of 10(-9)-10(-7) M, although specific binding was almost completely dose dependently inhibited by non-radiolabeled omega-CTX MVIIC (IC(50) value was about 0.1 nM). 20 mug protein/ml CaM did not inhibit specific binding. Therefore, these results suggest that BHKN101 cells have a typical Ca(v)2.2 channels which are also inhibited by CaM and have not specific binding sites for omega-CTX MVIIC, although omega-CTX MVIIC is a blocker for both Ca(v)2.1 (alpha(1A; )P/Q-type) and Ca(v)2.2 channels.

      Xenome Limited Launches MultiCentre Clinical Trial For Severe Cancer Pain

      BRISBANE, Australia and SAN DIEGO, Nov. 15 /PRNewswire/ -- Xenome Limited today announced it had commenced a Phase I/IIa trial of Xen2174, a new class of peptide therapeutic for the treatment of severe intractable pain. (see Story from BioSpace.com)

    1 November, 2005

      Video Clip: Conus textile and Conus geographus
      (NATURE The Venom Cures: Cone Shell Cures | PBS | Released October 29, 2005).

      Cone shell cures ( Printable Page)

      Script: When it comes to research on venom and converting it into useful drugs, studies involving exotic snakes or brightly colored frogs seem to attract the most attention. However, one of the most promising new venom-derived drugs actually comes from a very modest-looking sea snail.

      Conus textile envenomates a mollusk

      Nature video clip "Watch a cone shell hunt"

      Abstract:Worldwide, there are more than 600 kinds of cone shells found mostly in tropical waters around the Pacific. Collectors love them because their shells are decorated with an amazing array of intricate patterns.
      Biologists, however, have long been fascinated by the behavior of these clever hunters. Some cone shells target other snails, while others like to feast on fish. To sense food, cone shells filter water through a tubelike organ called a siphon, awaiting a whiff of the telltale chemicals emitted by their prey.
      Then, when its victim comes near, the cone shell extends a proboscis armed with a harpoonlike tip that injects venom filled with special chemicals called "conotoxins." These toxins stop nerve cells from communicating with each other, causing paralysis within seconds and, eventually, death. Cone shells have even killed people who pick them up, unaware of the danger. Indeed, cone snail venom is so powerful and painless that victims can die unaware that they've even been bitten.
      Conotoxins have long interested medical researchers because of their potential painkilling abilities. It turns out, however, that cone shell venom is very complex; each kind contains perhaps 50 or more different chemicals that target the brain and nervous system. Overall, researchers believe that more than 50,000 conotoxins may exist. That diversity has made it hard for them to isolate a specific chemical to work on.
      But over the last few decades, conotoxins have begun to give up their secrets. Researchers have published more than 2,500 papers on the chemicals, and have described and identified more than 100 specific toxins which show promise for treating everything from arthritis to cancer. But the first new drug derived from a conotoxin, approved in 2004, targets chronic pain. Researchers estimate that the drug, based on the venom from the delicate gray and ivory magician cone shell, is a thousand times stronger than morphine, the most powerful traditional painkiller.
      Even as cone shells show promise for medicine, however, their survival may be at stake. Collectors gather millions of the animals each year for the decorative shell trade. Demand from conotoxin researchers is growing too, since many shells may be needed to produce even small amounts of toxin. And coral reefs, which support more than half of all cone shell species, are under increasing threat from human activities.
      To protect cone shells, biologists are asking nations in tropical zones to take new steps to monitor the shell trade and protect reefs. "To lose these species would be a self-destructive act of unparalleled folly," researcher Eric Chivian of Harvard University in Cambridge, Massachusetts wrote in a 2003 paper published by the journal SCIENCE. "Tropical cone snails may contain the largest and most clinically important pharmacopoeia of any [group of animals] in nature."

    Check out the numerous and varied Cone-shell Fact bites

    31 October, 2005

      Toxic Snail Venoms Yielding New Painkillers
      (National Geographic Newsletter, October 2005).

        In this 2-part article, the subject of cone shells and their sometimes toxic venoms is discussed with Dr. Jon-Paul Bingham, and other researchers investigating their value as a source of novel drug leads. Click here for Part 1 and Part 2 (Toxin factories).

      Warren Smith, of Shoalhaven Heads N.S.W. Australia has photos of live Conus textile and Conus striatus to illustrate a page on Cone Shells - Deadly Australians.

      See also the article about Cone Shells on the Australian Institute of Marine Science (AIMS) site "Dangerous marine animals of Northern Australia".

      Kohn, A. J. and Almasi, K. N. (1993) Imposex in Australian Conus. J. Mar. Biol. Assoc. U.K. 73 (1), 241-244.

      Duda, T. F. and Rolan, E. (2005) Explosive radiation of Cape Verde Conus, a marine species flock. Molecular Ecology 14 , 267-272.

        Abstract: Nearly 50 species of the marine gastropod genus Conus are restricted to the Cape Verde archipelago. This unusual concentration of endemics within a single set of oceanic islands is extremely uncharacteristic of marine taxa. Here we used phylogenetic analyses of 90 Conus species, including 30 endemics from Cape Verde, to reveal the relationships and origins of the endemic Cape Verde Conus. Results show that these species group in two distinct clades and represent a marine species flock that is restricted to a very narrowly confined geographical area. Species' originations occurred in exceptionally limited parts of the archipelago and in some cases radiations took place solely within single islands. Finally, comparison of levels of divergence between Cape Verde endemics and other Conus species suggests that the radiation of Conus in Cape Verde occurred during the last few million years.

      Conotoxins: essential data (Toxins List, Biological Weapons Gateway).

      Wikipedia entry for Conotoxin

      Ziconotide not for patients with psychiatric symptoms - a review

      Wermeling, D.P. (2005) Ziconotide, an intrathecally administered N-type calcium channel antagonist for the treatment of chronic pain. Pharmacotherapy. 25:1084-1094.REVIEW.
      Department of Pharmacy Practice and Science, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, USA. dwermel@uky.edu

      Abstract: Ziconotide is a novel peptide that blocks the entry of calcium into neuronal N-type voltage-sensitive calcium channels, preventing the conduction of nerve signals. N-type calcium channels are present in the superficial laminae of the dorsal horn of the spinal cord. In various animal models of pain, intrathecal administration of ziconotide blocked nerve transmission and nociception. The United States Food and Drug Administration recently approved ziconotide intrathecal infusion for the management of severe chronic pain in patients who require intrathecal therapy and who are intolerant of or refractory to other treatment, such as systemic analgesics, adjunctive therapies, or intrathecal morphine. The drug has a narrow therapeutic window and a lag time for the onset and offset of analgesia and adverse events. In early clinical trials, frequent and severe psychiatric and central nervous system adverse effects were associated with rapid intrathecal infusion (0.4 microg/hr) and frequent up-titration (every 12 hrs). Therefore, patients with psychiatric symptoms are not candidates for this drug. Drug trials of external intrathecal catheters and microinfusion devices demonstrated a 3% risk of meningitis. A low initial infusion rate of 0.1 microg/hour and limiting infusion rate increases to 2-3 times/week are now recommended. Patients responsive to intrathecal ziconotide require an implanted infusion system to receive long-term therapy.

      Mechcatie, E. (2005) Marine Snail Toxin Approved to Treat Severe Pain. Internal Medicine News 38: (Issue 5), 75. or click to download the PDF version of the article.

      Outline: An intrathecal formulation of a synthetic version of a toxin used by a fish-eating marine snail to catch its prey has been approved as a treatment for severe, chronic pain. The Food and Drug Administration approved ziconotide for intrathecal (IT) infusion for managing severe chronic pain “in patients for whom intrathecal therapy is warranted and who are intolerant of or refractory to other treatment, such as systemic analgesics, adjunctive therapies, or IT morphine.” It is being marketed under the trade name Prialt by Elan Pharmaceuticals Inc. Ziconotide, which is not an opioid, is a synthetic version of a conopeptide used by a species of marine snail, Conus magus, to sting fish. In nature the toxin “is so powerful it stops the fish dead in its track, and the snail eats it,” said Mark Wallace, M.D., director of the center for pain and palliative medicine at the University of California, San Diego. The synthetic version of this “conotoxin” is an N-type calcium channel antagonist. N-type calcium channels are located mainly in the dorsal horn cells of the spinal cord, predominantly on the superficial layers, in the area of substantia gelatinosa where pain fibers synapse, Dr. Wallace explained. Ziconotide “blocks those calcium channels at the level where these pain fibers meet up,” essentially shutting them down, he said, noting that opioids also have the same effect. The three trials that led to the approval included patients with “really refractory” pain due to various causes, including low back pain, cancer pain, neuropathic pain, pain from nervous system injuries, and HIV-related pain, said Dr. Wallace, an investigator in the studies and a consultant to the manufacturer. The three pivotal trials used the Visual Analog Scale of Pain Intensity (VASPI), as the primary end point. The most recent trial was a multicenter study in 220 patients with severe chronic pain, described by most as refractory to treatments including IT morphine. Patients were first taken off IT medications and stabilized on analgesics that included opiates and then treated with placebo or ziconotide. At 3 weeks, VASPI scores had improved by a mean of 12% from baseline vs. a 5% mean improvement for patients on placebo, a highly significant difference. During treatment, the use of non-IT opioids dropped by 24% among patients on ziconotide, compared with 17% among those on placebo. Dr. Wallace said ziconotide treatment is not associated with addiction, withdrawal, or tolerance, and it is not a controlled substance. The most common side effects are neurologic, including neurocognitive impairment and dizziness. In ziconotide trials, dizziness, nausea, confusion, and headache were among the most common side effects, reported in at least 25% of patients. The drug comes with a black box warning that people can develop severe psychiatric symptoms and neurologic impairment during treatment, and it is contraindicated in people with a history of psychosis.

    • See also: The Sunday Times - UK Elan pain drug sells below expectations. ELAN is facing problems with one of its most important second-line drugs, the chronic pain-killer Prialt, which is posting weaker than expected sales. ...

      Chen, Z.J. and Minneman, K.P. (2005) Recent progress in alpha1-adrenergic receptor research. Acta Pharmacol Sin. 26: 1281-1287.
      Department of Pharmacology, School of Medicine, Emory University, Atlanta, GA 30322, USA. kminneman@pharm.emory.edu

      Abstract: alpha1-Adrenergic receptors (AR) play an important role in the regulation of physiological responses mediated by norepinephrine and epinephrine, particularly in the cardiovascular system. The three cloned alpha1-AR subtypes (alpha1A, alpha1B, and alpha1D) are G protein-coupled receptors that signal through the Gq/11 signaling pathway, each showing distinct pharmacological properties and tissue distributions. However, due to the lack of highly subtype-selective drugs, the functional roles of individual subtypes are still not clear. Development of new subtype-specific drugs will greatly facilitate the identification of the functions of each subtype. Conopeptide rho-TIA has been found to be a new alpha1B-AR selective antagonist with different modes of inhibition at alpha1-AR subtypes. In addition, recent studies using genetically engineered mice have shed some light on alpha1-AR functions in vivo, especially in the cardiovascular system and brain. Several proteins have been shown to interact directly with particular alpha1-AR, and may be important in regulating receptor function. Receptor heterodimerization has been shown to be important for cell surface expression, signaling and internalization. These new observations are likely to help elucidate the functional roles of individual alpha1-AR subtypes.

    21 October, 2005

      Ziconotide and palliative care

      Prommer, E.E. (2005) Ziconotide: can we use it in palliative care? Am J Hosp Palliat Care. 22: 369-374. Comment by Narayana, A.K. in: Am J Hosp Palliat Care. 2005 Nov-Dec;22(6):408.
      Division of Hematology/Oncology, UCLA School of Medicine, Los Angeles, California, USA.

      Abstract: Ziconotide (PRIALT) is a new nonopioid treatment for chronic pain. It is a peptide that is the synthetic analog of the omega-conotoxin, derived from the marine snail, Conus magus. The therapeutic benefit of ziconotide derives from its potent and selective blockade of neuronal N-type voltage-sensitive calcium channels. Interference with these channels inhibits input from pain-sensing primary nociceptors. A recent clinical trial demonstrated that ziconotide has a significant analgesic effect compared to placebo in patients considered intolerant or refractory to other treatment such as systemic analgesics, adjunctive therapies, or intrathecal (IT) morphine. Thus, ziconotide is the first of a new class of agents--N-type calcium channel blockers, or NCCBs. Ziconotide may represent another option for patients with refractory pain.

    ------------ Prommer, E.E. (2005) Ziconotide: can we use it in palliative care? Am J Hosp Palliat Care. 22 (5):369-374. Comment in: Am J Hosp Palliat Care. 2005 Nov-Dec;22(6):408.

    Division of Hematology/Oncology, UCLA School of Medicine, Los Angeles, California, USA.
    Abstract: Ziconotide (PRIALT) is a new nonopioid treatment for chronic pain. It is a peptide that is the synthetic analog of the omega-conotoxin, derived from the marine snail, Conus magus. The therapeutic benefit of ziconotide derives from its potent and selective blockade of neuronal N-type voltage-sensitive calcium channels. Interference with these channels inhibits input from pain-sensing primary nociceptors. A recent clinical trial demonstrated that ziconotide has a significant analgesic effect compared to placebo in patients considered intolerant or refractory to other treatment such as systemic analgesics, adjunctive therapies, or intrathecal (IT) morphine. Thus, ziconotide is the first of a new class of agents--N-type calcium channel blockers, or NCCBs. Ziconotide may represent another option for patients with refractory pain. -------------

    10 October, 2005

      An alpha-conotoxin from Conus victoriae prevents pain

      Satkunanathan, N., Livett, B., Gayler, K., Sandall, D., Down, J. and Khalil, Z. (2005) Alpha-conotoxin Vc1.1 alleviates neuropathic pain and accelerates functional recovery of injured neurones. Brain Res. 1059: 149-158.

      National Ageing Research Institute, and Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville Victoria 3010, Australia.

      Abstract: This paper demonstrates the capacity of the neuronal nicotinic acetylcholine receptor (nAChR) antagonist alpha-conotoxin Vc1.1 to inhibit pain responses in vivo. Vc1.1 suppressed pain behaviors when tested in two models of peripheral neuropathy of the rat sciatic nerve, the chronic constriction injury (CCI) and partial nerve ligation (PNL) models. Mechanical hyperalgesia was assessed using an Ugo Basile Analgesymeter. Vc1.1 was administered by intramuscular bolus injection near the site of injury at doses of 0.036 mug, 0.36 mug and 3.6 mug in CCI rats and at a dose of 0.36 mug in PNL rats. Vc1.1 was also administered contralaterally in CCI rats at doses of 0.36 mug and 3.6 mug. Treatment started after the development of hyperalgesia and continued for 7 days. Vc1.1 significantly attenuated mechanical hyperalgesia in both CCI and PNL rats for up to a week following cessation of treatment. Vc1.1 also accelerated functional recovery of injured neurones. A blister was raised over the footpad innervated by the peripheral terminals of the injured nerve. The ability of these terminals to mount an inflammatory vascular response upon perfusion of the blister base with substance P provided a measure of functional recovery. This study shows that alpha-conotoxin Vc1.1, a neuronal nAChR antagonist, suppressed mechanical pain responses associated with peripheral neuropathy in rats in vivo and accelerated functional recovery of the injured neurones. A role for neuronal nAChRs in the analgesic activity of Vc1.1 is proposed.

      Novel pain relief via marine snails

      Sharp, D. Novel pain relief via marine snails. The Lancet 366 (9484)439-440, 2005.
      c/- The Lancet, London NW1 7BY, UK

      Abstract: Most conopeptides (conotoxins and related compounds) with potential clinical applications are in the early stage of development (Table 1). The exception is ziconotide,(6) a synthetic drug but with a 25-aminoacid sequence identical to that of the parent venom constituent in C magus. These peptides have a wide range of targets. Listing eight conopeptides under development for neurological conditions, including pain, [Livett BG] et al(5) noted competitive blockade of N-type nicotinic acetylcholine receptors, N-type calcium-channel blockade, inhibition of the neuronal noradrenaline transporter, binding to the neurotensin receptor and selective inhibition of N-methyl-D-aspartate receptors. Ziconotide is a selective blocker of N-type voltage-sensitive calcium-channels, which are abundant in dorsal root ganglion cells, so the spinal transmission of pain messages is interrupted. It has no effect on opioid receptors. This drug has been approved in the USA and Europe for the management, via the intrathecal route only, of severe resistant pain; it is on the market in the USA. Animal studies did not encourage the development of ziconotide as an epilepsy drug and clinical studies of its neuroprotective properties were stopped because of a lowering of blood pressure. Severe pain, in which the patient will usually be at the limit of morphine intake, is the only approved indication.

      To date only one controlled clinical trial on ziconotide has been fully published (8) however, trial data are available (9) and the results of three pivotal controlled studies are summarised in Table 2. Clinical information on other conopeptides is not abundant so far-eg, for the Amrad product, AM336, there is just one poster presentation on findings in seven patients (10). Trials have involved cautious dose-titration, and the results for ziconotide in Table 2 are those for the end of the initial titration phase. From the ten controlled longer-term open-label, and other studies of intrathecal ziconotide, in well over 1000 patients, a picture of the drug's adverse reactions profile is emerging. Serious reactions in three closely monitored trial patients were reported in detail 5 years ago (11). The most frequent reactions (recorded in 10% or more of patients) are confusion, dizziness, nystagmus, memory impairment, headache, blurred vision, nausea and vomiting, abnormal gait, and asthenia. A novel non-opioid class of drugs for the management of severe pain would be welcome. Whether synthetic conopeptides or new chemical compounds built round their structures fit the bill remains to be seen.

      (1) Bonnemain B. Helix and drugs: snails for western health care from antiquity to the present. Evid Based Complement Alternat Med 2005; 2: 25-28. (2) Fegan D, Andresen D. Conus geographus envenomation. Lancet 1997; 349: 1672. (3) Soiled BL, Wilson D, Zhaxybayeva O, Gogarten JP, Drinkwater R, King GF. Were arachnids the first to use combinatorial peptide libraries? Peptides 2005; 26: 131-139. (4) Stix G. A toxin against pain. Sci Am 2005; 292: 70-75. (5) Livett BG, Gayler KR, Khalil Z. Drugs from the sea: conopeptides as potential therapeutics. CurrMed Chem 2004; 11: 1715-23. (6) Miljanich GP. Ziconotide: neuronal calcium channel blocker for treating severe chronic pain. CurrMed Chem 2004; 11: 3029-40. (7) Duda TF Jr, Bingham J-P, Livett BG, et al. How much at risk are cone snails? Science 2004; 303: 955.(8) Staats PS, Yearwood T, Charapata SG, et al. Intrathecal ziconotide in the treatment of refractory pain in patients with cancer or AIDS: a randomized controlled trial. JAMA 2004; 291: 63-70. (9) European Medicines Evaluation Agency. Priait: European public assessment report. 2005 : (accessed June 6, 2005). (10) Cousins MJ, Goucke CR, Cher LM, et al. A phase I clinical trial of AM336, a novel N-type calcium channel blocker. 10th World Congress on Pain, San Diego, California, August, 2002: poster 615-P249. (11) Penn RD, Paice JA. Adverse effects associated with the intrathecal administration of ziconotide. Pain 2000; 85: 291-96

      Novel "M"-Superfamily conotoxin from Conus vexillum

      Jiang H, Wang CZ, Xu CQ, Fan CX, Dai XD, Chen JS, Chi CW (2005) A novel M-superfamily conotoxin with a unique motif from Conus vexillum. Peptides. 2005, Sept. 19 [ahead of publication]
      Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; Research Institute of Pharmaceutical Chemistry, Beijing 102005, China.

      Abstract: Cone snails are tropical marine mollusks that envenomate prey with a complex mixture of neuropharmacologically active compounds for the purpose of feeding and defence, each evolved to act in a highly specific manner on different parts of the nervous system. Here, we report the peptide purification, molecular cloning, chemical synthesis, and functional characterization of a structurally unique toxin isolated from the venom of Conus vexillum. The novel peptide, designated Vx2, was composed of 21 amino acid residues cross-linked by 3 disulfide bonds (WIDPSHYCCCGGGCTDDCVNC). Intriguingly, its mature peptide sequence shows low level of similarity with other identified conotoxins, and its unique motif (-CCCGGGC-) was not reported in other Conus peptides. However, its signal peptide sequence shares high similarity with those of the M-superfamily conotoxins. Hence, Vx2 could be classified into a new family of the M-superfamily.

      Binding of alpha-conotoxin ImI from Conus imperialis to Aplysia acetylcholine binding protein

      Hansen, S.B., Sulzenbacher, G., Huxford, T., Marchot, P., Taylor, P. and Bourne, Y. (2005) Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations. EMBO J. 24: 3635-3646
      [1] Department of Pharmacology, University of California at San Diego, La Jolla, CA, USA [2] Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA, USA.

      Abstract: Upon ligand binding at the subunit interfaces, the extracellular domain of the nicotinic acetylcholine receptor undergoes conformational changes, and agonist binding allosterically triggers opening of the ion channel. The soluble acetylcholine-binding protein (AChBP) from snail has been shown to be a structural and functional surrogate of the ligand-binding domain (LBD) of the receptor. Yet, individual AChBP species display disparate affinities for nicotinic ligands. The crystal structure of AChBP from Aplysia californica in the apo form reveals a more open loop C and distinctive positions for other surface loops, compared with previous structures. Analysis of Aplysia AChBP complexes with nicotinic ligands shows that loop C, which does not significantly change conformation upon binding of the antagonist, methyllycaconitine, further opens to accommodate the peptidic antagonist, alpha-conotoxin ImI, but wraps around the agonists lobeline and epibatidine. The structures also reveal extended and nonoverlapping interaction surfaces for the two antagonists, outside the binding loci for agonists. This comprehensive set of structures reflects a dynamic template for delineating further conformational changes of the LBD of the nicotinic receptor.

      Mechanisms leading to conotoxin diversity in Conus striatus

      Pi, C., Liu, Y., Peng, C., Jiang, X., Liu, J., Xu, B., Yu, X., Yu, Y., Jiang, X., Wang, L., Dong, M., Chen, S. and Xu, A.L. (2006). Analysis of expressed sequence tags from the venom ducts of Conus striatus: focusing on the expression profile of conotoxins. Biochimie 88: 131-140.
      State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Therapeutic Functional Genes, The Open Laboratory for Marine Functional Genomics of State High-Tech Development Program, Department of Biochemistry, College of Life Sciences, Sun Yat-sen (Zhongshan) University, 135 Xingangxi Road, 510275 Guangzhou, China.

      Abstract: Cone snails (genus Conus) are predatory marine gastropods that use venom peptides for interacting with prey, predators and competitors. A majority of these peptides, generally known as conotoxins demonstrate striking selectivity in targeting specific subtypes of ion channels and neurotransmitter receptors. So they are not only useful tools in neuroscience to characterize receptors and receptor subtypes, but offer great potential in new drug research and development as well. Here, a cDNA library from the venom ducts of a fish-hunting cone snail species, Conus striatus is described for the generation of expressed sequence tags (ESTs). A total of 429 ESTs were grouped into 137 clusters or singletons. Among these sequences, 221 were toxin sequences, accounting for 52.1% (corresponding to 19 clusters) of all transcripts. A-superfamily (132 ESTs) and O-superfamily conotoxins (80 ESTs) constitute the predominant toxin components. Some non-disulfide-rich Conus peptides were also found. The expression profile of conotoxins also explained to some extent the pharmacological and physiological reactions elicited by this typical piscivorous species. For the first time, a nonstop transcript of conotoxin was identified, which is suggestive that alternative polyadenylation may be a means of post-transcriptional regulation of conotoxin production. A comparison analysis of these conotoxins reveals the different variation and divergence patterns in these two superfamilies. Our investigations indicate that focal hyper-mutation, block substitution and exon shuffling are three main mechanisms leading to the conotoxin diversity in a species . The comprehensive set of Conus gene sequences allowed the identification of the representative classes of conotoxins and related components, which may lay the foundation for further research and development of conotoxins.

      Production of an isotopically (15)N/(13)C-enriched conopeptide

      Kumar, G.S., Ramasamy, P., Sikdar, S.K. and Sarma, S.P. (2005). Overexpression, purification, and pharmacological activity of a biosynthetically derived conopeptide. Biochem Biophys Res Commun. 335:965-972.
      Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.

      Abstract: A high yielding fusion protein system based on the protein cytochrome b(5) has been used for the production of novel 13-residue acyclic conopeptide. This peptide, Mo1659, can be liberated from the carrier protein using CNBr cleavage and subsequent purification using RP-HPLC methods. The yield of isotopically enriched peptides is high, ranging from 3 to 4mg of purified peptide from a 500ml culture, indicating that this system can be widely used for peptide production. Biosynthetic Mo1659 is active on non-inactivating K(+) channel much like the natural Mo1659, despite the absence of C-terminal amidation. Heteronuclear NMR studies show that the peptide exists in a conformational equilibrium involving proline-10. To our knowledge this is the first report of the production of an isotopically (15)N/(13)C-enriched conopeptide.

    4 October, 2005

      Mechanism of action of Contulakin-G

      Cognetix Inc. of Salt Lake City, Utah, have announced in a Business Wire dated 27 September 2005 that it has identified the novel mechanism of action for CGX-1160, its lead compound in clinical development for the treatment of chronic intractable pain.
      Synopsis: Following extensive studies, Cognetix scientists used neurotensin receptor knock-out mice to conclusively identify the neurotensin NTR1 receptor as the specific site through which CGX-1160 produces analgesia. A series of in vitro experiments has revealed that CGX- 1160 is able to produce a stronger activation of the NTR1 receptor than neurotensin. Cognetix is the only company in the world working on the NTR1 target for pain and will continue to examine whether the uniquely high efficacy of CGX-1160 at the NTR1 receptor may have applicability to the development of anti-psychotic drugs. As a result of the discovery of the mechanism of action, Cognetix has filed a provisional patent to protect the methods of screening for peptidomimetics or small molecules, the composition of identified compounds and the uses of the identified compounds for pain and other indications.

    • About CGX-1160. CGX-1160 is a broad spectrum non-opioid analgesic. It is the synthetic form of a natural peptide extracted from the venom of the Conus Geographus sea snail. The discovery of the mechanism of action provides further evidence of the differentiation between CGX-1160 and Elan's (NYSE:ELN) conopeptide based drug Prialt(R), which is currently marketed and sold in the United States and Europe.
    • CGX-1160 operates by activating the G-protein-coupled neurotensin NTR1 receptor (Prialt(R) inhibits the unrelated ion channel, n-type calcium channel), is a linear and smaller peptide, and has a significantly superior safety profile and therapeutic index than Prialt(R). Cognetix has successfully completed its CGX-1160 Phase 1b clinical trial at Brigham and Women's Hospital in Boston, Mass. The trial was conducted in a small population of spinal cord injured patients. The results support the Company's opinion that CGX-1160 will be a safe and effective drug for the treatment of chronic intractable pain. Phase II clinical trials are forecast to commence in 2006.
    • In July 2005, the U.S. Food and Drug Administration (FDA) granted an Orphan Drug designation to CGX-1160 for the intrathecal treatment of neuropathic pain associated with spinal cord injury.

      About Cognetix. Cognetix, Inc. is a private drug discovery and development company focused on the commercialization of conopeptide-based CNS pharmaceuticals. Conopeptides are a highly specialized, but structurally and functionally diverse group of peptides produced by venomous Conus sea snails, or cone snails These snails use a complex venom "cocktail" composed of between 50 and 200 distinct and unique peptides. These peptides target a variety of therapeutically important ion channels and receptors with unprecedented potency and selectivity and represent a large and diverse pharmacopeia of bioactive molecules. This potent and subtype selective activity at many molecular targets is of primary interest in Cognetix' pharmaceutical drug development program. . The Company has a strong drug discovery program in the field of pain, powered by a platform consisting of the most extensive conopeptide libraries and conopeptide-related intellectual property position in the world. Cognetix believes it has the largest patent portfolio in the world on conopeptide sequences, uses, synthesis, and methods. This patent portfolio includes more than 80 issued or allowed U.S. and international patents and an additional 80 patents pending.

    Further information: Contact Cognetix, Inc. Roger L. Flowerdew, 801-581-0400 rflowerdew@cognetix.com www.cognetix.com .

    21 September, 2005

      Cone shells on display

      Sonia Fuschi from the Shelline Group, Località Camposcala, Pod. 272 – 01014 Montalto di Castro (VT) – ITALY, is adding new images to her display of cone shell images all the time and presently has 179 specimens on display from 43 species of CONIDAE. Click here for the photogallery of the following 43 species of Conus ( araneosus nicobaricus; arenatus f. aequipunctatus; arenatus bizona; ateralbus; aurisiacus; bandanus f. vidua; barthelemyi; bengalensis; bullatus; caracteristicus; coronatus; crocatus f. thailandis; dalli; dusaveli; ebraeus; emaciatus; glaucus; gubernator leehmani; hyaena; ichinoseana; inscriptus; locumtenens; madagascarensis; malacanus; miliaris; milneedwardsii; natalis; pennaceus; pennaceus f. behelokensis; pennaceus f. elisae; purpurascens; rattus; sieboldii; sowerbyi; sulcatus f. samiae; sulcocastaneus; telatus; tessulatus; textile; textile f. archiepiscopus; textile f. euetrios; tulipa; ventricosus; venulatus; victor; vicweii; zeylanicus).

    20 September, 2005

      Review on pharmaceutical potential of marine toxins

      Watters, M.R. (2005) Tropical marine neurotoxins: venoms to drugs. Semin Neurol. 25: 278-289. REVIEW
      Professor of Medicine, Division of Neurology, University of Hawaii, Honolulu, Hawaii.

      Abstract: Neurotoxic venoms are common among tropical marine creatures, which have specialized apparatuses for delivery of the venoms. These include jellyfish and anemones, venomous cone snails, venomous fish, stingrays, sea snakes, and venomous octopuses. Numerous toxic neuropeptides are found within these venoms, and some can discriminate between closely related intracellular targets, a characteristic that makes them useful to define cation channels and attractive for drug development. A synthetic derivative of an omega-conotoxin is now available, representing a new class of analgesics. In general, toxic marine venoms contain proteins that are heat labile, providing opportunity for therapeutic intervention following envenomation, while ingestible seafood toxins are thermostable toxins. Ingestible toxins found in the tropics include those associated with reef fish, pufferfish, and some shellfish, which serve as food-chain vectors for toxins produced by marine microorganisms.

      Engineered Peptides

      Clark, R.J., Fischer, H., Dempster, L., Daly, N.L., Rosengren, K.J., Nevin, S.T., Meunier, F.A., Adams, D.J. and Craik, D.J. (2005) Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the {alpha}-conotoxin MII. Proc Natl Acad Sci U S A. 102: 13767-13772.
      Institute for Molecular Bioscience and School of Biomedical Sciences, University of Queensland, Brisbane QLD 4072, Australia.

      Abstract: Conotoxins (CTXs), with their exquisite specificity and potency, have recently created much excitement as drug leads. However, like most peptides, their beneficial activities may potentially be undermined by susceptibility to proteolysis in vivo. By cyclizing the alpha-CTX MII by using a range of linkers, we have engineered peptides that preserve their full activity but have greatly improved resistance to proteolytic degradation. The cyclic MII analogue containing a seven-residue linker joining the N and C termini was as active and selective as the native peptide for native and recombinant neuronal nicotinic acetylcholine receptor subtypes present in bovine chromaffin cells and expressed in Xenopus oocytes, respectively. Furthermore, its resistance to proteolysis against a specific protease and in human plasma was significantly improved. More generally, to our knowledge, this report is the first on the cyclization of disulfide-rich toxins. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine.

      Conotoxin ImI from Conus imperialis

      Kang, T.S., Vivekanandan, S., Jois, S.D., Kini, R.M. (2005) Effect of C-Terminal Amidation on Folding and Disulfide-Pairing of alpha-Conotoxin ImI. Angew Chem Int Ed Engl. 44 : 6333-6637.
      Department of Biological Sciences National University of Singapore 14 Science Drive 4, Block S3 #03-17, Singapore 117543, Singapore, Fax: (+65) 6779-2486.

      Abstract: No abstract

      Chi-Conotoxin Xen2174, from Conus marmoreus

      Nielsen, C.K., Lewis, R.J., Alewood, D., Drinkwater, R., Palant, E., Patterson, M., Yaksh, T.L., McCumber, D., Smith, M.T. (2005) Anti-allodynic efficacy of the chi-conopeptide, Xen2174, in rats with neuropathic pain. Pain 118: 112-124.
      School of Pharmacy, The University of Queensland, Brisbane, Qld, Australia.

      Abstract: Xen2174 is a structural analogue of Mr1A, a chi-conopeptide recently isolated from the venom of the marine cone snail, Conus marmoreus. Although both chi-conopeptides are highly selective inhibitors of the norepinephrine transporter (NET), Xen2174 has superior chemical stability relative to Mr1A. It is well-known that tricyclic antidepressants (TCAs) are also potent NET inhibitors, but their poor selectivity relative to other monoamine transporters and various G-protein-coupled receptors, results in dose-limiting side-effects in vivo. As TCAs and the alpha(2)-adrenoceptor agonist, clonidine, have established efficacy for the relief of neuropathic pain, this study examined whether intrathecal (i.t.) Xen2174 alleviated mechanical allodynia in rats with either a chronic constriction injury of the sciatic nerve (CCI-rats) or an L5/L6 spinal-nerve injury. The anti-allodynic responses of i.t. Mr1A and i.t. morphine were also investigated in CCI-rats. Paw withdrawal thresholds were assessed using calibrated von Frey filaments. Bolus doses of i.t. Xen2174 produced dose-dependent relief of mechanical allodynia in CCI-rats and in spinal nerve-ligated rats. Dose-dependent anti-allodynic effects were also produced by i.t. bolus doses of Mr1A and morphine in CCI-rats, but a pronounced 'ceiling' effect was observed for i.t. morphine. The side-effect profiles were mild for both chi-conopeptides with an absence of sedation. Confirming the noradrenergic mechanism of action, i.t. co-administration of yohimbine (100nmol) with Xen2174 (10nmol) abolished Xen2174s anti-allodynic actions. Xen2174 appears to be a promising candidate for development as a novel therapeutic for i.t. administration to patients with persistent neuropathic pain.

    26 July, 2005

      Updates to Conus Biodiversity Website

      Dr. Alan Kohn has announced some additions and updates to his Conus Biodiversity Website.
      Updates: "Previously we had posted types of 176 species-group taxa of Conus described between 1758 and 1840, representing about 75% of the species whose types are known to exist. We have continued our chronological approach, and have now posted images of primary types of 146 of the 1,436 species described during the remainder of the 20th Century, 1841-1900.
      In addition to the images, we have added a link to all Conus gene sequences that have been deposited in GenBank. You will see the NCBI icon next to the camera icon that indicates the presence of a primary type image. Clicking on the NCBI icon will take you to the GenBank nucleotide and protein sequences for that species as well as citations of publications that have used them.

      We have also added a new menu labeled “Information.” New pages here include:

    • Publications of the project since its inception in 2003;
    • Corrections and Updates to the Manual of the Living Conidae, Vol. 1. Indo-Pacific Region, by Röckel, Korn and Kohn (1995), including additional systematic information on Indo-West Pacific Conus subsequent to that publication. We thank Ross Mayhew for suggesting this addition to the website.
    • Recent Additions such as this notice of the latest enhancements to the website.

      In the near future we hope to add additional images of primary types of species described through 1900, and images of the shells of specimens whose genes and proteins have now been sequenced."

    25 July, 2005

      Conus victoriae venom component, alpha-conotoxin Vc1.1 (ACV1) enters Phase I clinical trials

      Two articles about our cone shell work have appeared in the local press.

      Pain-killer comes out of its shell
      The Age (subscription) - Melbourne,Victoria,Australia
      ... Elan's Prialt omega conotoxin now offers a superior alternative, but must be ... Injected beneath the skin, or into muscle, ACV1 quells chronic neuropathic pain ...

      Another article "Molecular miners find pain relief drugs from the sea" [With an increasing age demographic in our society the need for more effective pain suppressing compounds is a priority. ACV1 may fill this unmet need] This appeared in Yztimes ("YZage" web bulletin)

    4 July, 2005

      alpha-conotoxin discriminates between alpha9alpha10 and alpha7 nicotinic receptors

      McIntosh, J.M., Plazas, P.V., Watkins, M., Gomez-Casati, M.E,, Olivera, B.M. and Elgoyhen, A.B (2005). A novel alpha-conotoxin, PeIA, cloned from Conus pergrandis discriminates between rat alpha9alpha10 and alpha 7 nicotinic cholinergic receptors. J Biol Chem. 280: 30107-30112
      Departments of Psychiatry and Biology, University of Utah, Salt Lake City, UT 84112.

      Abstract: The alpha9 and alpha10 nicotinic cholinergic subunits assemble to form the receptor believed to mediate synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea, one of the few examples of postsynaptic function for a non-muscle nicotinic acetylcholine receptor (nAChR). However, it has been suggested that the expression profile of alpha9 and alpha10 overlaps with that of alpha7 in the cochlea and in sites like dorsal root ganglion neurons, peripheral blood lymphocytes, developing thymocytes and skin. We now report the cloning, total synthesis and characterization of a novel toxin alpha-conotoxin PeIA that discriminates between alpha9alpha10 and alpha7 nAChRs. This is the first toxin to be identified from Conus pergrandis, a species found in deep waters of the Western Pacific. alpha-Conotoxin PeIA displayed a 260-fold higher selectivity for alpha-bungarotoxin-sensitive alpha9alpha10 nAChRs compared to alpha-bungarotoxin-sensitive alpha7 receptors. The IC(50) of the toxin was 6.9 +/- 0.5 nM and 4.4 +/- 0.5 nM, for recombinant alpha9alpha10 and wild-type hair cell nAChRs, respectively. alpha-Conotoxin PeIA bears high resemblance to alpha-conotoxins MII and GIC isolated from Conus magus and Conus geographus, respectively. However, neither alpha-conotoxin MII nor alpha-conotoxin GIC at concentrations of 10 muM, blocked acetylcholine responses elicited in Xenopus oocytes injected with the alpha9 and alpha10 subunits. Among neuronal non-alpha-bungarotoxin sensitive receptors, alpha-conotoxin PeIA was also active at alpha3beta2 receptors and chimeric alpha6/alpha3beta2beta3 receptors. alpha-Conotoxin PeIA represents a novel probe to differentiate responses mediated either through alpha9alpha10 or alpha7 nAChRs in those tissues where both receptors are expressed.

      Are cone snail venoms of danger to fish ?

      Mebs, D. and Kauferstein, S. (2005) Ichthyotoxicity caused by marine cone snail venoms? Toxicon. 46: 355-356

      Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyallee 104, D-69596 Frankfurt am Main, Germany.

      Abstract: Ten venoms from marine cone snails were tested for ichthyotoxic effects on zebra fish (Brachydanio rerio) when added to the water. Only two venoms, from Conus capitaneus and Conus episcopatus, produced lethal effects at high concentrations (50-300mug/ml) within 20-90min. No sedative or hypnotic symptoms were observed. The experiments confirm that Conus venoms exert a quick and prompt activity only by parenteral injection into the prey as it is performed by the snail.

    24 June, 2005

      Human trials on alpha-conotoxin Vc1.1 (ACV1) from Conus victoriae

      Human trials for pain relief drugs from sea
      A cone snail toxin with great potential for easing pain, discovered by University of Melbourne scientists, could provide an improved treatment for neuropathic pain associated with diabetes.

      Dr. Livett with a selection of cone shells.

      See these links for further details.

      University of Melbourne: UniNews article

      ASX announcement from Metabolic Pharmaceuticals Please click on the hyperlink below: http://www.metabolic.com.au/?lo2mt&4z6U&1dtY&mBg

      Armishaw, C.J. and Alewood, P.F.(2005) Conotoxins as research tools and drug leads. Curr Protein Pept Sci. 6: 221-240.
      Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.

      Abstract: The complex mixture of biologically active peptides that constitute the venom of Conus species provides a rich source of ion channel neurotoxins. These peptides, commonly known as conotoxins, exhibit a high degree of selectivity and potency for different ion channels and their subtypes making them invaluable tools for unravelling the secrets of the nervous system. Furthermore, several conotoxin molecules have profound applications in drug discovery, with some examples currently undergoing clinical trials. Despite their relatively easy access by chemical synthesis, rapid access to libraries of conotoxin analogues for use in structure-activity relationship studies still poses a significant limitation. This is exacerbated in conotoxins containing multiple disulfide bonds, which often require synthetic strategies utilising several steps. This review will examine the structure and activity of some of the known classes of conotoxins and will highlight their potential as neuropharmacological tools and as drug leads. Some of the classical and morerecent approaches to the chemical synthesis of conotoxins, particularly with respect to the cont rolled formation of disulfide bonds will be discussed in detail. Finally, some examples of structure-activity relationship studies will be discussed, as well as some novel approaches for designing conotoxin analogues.

    23 June, 2005

      delta-conotoxin SVIE from Conus striatus: interaction with the voltage sensor of NaV channels

      *Leipold, E., *Hansel, A., **Olivera, B.M., ***Terlau, H. and *Heinemann, S.H. (2005) Molecular interaction of delta-conotoxins with voltage-gated sodium channels. FEBS Letters, 579 : 3881-3884.

      *Institute of Molecular Cell Biology, Research Unit Molecular and Cellular Biophysics, Friedrich Schiller University Jena, Drackendorfer Strasse 1, D-07747 Jena, Germany
      **Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA
      ***Max Planck Institute for Experimental Medicine, Research Unit Molecular and Cellular Neuropharmacology, Hermann-Rein-Strasse 3, D-37075 Göttingen, Germany.

      Abstract: Various neurotoxic peptides modulate voltage-gated sodium (NaV) channels and thereby affect cellular excitability. delta-Conotoxins from predatory cone snails slow down inactivation of NaV channels, but their interaction site and mechanism of channel modulation are unknown. Here, we show that delta-conotoxin SVIE from Conus striatus interacts with a conserved hydrophobic triad (YFV) in the domain-4 voltage sensor of NaV channels. This site overlaps with that of the scorpion alpha-toxin Lqh-2, but not with the alpha-like toxin Lqh-3 site. delta-SVIE functionally competes with Lqh-2, but exhibits strong cooperativity with Lqh-3, presumably by synergistically trapping the voltage sensor in its “on” position.
      Keywords: Conotoxin; Inactivation; Receptor site; Scorpion toxin; Sodium channel; Neurotoxin

    22 June, 2005

      Gla-containing conotoxins are post-translationally directed.

      Brown, M.A., Begley, G.S., Czerwiec, E., Stenberg, L.M., Jacobs, M., Kalume, D.E., Roepstorff, P., Stenflo, J., Furie, B.C. and Furie, B. (2005) Precursors of novel Gla-containing conotoxins contain a carboxy-terminal recognition site that directs gamma-carboxylation. Biochemistry 44: 9150-9159.

      Marine Biological Laboratory, Woods Hole, Massachusetts 02543, Center for Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark, and Department of Clinical Chemistry, Lund University, University Hospital, Malmo S-205 02, Sweden.

      Abstract: Vitamin K-dependent gamma-glutamyl carboxylase catalyzes the conversion of glutamyl residues to gamma-carboxyglutamate. Its substrates include vertebrate proteins involved in blood coagulation, bone mineralization, and signal transduction and invertebrate ion channel blockers known as conotoxins. Substrate recognition involves a recognition element, the gamma-carboxylation recognition site, typically located within a cleavable propeptide preceding the targeted glutamyl residues. We have purified two novel gamma-carboxyglutamate-containing conotoxins, Gla-TxX and Gla-TxXI, from the venom of Conus textile. Their cDNA-deduced precursors have a signal peptide but no apparent propeptide. Instead, they contain a C-terminal extension that directs gamma-carboxylation but is not found on the mature conotoxin. A synthetic 13-residue "postpeptide" from the Gla-TxXI precursor reduced the K(m) for the reaction of the Conus gamma-carboxylase with peptide substrates, including FLEEL and conantokin-G, by up to 440-fold, regardless of whether it was positioned at the N- or C-terminal end of the mature toxin. Comparison of the postpeptides to propeptides from other conotoxins suggested some common elements, and amino acid substitutions of these residues perturbed gamma-carboxylation of the Gla-TxXI peptide. The demonstration of a functional and transferable C-terminal postpeptide in these conotoxins indicates the presence of the gamma-carboxylation recognition site within the postpeptide and defines a novel precursor structure for vitamin K-dependent polypeptides. It also provides the first formal evidence to prove that gamma-carboxylation occurs as a post-translational rather than a cotranslational process.

    22 June, 2005

    21 June, 2005

      New Kunitz-type neurotoxin family from Conus striatus.

      Monika Bayrhuber and colleagues, from Max-Plank institute, Gottingen, Germany, describe a new 60-residue neurotoxin from the venom of Conus striatus that retains 2 out of the 3 disufide links typically found in these small, basic protein module which targets voltage-gated potassium channels.

      Bayrhuber, M., Vijayan, V., Ferber, M., Graf, R., Korukottu, J., Imperial, J., Garrett, J.E., Olivera, B.M., Terlau, H., Zweckstetter, M. and Becker, S. (2005) Conkunitzin-S1 Is the first member of a new Kunitz-type neurotoxin family: Structural and functional characterization. J. Biol. Chem. 280: 23766-23770
      Molecular and Cellular Neuropharmacology Group, Max-Planck-Institute for Experimental Medicine, Hermann-Rein-Strasse 3, 37075 Göttingen, Germany, the Department of Biology, University of Utah, Salt Lake City, Utah 84112, and Cognetix, Inc., Salt Lake City, Utah 84108

      Abstract: Conkunitzin-S1 (Conk-S1) is a 60-residue neurotoxin from the venom of the cone snail Conus striatus that interacts with voltage-gated potassium channels. Conk-S1 shares sequence homology with Kunitz-type proteins but contains only two out of the three highly conserved cysteine bridges, which are typically found in these small, basic protein modules. In this study the three-dimensional structure of Conk-S1 has been solved by multidimensional NMR spectroscopy. The solution structure of recombinant Conk-S1 shows that a Kunitz fold is present, even though one of the highly conserved disulfide cross-links is missing. Introduction of a third, homologous disulfide bond into Conk-S1 results in a functional toxin with similar affinity for Shaker potassium channels. The affinity of Conk-S1 can be enhanced by a pore mutation within the Shaker channel pore indicating an interaction of Conk-S1 with the vestibule of potassium channels.

    21 June, 2005

    20 June, 2005

      kM-conotoxin RIIIK - interaction with fish K+ channel.

      Al-Sabi, Ahmed (2004): Structural and functional studies of kM-conotoxin RIIIK interaction with Shaker-related potassium channels from trout fish (tSha 1). Dissertation: Bremen, November 2004.

    19 June, 2005

    16 June, 2005

      Conus pennaceus alpha-toxin binding to ACh-binding protein.

      Celie, P.H., Kasheverov, I.E., Mordvintsev, D.Y., Hogg, R.C., van Nierop, P., van Elk, R., van Rossum-Fikkert, S.E., Zhmak, M.N., Bertrand, D., Tsetlin, V., Sixma, T.K. and Smit, A.B. (2005) Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant. Nat Struct Mol Biol, 12: 582-588
      [1] Division of Molecular Carcinogenesis, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. [2] These authors contributed equally to this work.

      Abstract: Conotoxins (Ctx) form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. The subgroup alpha-Ctx specifically and selectively binds to subtypes of nicotinic acetylcholine receptors (nAChRs), which are targets for treatment of several neurological disorders. Here we present the structure at a resolution of 2.4 A of alpha-Ctx PnIA (A10L D14K), a potent blocker of the alpha(7)-nAChR, bound with high affinity to acetylcholine binding protein (AChBP), the prototype for the ligand-binding domains of the nAChR superfamily. alpha-Ctx is buried deep within the ligand-binding site and interacts with residues on both faces of adjacent subunits. The toxin itself does not change conformation, but displaces the C loop of AChBP and induces a rigid-body subunit movement. Knowledge of these contacts could facilitate the rational design of drug leads using the Ctx framework and may lead to compounds with increased receptor subtype selectivity.

    15 June, 2005

      Dr. Jon-Paul Bingham and Eric Chivian speak on Cone Shell Conservation

      John Roach for Pulse of the Planet reports in National Geographic News June 14, 2005, about "Toxic Snail Venoms Yielding New Painkillers, Drugs"
      Extract: In chronic pain? Don't be surprised if you find yourself at a corner pharmacy filling a prescription for synthetic snail venom sometime soon. Last December the U.S. Food and Drug Administration approved the first painkiller derived from a cocktail of potent chemicals produced by cone snails. The creatures, which are also known as cone shells, inhabit the world's dwindling coral reefs. There are more than 500 known cone snail species.

      Studying cone snail venom, researchers have derived other new treatments for pain, epilepsy, and incontinence. The drugs are in clinical and preclinical trials. Scientists say more experimental drugs are in development at research labs around the world. "They could have a cure to prostate cancer, a cure to AIDS. I have no idea," said Jon-Paul Bingham, a biochemist at Clarkson University in Potsdam, New York. Bingham is a leading researcher on cone snails. He said there are more than 500 cone snail species, each able to produce more than a hundred unique toxins. Each toxin is a potential new drug. Scientists have studied less than one percent of them.

      Bingham worries that "it's getting harder and harder to [collect] these snails." Cone snails are found primarily in coral reefs in warm, tropical waters. But as Eric Chivian, the founder and director of the Center for Health and the Global Environment at the Harvard Medical School in Cambridge, Massachusetts, noted, "We are destroying coral reefs." According to Chivian, some 26 percent of the world's reefs are damaged beyond repair, and another 30 to 50 percent are severely degraded. The proven potential of drugs derived from cone snail venom is the best example of the cost associated with the loss of the world's coral reefs, Chivian said. "I'm totally convinced cone snails as a group may have more potential for new medicines than any other genus in nature," Chivian said. (A genus is a group of closely related species.) "The number of different toxins they have developed over some 30 to 50 million years of evolution is unparalleled in nature."

      Toxin Factories

      In nature, cone snails use their venom to immobilize prey, such as fish, mollusks, and worms. Cone snails harpoon their prey with pointed tongues that are shaped like hypodermic needles. The snails then pump their prey's flesh full of toxins. "The poor animal, even if it gets away, [it] breaks off the harpoon. The likelihood of it being fatal is 100 percent, basically," Bingham said. "For any [human] who has been stung by a cone shell, the chance of mortality is 90 percent." The potency and complexity of the venom fascinates scientists. Over millions of years cone snails have evolved toxins that target specific species in specific environments. No one toxin is exactly like another. In addition, any cone snail can alter the compounds in its venom at will. Scientists are studying how each cone snail toxin affects its victim. The results are improving our understanding of how cells, such as those that make up the nervous system, interact and communicate. The research is also leading to the development of new drugs. "We are learning what nature has put in these animals and applying the strategy to other organisms," Bingham said. Consider, for example, the painkiller ziconotide, the cone-snail-venom-derived drug that was approved by the FDA in December.

      The painkiller (brand name: Prialt) is injected through a special pump into the fluid surrounding a patient's spinal cord. The drug blocks nerve channels that ordinarily transmit pain. Unlike opium-derived painkillers, such as morphine, ziconotide is said to pose a low risk of addiction or increased tolerance and has few side effects. "If you stop using the drug, the pain comes back. And no matter how long you've used [the drug], you don't have to increase the dosage to kill the pain," Bingham said. Given the potential of snail venom in the development of new drugs, Bingham said he is concerned that the rush to discover more promising toxins may contribute to the decline the world's cone snail species.

      Cone Snail Conservation

      Chivian, of Harvard Medical School, said the main threat to cone snails is the loss of coral reefs, which he attributes to bleaching (a type of slow death evident when multihued coral reefs turn a ghostly white) and disease outbreaks induced by global warming. Coastal development, pollution, and destructive fishing practices also negatively affect coral reefs. As for cone snails, their shells are collected and sold by the thousands at curio shops to tourists. Chivian said that additional collecting for biomedical research, if done irresponsibly, could further imperil the snails. But by far the greatest impact on the snails has been caused by the loss of coral reefs, Chivian said.

      Bingham, the Clarkson University researcher, is taking no chances. He advocates responsible use of cone snails, including minimal collection for research. To that end, Bingham and his colleagues are among the few scientists in the world who "milk" cone snails for venom. Milking involves a tricky—and potentially deadly—process: enticing a captive cone snail to harpoon a condom-covered test tube opening. (Other scientists typically retrieve venom by dissecting the venom glands of dead snails.) Bingham notes that milking reduces the number of cone snails he needs to take from the wild for research. The process also allows him to study how and why the snails change the compounds in their venom over time. Each change in venom compounds yields a new toxin with unknown potential. Once scientists identify the compounds in any given cone snail toxin, researchers can readily make it in synthetic form, Bingham added, noting that this eliminates the need to extract more venom from the snails.

      Chivian, meanwhile, said any measure to protect cone snails is appreciated. "We are losing species at a great rate without identifying them and without any knowledge of what value they may have for our health and our lives," he said.

    15 June, 2005

    14 June, 2005

      ACV1 (conotoxin Vc1.1 from Conus victoriae) to enter Phase I trials for diabetic neuropathy

      The University of Melbourne posted a Press Release on Friday 10 June about progress by Metabolic Pharmaceuticals Limited, Melbourne, Australia, who are developing ACV1 (conotoxin Vc1.1 from Conus victoriae) for clinical use and announced that it is to the stage where it is ready to enter Phase I clinical trials.

      Metabolic Pharmaceuticals Limited received an AusIndustry "Commercial Ready" Grant of AUD$449,902 to support a Phase 1 Clinical Trial of ACV1. This alpha-conotoxin has recently been shown to block ion transport through neuronal nicotinic receptors in excised segments of human sural nerve.

    ACV1 (conotoxin Vc1.1) to enter clinical trials for diabetic neuropathy

    Melbourne University researchers have found snails could be a pain cure for diabtes patients. Trials of a cone shell peptide from the cone snail (Conus victoriae) have been successful in preventing pain in animal models of human neuropathic pain. University researcher Assoc. Prof. Bruce Livett and colleagues at the Bio21 Molecular Science & Biotechnology Institute and at the National Ageing Research Institute (NARI), Parville, Victoria, Australia, have licensed Metabolic Pharmaceuticals Limited, Melbourne to develop ACV1 for clinical use. Metabolic have successfully completed the formal preclinical toxicity tests on ACV1 and are now ready to test ACV1 on humans. ACV1, the conopeptide alpha-conotoxin Vc1.1 from Conus victoriae, could be used to treat neuropathic pain related to diabetes (diabetic neuropathy). It could also be useful for treating other painful conditions such as the pain associated with multiple sclerosis, shingles and sciatica. Click for Press Release: Molecular miners find pain relier drugs from the sea. A cone snail toxin discovered by Melbourne researchers has proven to have great potential for easing pain and could provide an improved treatment for neuropathic pain associated with diabetes.

    6 June, 2005

      Toxins from Molluscivorous Cone Shells

      Corpuz, G.P., Jacobsen, R.B., Jimenez, E.C., Watkins M., Walker, C., Colledge, C., Garrett, J.E., McDougal, O., Li, W., Gray, W.R., Hillyard, D.R., Rivier, J., McIntosh, J.M., Cruz, L.J. and Olivera, B.M. (2005) Definition of the M-Conotoxin Superfamily: Characterization of novel peptides from molluscivorous Conus venoms. Biochemistry. 44: 8176-8186.
      Departments of Biology, Pathology, Chemistry, and Psychiatry, University of Utah, Salt Lake City, Utah 84112, Marine Science Institute, University of the Philippines, Diliman, Quezon City, Philippines, Department of Physical Sciences, College of Science, University of the Philippines Baguio, Baguio City, Philippines, The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California, and Cognetix, Inc., 421 Wakara Way, Salt Lake City, Utah 84108.

      Abstract: Most of the >50 000 different pharmacologically active peptides in Conus venoms belong to a small number of gene superfamilies. In this work, the M-conotoxin superfamily is defined using both biochemical and molecular criteria. Novel excitatory peptides purified from the venoms of the molluscivorous species Conus textile and Conus marmoreus all have a characteristic pattern of Cys residues previously found in the mu-, kappaM-, and psi-conotoxins (CC-C-C-CC). The new peptides are smaller (12-19 amino acids) than the mu-, kappaM-, and psi-conotoxins (22-24 amino acids). One peptide, mr3a, was chemically synthesized in a biologically active form. Analysis of the disulfide bridges of a natural peptide tx3c from C. textile and synthetic peptide mr3a from C. marmoreus showed a novel pattern of disulfide connectivity, different from that previously established for the mu- and psi-conotoxins. Thus, these peptides belong to a new group of structurally and pharmacologically distinct conotoxins that are particularly prominent in the venoms of mollusc-hunting Conus species. Analysis of cDNA clones encoding the novel peptides as well as those encoding mu-, kappaM-, and psi-conotoxins revealed highly conserved amino acid residues in the precursor sequences; this conservation in both amino acid sequence and in the Cys pattern defines a gene superfamily, designated the M-conotoxin superfamily. The peptides characterized can be provisionally assigned to four distinct groups within the M-superfamily based on sequence similarity within and divergence between each group. A notable feature of the superfamily is that two distinct structural frameworks have been generated by changing the disulfide connectivity on an otherwise conserved Cys pattern.

      Dutertre, S., Nicke, A., Lewis, R..J. (2005) beta 2 subunit contribution of 4/7 alpha -conotoxin binding to the nicotinic acetylcholine receptor. J Biol Chem. 280: 30460-30468.
      Institute for Molecular Bioscience, Brisbane, Queensland 4072.

      Abstract: The structures of AChBP and nAChR homology models have been used to interpret data from mutagenesis experiments at the nAChR. However, little is known about AChBP derived structures as predictive tools. Molecular surface analysis of nAChR models has revealed a conserved cleft as the likely binding site for the 4/7 a-conotoxins. Here, we used an a3ss2 model to identify ss2 subunit residues in this cleft and investigated their influence on the binding of a-conotoxins MII, PnIA and GID to the a3ss2 nAChR by two-electrode voltage-clamp analysis. While a ss2-L119Q mutation strongly reduced the affinity of all three a-conotoxins, ss2-F117A, ss2-V109A and ss2-V109G mutations selectively enhanced the binding of MII and GID. An increased activity of a-conotoxins GID and MII was also observed when the ss2-F117A mutant was combined with the a4 instead of the a3 subunit. Investigation of A10L-PnIA indicated that high affinity binding to ss2-F117A, ss2-V109A and ss2-V109G mutants was conferred by amino acids with a long side chain in position 10 (PnIA numbering). Docking simulations of 4/7 a-conotoxin binding to the a3ss2 model supported a direct interaction between mutated nAChR residues and a-conotoxin residues 6, 7 and 10. Taken together, these data provide evidence that the ss subunit contributes to a-conotoxin binding and selectivity, and demonstrate that a small cleft leading to the agonist binding site is targeted by a-conotoxins to block the nAChR.

      Nilsson, K.P., Lovelace, E.S., Caesar, C.E., Tynngard, N., Alewood, P.F., Johansson, H.M., Sharpe, I.A., Lewis, R.J., Daly, N.L. and Craik, D.J. (2005). Solution structure of chi-conopeptide MrIA, a modulator of the human norepinephrine transporter. Biopolymers 80 : 815-823.
      Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Australia.

      Abstract: The chi-conopeptides MrIA and MrIB are 13-residue peptides with two disulfide bonds that inhibit human and rat norepinephrine transporter systems and are of significant interest for the design of novel drugs involved in pain treatment. In the current study we have determined the solution structure of MrIA using NMR spectroscopy. The major element of secondary structure is a beta-hairpin with the two strands connected by an inverse gamma turn. The residues primarily involved in activity have previously been shown to be located in the turn region (Sharpe, I.A.; Palant, E.; Schroder, C.I.; Kaye, D.M.; Adams, D.J.; Alewood, P.F.; Lewis, R.J. J Biol Chem 2003, 278, 40317-40323), which appears to be more flexible than the beta-strands based on disorder in the ensemble of calculated structures. Analogues of MrIA with N-terminal truncations indicate that the N-terminal residues play a role in defining a stable conformation and the native disulfide connectivity. In particular, non-covalent interactions between Val3 and Hyp12 are likely to be involved in maintaining a stable conformation. The N-terminus also affects activity, as a single N-terminal deletion introduced additional pharmacology at rat vas deferens, while deleting the first two amino acids reduced chi-conopeptide potency. (c) 2005 Wiley Periodicals, Inc. Biopolymers (Peptide Science), 2005.

    31 May, 2005

      Selective preservation of {alpha}6* nicotinic ACh receptor-mediated function in an animal model of Parkinsons

      Sarah E McCallum[1] , Neeraja Parameswaran[1] , Tanuja Bordia[1] , J. Michael McIntosh[2], Sharon R. Grady[3], and Maryka Quik[1]* (2005) Decrease in {alpha}3*/{alpha}6* nicotinic receptors but not nicotine-evoked dopamine release in monkey brain after nigrostriatal damage. Mol. Pharmacol. 68 :737-746
      [1] The Parkinson's Institute; [2] University of Utah ;[3] Institute for Behavioral Genetics
      * Address correspondence to: E-mail: mquik@parkinsonsinstitute.org

      Abstract: Nicotinic receptors (nAChRs) are decreased in the striatum of Parkinson's disease (PD) patients or in experimental models following nigrostriatal damage. Since presynaptic nAChRs on striatal dopamine terminals mediate dopamine release, receptor loss may contribute to behavioral deficits in PD. The present experiments were done to determine whether nAChR function is affected by nigrostriatal damage in nonhuman primates as this model shares many features with PD. Initial characterization of nicotine-evoked [3H]dopamine release from monkey striatal synaptosomes revealed that release was calcium-dependent and inhibited by selective nAChR antagonists. Interestingly, a greater proportion (~ 70%) of release was inhibited by the {alpha}6* antagonist {alpha}-CtxMII) compared to rodents. Next, monkeys were lesioned with MPTP, and [3H]dopamine release, dopamine transporter and nAChRs measured. As anticipated, lesioning decreased the transporter and {alpha}6* nAChRs in caudate and putamen. In contrast, {alpha}6* nAChR-evoked [3H]dopamine release was reduced in caudate, but not putamen demonstrating a dissociation between nAChR sites and function. A different pattern was observed in the mesoolimbic dopamine system. Dopamine transporter levels were not reduced in nucleus accumbens after MPTP, as expected, however, there was a 50% decline in {alpha}6* nAChR sites with no decrease in {alpha}6* receptor-evoked dopamine release. No declines in {alpha}4*) binding or nicotine-evoked release were observed in any region. These results show a selective preservation in {alpha}6* nAChR-mediated function in the nigrostriatal and mesolimbic dopamine systems following nigrostriatal damage. Maintenance of function in putamen, a region with selective loss of dopaminergic terminals, may be important in PD.
      Key words: Dopamine, Nicotinic cholinergic, Receptor binding studies, Excitotoxicity, neurodegeneration

    31 May, 2005

      Beautiful Cone Shells on the Web

      Eduardo J. Pagobo known as Eddie from Punta Engaño Proper, Lapu-Lapu City, Cebu, Philippines has long been a developer on the web. Now he has outdone himself with many Outstanding and sharp photos of Beautiful shells. Among these are some 90 species of Conus. Well worth a visit.

    30 May, 2005

      alpha-Conotoxins: probes for nicotinic receptor. REVIEW

      Janes RW. (2005) alpha-Conotoxins as selective probes for nicotinic acetylcholine receptor subclasses. Curr Opin Pharmacol. 5: 280-292.
      School of Biological Sciences, Queen Mary, University of London, Mile End Road, London, E1 4NS, UK.

      Abstract: alpha-Conotoxins are selective antagonists of neuromuscular or neuronal nicotinic acetylcholine receptors. Individual family members are often highly selective towards distinct receptor subclasses, most notably within neuronal nicotinic acetylcholine receptors. As such they are being used as tools to probe for the type and diversity of receptor subclasses in distinct parts of the central and peripheral nervous systems. Many new alpha-conotoxins are being identified every year, broadening the available armoury because small variations in their sequences and structures often confer altered selectivity towards receptor subunits and subclasses. Many neurological diseases are being associated wholly or in part with functional changes within specific subclasses of nicotinic acetylcholine receptors. Significantly, with more structures of alpha-conotoxins also becoming available this enables ready comparison of their similarities and, more notably, of their subtle differences, which dictate subclass selectivity. As such, alpha-conotoxins offer the potential to become templates for the creation, through rational drug design strategies, of pharmaceuticals highly selective for specific subclasses of nicotinic acetylcholine receptors.

      alphaS-Conotoxin RVIII targets nicotinic receptors

      Teichert, R.W., Jimenez, E.C. and Olivera, B.M. (2005) alphaS-Conotoxin RVIIIA: A structurally unique conotoxin that broadly targets nicotinic acetylcholine receptors. Biochemistry. 44: 7897-7902.
      Department of Biology, University of Utah, Salt Lake City, Utah 84112, Department of Physical Sciences, College of Science, University of the Philippines Baguio, Baguio City, Philippines.

      Abstract: We report the purification and characterization of a new conotoxin from the venom of Conus radiatus. The peptide, alphaS-conotoxin RVIIIA (alphaS-RVIIIA), is biochemically unique with respect to its amino acid sequence, post-translational modification, and molecular targets. In comparison to other nicotinic antagonists from Conus venoms, alphaS-RVIIIA exhibits an unusually broad targeting specificity for nicotinic acetylcholine receptor (nAChR) subtypes, as assayed by electrophysiology. The toxin is paralytic to mice and fish, consistent with its nearly irreversible block of the neuromuscular nAChR. Similar to other antagonists of certain neuronal nAChRs, the toxin also elicits seizures in mice upon intracranial injection. The only previously characterized conotoxin from the S superfamily, sigma-conotoxin GVIIIA, is a specific competitive antagonist of the 5-HT(3) receptor; thus, alphaS-RVIIIA defines a novel family of nicotinic antagonists within the S superfamily. All previously characterized competitive conotoxin nAChR antagonists have been members of the A superfamily of conotoxins. Our working hypothesis is that the particular group of fish-hunting Conus species that includes Conus radiatus uses the alphaS-conotoxin family to target the muscle nAChR and paralyze prey.

    20 May, 2005

      Live Conus images

      Philippe Poppe & Guido announce the start of http://www.poppe-images.com. This site replaces the marine life section on http://www.conchology.be/, and will consist of two emails a week, similar to that sent by Conchology Inc., will also be applied by poppe-images for all registered members. Register on POPPEIMAGES&trade to receive the new mails. Presently their files contain more than 15,000 digital photos on the marine fauna of the Philippines. A little more than 2300 go online today and as photos are processed and documented they will be joined to the homepage at an average rate of 200 a week. A lot of technological and research work has been achieved during the last 6 months by their webmasters, graphics designers and data encoders. It is natural that the system will have some defects and that a lot of these can be corrected. We count on your collaboration to email us any possible changes. Thanks.

    17 May, 2005

      Fossil Conus

      The June 2005 Scientific American has images of shell beads from Blombos Cave in South Africa, dated 75,000 years ago. The reference is Francesco d’Errico, Christopher Henshilwood, Marian Vanhaeren and Karen van Niekerk, "Nassarius kraussianus Shell Beads from Blombos Cave: Evidence for Symbolic Behavior in the Middle Stone Age", in Journal of Human Evolution, 48: 3-24 January 2005.(Thanks to John W. Lancaster, PA, who posted this on CONCH-L).

    16 May, 2005

      Novel sodium channel blocking toxins from Conus striatus and Conus kinoshitai.

      Bulaj, G., West, P.J., Garrett, J.E., Marsh, M., Zhang, M.M., Norton, R.S., Smith, B.J., Yoshikami, D. and Olivera, B.M. (2005) Novel conotoxins from Conus striatus and Conus kinoshitai selectively block TTX-resistant sodium channels. Biochemistry 44: 7259-7265.
      Departments of Biology and Pathology, University of Utah, Salt Lake City, Utah 84112, Cognetix, Inc., 421 Wakara Way, Suite 201, Salt Lake City, Utah 84108, and The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville 3050, Australia.

      Abstract: The peptides isolated from venoms of predatory marine Conus snails ("conotoxins") are well-known to be highly potent and selective pharmacological agents for voltage-gated ion channels and receptors. We report the discovery of two novel TTX-resistant sodium channel blockers, mu-conotoxins SIIIA and KIIIA, from two species of cone snails. The two toxins were identified and characterized by combining molecular techniques and chemical synthesis. Both peptides inhibit TTX-resistant sodium currents in neurons of frog sympathetic and dorsal root ganglia but poorly block action potentials in frog skeletal muscle, which are mediated by TTX-sensitive sodium channels. The amino acid sequences in the C-terminal region of the two peptides and of the previously characterized mu-conotoxin SmIIIA (which also blocks TTX-resistant channels) are similar, but the three peptides differ in the length of their first N-terminal loop. We used molecular dynamics simulations to analyze how altering the number of residues in the first loop affects the overall structure of mu-conotoxins. Our results suggest that the naturally occurring truncations do not affect the conformation of the C-terminal loops. Taken together, structural and functional differences among mu-conotoxins SmIIIA, SIIIA, and KIIIA offer a unique insight into the "evolutionary engineering" of conotoxin activity.

      Dong H, Hu SJ.(2005) [The role of voltage-gated sodium channels in neuropathic pain] Sheng Li Ke Xue Jin Zhan.36(1):61-63. Chinese. No abstract available.

    11 May, 2005

      Conotoxin SO-3 inhibits N-type calcium channels.

      Wen L, Yang S, Qiao H, Liu Z, Zhou W, Zhang Y, Huang P. (2005) SO-3, a new O-superfamily conopeptide derived from Conus striatus, selectively inhibits N-type calcium currents in cultured hippocampal neurons. Br J Pharmacol 145: 728-739.
      1 Beijing Institute of Pharmacology and Toxicology, 27 Tai-Ping Road, Haidian District, Beijing 100850, China.

      Abstract: Whole-cell currents in cultured hippocampal neurons were recorded to investigate the effects of SO-3, a new O-superfamily conopeptide derived from Conus striatus, on voltage-sensitive channels.SO-3 had no effect on voltage-sensitive sodium currents, delayed rectifier potassium currents, and transient outward potassium currents.Similar to the selective N-type calcium channel blocker omega-conotoxin MVIIA (MVIIA), SO-3 could concentration-dependently inhibit the high voltage-activated (HVA) calcium currents (I(Ca)).MVIIA(3 muM), 10 muM nimodipine, and 0.5 muM omega-agatoxin IVA (Aga) could selectively block the N-, L-, and P/Q-type I(Ca), which contributed approximately 32, approximately 38, and approximately 21% of the HVA currents in hippocampal neurons, respectively. About 31% of the total HVA currents were inhibited by 3 muM SO-3. SO-3 (3 muM) and 3 muM MVIIA inhibited the overlapping components of HVA currents, whereas no overlapping component was inhibited by 3 muM SO-3 and 10 muM nimodipine, or by 3 muM SO-3 and 0.5 muM Aga. Also, 3 muM SO-3 had no effect on R-type currents.SO-3 had less inhibitory effects on non-N-type HVA currents than MVIIA at higher concentrations (30 and 100 muM).The inhibitory effects of SO-3 and MVIIA on HVA currents were almost fully reversible. However, the recovery from block by MVIIA was more rapid than recovery from block by SO-3.It is concluded that SO-3 is a new omega-conotoxin selectively targeting N-type voltage-sensitive calcium channels. Considering the significance of N-type calcium channels for pain transduction, SO-3 may have therapeutic potential as a novel analgesic agent.

      Xenome applies to export Conus tissues.

      Xenome Ltd. to export tissue samples from Conus species.

      The Department of the Environment and Heritage (DEH) received an application for approval of a Wildlife Trade Operation under Part 13A, section 303FN of the Environment Protection and Biodiversity Conservation Act 1999 (EPBC Act) to export tissue samples from Conus species collected from the Great Barrier Reef to the USA for DNA (cDNA) library construction. The completed cDNA libraries will then be returned to Xenome Ltd for analysis as part of Xenome Ltd's research and development program. Conus species are not listed as threatened species under the EPBC Act or listed on Appendix I of the Convention on International Trade in endangered species of Wild Fauna and Flora (CITES). It is proposed that should the operation be approved it would be in force for up to three (3) years from the date of declaration.

      In accordance with the provisions of secton 303FR of the Act, the public was invited to comment on this proposal (closing date 7 April)under the following headings:

      • Management and methodology
      • Ecological sustainability of the operation.

      For further information or to send comments after the date above contact The Director, Susatainable Wildlife Industries, Dept. of the Environment and Heritage, GPO Box 787, Canberra, ACT 2601. wsm@deh.gov.au

      Download Construction of Conoidea cDNA libraries

    9 May, 2005

      Use of alpha1B-antagonist conotoxin rho-TIA to distinguish alpha1-adrenergic receptor subtypes.

      Kamikihara SY, Mueller A, Lima V, Silva AR, Costa IB, Buratini Jr J, Pupo AS. (2005) Differential distribution of functional {alpha} 1-adrenergic receptor subtypes along the rat tail artery. J Pharmacol Exp Ther. 314: 753-761
      Depto de Farmacologia, Instituto de Biociencias - UNESP, Botucatu.

      Abstract:The rat tail artery has been used for the study of vasoconstriction mediated by alpha1A- adrenoceptors(ARs). However, rings from proximal segments of the tail artery (within the initial 4 cm, PRTA) were at least 3-fold more sensitive to methoxamine and phenylephrine (n=6 to 12; p<0.05) than rings from distal parts (between the 6(th) and 10(th) cm, DRTA). Interestingly, the imidazolines N-[5-(4,5-Dihydro-1H-imidazol-2-yl)-2- hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl] methanesulfonamide hydrobromide (A-61603) and oxymetazoline, which activate selectively alpha1A-ARs, were equipotent in PRTA and DRTA (n=4 to 12), while buspirone, which activates selectively alpha1D-AR, was approximately 70-fold more potent in PRTA than in DRTA (n=8; p<0.05). The selective alpha1D-AR antagonist 8-[2-[4- (methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5] decane-7,9-dione dihydrochloride (BMY-7378) was approximately 70-fold more potent against the contractions induced by phenylephrine in PRTA (pKB approximately 8.45, n=6) than in DRTA (pKB approximately 6.58, n=6), although the antagonism was complex in PRTA. 5- methylurapidil, a selective alpha1A- antagonist, was equipotent in PRTA and DRTA (pKB approximately 8.4), but the Schild slope in DRTA was 0.73+/-0.05 (n=5). The non-competitive alpha1B-antagonist conotoxin rho-TIA reduced the maximal contraction induced by phenylephrine in DRTA, but not in PRTA. These results indicate a predominant role for alpha1A-ARs in the contractions of both PRTA and DRTA, but with significant co-participations of alpha1D-ARs in PRTA and alpha1B-ARs in DRTA. Semi-quantitative RT-PCR revealed that mRNA encoding alpha1A- and alpha1B-ARs are similarly distributed in PRTA and DRTA while mRNA for alpha1D- ARs is twice more abundant in PRTA. Therefore, alpha1-ARs subtypes are differentially distributed along the tail artery. It is important to consider the segment from which the tissue preparation is taken to avoid misinterpretations on receptor mechanisms and drug selectivities.

    6 May, 2005

      Evolution of conotoxins - and more!

      Sollod, B.L., Wilson, D., Zhaxybayeva, O., Gogarten, J.P., Drinkwater, R., King, G.F. (2005) Were arachnids the first to use combinatorial peptide libraries? Peptides. 26: 131-139. Review.
      Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06032-3305, USA.

      Abstract: Spiders, scorpions, and cone snails are remarkable for the extent and diversity of gene-encoded peptide neurotoxins that are expressed in their venom glands. These toxins are produced in the form of structurally constrained combinatorial peptide libraries in which there is hypermutation of essentially all residues in the mature-toxin sequence with the exception of a handful of strictly conserved cysteines that direct the three-dimensional fold of the toxin. This gene-based combinatorial peptide library strategy appears to have been first implemented by arachnids almost 400 million years ago, long before cone snails evolved a similar mechanism for generating peptide diversity.

    24 April, 2005

      Conus peptides containing D-gamma-hydroxyvaline

      Pisarewicz, K., Mora, D., Pflueger, F.C., Fields, G.B. and Marí, F.(2005) Polypeptide Chains Containing D-gamma-Hydroxyvaline. J. Am. Chem. Soc., 127: 6207 - 6215.
      Department of Chemistry & Biochemistry and Center of Excellence in Biomedical & Marine Biotechnology, Florida Atlantic University, 777 Glades Road, Boca Raton, Florida 33431 Frank Mari.

      Abstract: Life has an unexplained and distinct L-homochirality. Proteins typically incorporate only L-amino acids into their sequences. In the present study, D-Val and D-gamma-hydroxyvaline (D-Hyv; V*) have been found within ribosomally expressed polypeptide chains. Four conopeptides were initially isolated, gld-V*/gld-V*' from the venom of Conus gladiator and mus-V*/mus-V*' from the venom of Conus mus. Their complete sequences (gld-V*/gld-V*' = Ala-Hyp-Ala-Asn-Ser-D-Hyv-Trp-Ser and mus-V*/mus-V*' = Ser-Hyp-Ala-Asn-Ser-D-Hyv-Trp-Ser) were determined by a combination of nano/pico-NMR and MS/MS methods. The amino acid triad that contains the gamma-hydroxylated residue, Ser-D-Hyv-Trp, is a novel structural motif that is stabilized by specific interactions between the D-amino acid and its neighboring L-counterparts. These interactions inhibit lactonization, a peptide backbone scission process that would normally be initiated by gamma-hydroxylated residues. Conopeptides possessing the Ser-D-Hyv-Trp motif have been termed gamma-hydroxyconophans. We have also isolated analogous conopeptides (gld-V and mus-V) containing D-Val instead of D-Hyv; these are termed conophans. gamma-Hydroxyconophans and conophans are particularly atypical because (i) they are not constrained as most conopeptides, (ii) they are extremely short in length, (iii) they have a high content of hydroxylated residues, and (iv) their sequences have no close match with other peptides in sequence databases. Their modifications appear to be part of a novel hyperhydroxylation mechanism found within the venom of cone snails that enhances neuronal targeting. The finding of D-Val and D-Hyv within this family of peptides suggests the existence of a corresponding D-stereospecific enzyme capable of D-Val oxidation.

      Mari, F. and Fields, G.B. (2003) Conopeptides: Unique pharmacological agents that challenge current peptide methodologies. CHIMICA OGGI Chemistry today, June 2003, 43-48. REVIEW
      Department of Chemistry & Biochemistry, Center of Excellence in Biomedical & Marine Biotechnology, Florida Atlantic University, 777 Glades Road, Boca Raton, Florida, 33431

      Abstract: Conopeptides provide an awe-inspiring combinatorial library of modified peptides that have been refined and perfected for 55 million years to exquisitely target a vast array of neuronal receptors, including human ones. While the original intention of these marvelous peptide engineers was secure their next meal, cone snails have provided us with novel therapeutic agents that we have just begun to explore. The vast conopeptide library will lead to many opportunities for discovery, which will in turn challenge current peptide technologies. While significant advances have been made in the past 30 years, these represent just the beginning as tens of thousands of conopeptides remained to be investigated.

    20 April, 2005

      Conus peptides as drug leads - MINI-REVIEW

      Wang CZ, Chi CW.(2004) Conus peptides - a rich pharmaceutical treasure. Acta Biochim Biophys Sin (Shanghai).36 :713-723. Full Text
      Institute of Biochemistry and Cell Biology, Shanghai Institute of Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China.

      Abstract: Marine predatory cone snails (genus Conus) with over 500 species represent what is arguably the largest single genus of marine animals alive today. All Conus are venomous and utilize a complex mixture of Conus peptides to capture their preys and for other biological purposes. Each component of Conus peptides selectively targets a specific subtype of ion channels, neurotransmitter receptors or transporters. Owing to their diversity, more than 50,000 distinct active peptides are theoretically estimated in Conus venoms. These diversified toxins are generally categorized into several superfamilies and/or families based on their characteristic arrangements of cysteine residues and pharmacological actions. Some mechanisms underlying the remarkable diversity of Conus peptides have been postulated: the distinctive gene structure, gene duplication and/or allelic selection, genus speciation, and sophisticated expression pattern and post-translational modification of these peptides. Due to their highly pharmacological potency and target selectivity, Conus peptides have attracted extensive attention with their potentials to be developed as new research tools in neuroscience field and as novel medications in clinic for pain, epilepsy and other neuropathic disorders. Several instructive lessons for our drug development could be also learnt from these neuropharmacological "expertises". Conus peptides comprise a rich resource for neuropharmacologists, and most of them await to be explored.

      More on Pain Management:
      Researchers grapple with the intricacy of pain perception at the peripheral level By Megan M. Stephan

      Signal transduction and the immune system. By Jill U. Adams

      Brain imaging works to capture the many natures of the pain experience. By Eugene Russo

      Courtesy of the John C. Liebeskind History of Pain Collection, History & Special Collections Division, Louise M. Darling Library, UCLA

    19 April, 2005

      Conus striatus venom reveals a novel toxin that targets potassium channels

      Monika Bayrhuber, Vinesh Vijayan, Michael Ferber, Roland Graf, Jegannath Korukottu, Julita Imperial, James E. Garrett, Baldomero M. Olivera, Heinrich Terlau, Markus Zweckstetter, and Stefan Becker (2005) Conkunitzin-S1 is the first member of a new Kunitz-type neurotoxin family - structural and functional characterization. J. Biol. Chem 280: 23766-23770 Corresponding Author: sabe@nmr.mpibpc.mpg.de
      NMR based Structural Biology, Max-Planck-Institute for Biophysical Chemistry, Göttingen 37077

      Abstract: Conkunitzin-S1 (Conk-S1) is a 60-residue neurotoxin from the venom of the cone snail Conus striatus that interacts with voltage gated potassium channels. Conk-S1 shares sequence homology with Kunitz type proteins but contains only two out of the three highly conserved cysteine bridges, which are typically found in these small, basic protein modules. In this study the three-dimensional structure of Conk-S1 has been solved by multidimensional NMR spectroscopy. The solution structure of recombinant Conk-S1 shows that a Kunitz fold is present, even though one of the highly conserved disulfide crosslinks is missing. Introduction of a third, homologous disulfide bond into Conk-S1 results in a functional toxin with similar affinity for Shaker K channels. The affinity of Conk-S1 can be enhanced by a pore mutation within the Shaker channel pore indicating an interaction of Conk-S1 with the vestibule of K channels.

      Involvement of N-type and P/Q-type calcium channels in pain response to peripheral neuropathy

      Urban,M.O., Ren,K., Sablad,M. and Park, K.T. (2005) Medullary N-type and P/Q-type calcium channels contribute to neuropathy-induced allodynia. Neuroreport, 16: 563-566.
      Department of Pharmacology, Merck Research Laboratories, San Diego, CA 92121, USA.

      Abstract:The present study was designed to determine the contribution of N-type, P/Q-type and L-type calcium channels in the rostral ventromedial medulla to tactile allodynia following peripheral nerve injury. L5/L6 spinal nerve ligation in rats produced tactile allodynia, which was dose-dependently inhibited by intrarostral ventromedial medulla microinjection of the N-type calcium channel antagonist omega-conotoxin MVIIA. Similarly, intrarostral ventromedial medulla microinjection of the P/Q-type calcium channel antagonist omega-agatoxin IVA inhibited spinal nerve ligation-induced tactile allodynia, whereas intrarostral ventromedial medulla microinjection of the L-type calcium channel antagonist nimodipine had no effect. These results demonstrate that N-type and P/Q-type calcium channels in the rostral ventromedial medulla contribute to tactile allodynia following peripheral neuropathy, likely via neurotransmitter-mediated activation of descending facilitatory systems from the rostral ventromedial medulla.

      The alpha6beta2* subunit combination of the neuronal nicotinic acetylcholine receptor has a unique pharmacology

      Salminen, O., Whiteaker, P., Grady, S.R., Collins, A.C., McIntosh, J.M. and Marks, M.J. (2005) The subunit composition and pharmacology of alpha-Conotoxin MII-binding nicotinic acetylcholine receptors studied by a novel membrane-binding assay. Neuropharmacology. 48:696-705.
      Institute for Behavioral Genetics, University of Colorado, Boulder, CO, USA.

      Abstract: The subunit composition and pharmacology of alpha-Conotoxin MII-binding (alpha-CtxMII) nicotinic acetylcholine receptors (nAChR) was studied by an improved [(125)I]-alpha-CtxMII membrane binding method. This binding method facilitates pharmacological studies that have been difficult to accomplish with [(125)I]-alpha-CtxMII autoradiography or alpha-CtxMII inhibition of [(125)I]-epibatidine binding. Binding densities and K(d)-values obtained by this [(125)I]-alpha-CtxMII membrane binding were similar to the values obtained by autoradiography or alpha-CtxMII inhibition of [(125)I]-epibatidine binding, verifying that each of these approaches measures the same nAChR population. Binding results with nAChR subunit-null mutant mice confirm and extend observations from earlier studies: [(125)I]-alpha-CtxMII binding measures two sets of alpha6beta2* nAChR (alpha4alpha6beta2beta3 or alpha6beta2beta3). Most nicotinic agonists and antagonists show monophasic inhibition of [(125)I]-alpha-CtxMII binding, indicating that alpha4alpha6beta2beta3 and alpha6beta2beta3 have similar binding properties. Comparison of the binding and activation profiles of alpha6beta2* nAChR to those of other nAChR subtypes (alpha4beta2* and beta4*) indicates that these receptors have distinctly different pharmacology indicating that it may be possible to target alpha6beta2* nAChR selectively to develop compounds that might be therapeutically useful.

    9 April, 2005

      Supplement on "Pain" in 'The Scientist'
      Secko, D. (2005) An Onus on Conus. The Scientist 19 (Supplement 1): 32 (28 March 2005)
      Abstract: This past December, the US Food and Drug Administration approved Prialt, Elan Corporation's synthetic version of a peptide from the venomous sea snail Conus magus. The drug, ziconotide (Prialt) is the first of its kind and appears to allevieate severe pain by blocking N-type Ca++-channels.At least seven additional compounds are in clinical or preclinical trials at the moment.Those featured in this brief but topical account include ziconotide (Prialt; omega-conotoxin MVIIA from Conus magus; ACV1, alpha-conotoxin Vc1.1 from Conus victoriae; AM336, omega-conotoxin CVID from Conus catus; and alpha-conotoxin BuIA, a novel peptide from Conus bullatus that shows preference for the beta2 over beta4 nAChR receptor subunit and can distinguish between different nAChR receptor subunit combinations containing these neuronal nicotinic receptors.),

      This Supplement contains many diverse and interesting articles on Pain, including:
      Nicotine to Snuff Out Pain,
      Attuning to a live in Pain - How a chronic syndrome altered a life and shaped a mission - by Laura M. Hraster.
      - and many other interesting articles.

      kappaM-conotoxin RIIIK binds to the K+ channel via a basic ring
      Verdier, L., Al-Sabi, A., Rivier, J.E., Olivera, B.M., Terlau, H. and Carlomagno, T. (2005) Identification of a novel pharmacophore for peptide toxins interacting with K+ channels. J Biol Chem. 280: 21246-21255
      NMR Based Structural Biology, Max Planck Institute for Biophysical Chemistry, Gottingen 37077.

      Abstract: kappaM-Conotoxin RIIIK blocks TSha1 K+ channels from trout with high affinity by interacting with the ion channel pore. As opposed to many other peptides targeting K+ channels, kappaM-RIIIK does not possess a functional dyad. In this study we combine thermodynamic mutant cycle analysis and docking calculations to derive the binding mode of kappaM-conotoxin RIIIK to the TSha1 channel. The final model reveals a novel pharmacophore, where no positively charged side chain occludes the channel pore. Instead the positive charged residues of the toxin form a basic ring: kappaM-RIIIK is anchored to the K+ channel via electrostatic interactions of this basic ring with the loop and pore helix residues of the channel. The channel amino acid E354 is likely to be a fundamental determinant of the selectivity of kappaM-RIIIK for the TSha1 channel. The C-OH of O15 is in contact with the carbonyls of the selectivity filter, disturbing the charge distribution pattern necessary for the coordination of K+ ions. This novel, experimentally-based pharmacophore model proves the existence of diverse binding modes of peptidic toxins to K+ channels and underlines the role of intermolecular electrostatic interactions involving channel loop side chains in determining the selectivity of toxins for specific K+ channel types.

      The "Agonist Paradox" for acetylcholine and mu-opioid receptors

      Kukhovich, F.S., Darkhovskii, M.B., Gorbatova, E.N. and Polyakov, V.S. (2005) The Agonist Paradox: Agonists and Antagonists of Acetylcholine Receptors and Opioid Receptors. Chemistry & Biodiversity 2: 354-366.
      Scientific Research Institute of Organic Chemistry and Technology, Shosse Entuziastiov 23, Moscow, 111024, Russia (fax: +7-095-273 3440, +7-095-273 2218) email: Michael B. Darkhovskii.

      Abstract: In contrast to antagonists, agonists tend to induce considerable conformational changes in their receptors, resulting in opening of ion channels, either directly or via secondary messengers. These conformational transformations require great energy expenses. However, the experimentally determined free energies of complexation between agonists and receptors are often relatively smaller than those for the corresponding antagonists. To rationalize this so-called "agonist paradox", which has not been clarified in the literature, we have developed an alternative model. Our model may help to discriminate between agonists and antagonists of the acetylcholine (ACh) and mu-opioid receptors. For this purpose, a series of ligands (1-18) have been analyzed both in structural terms and with respect to complexation geometry within the anionic binding sites of these two receptor types.

    6 April, 2005

      Molecular Prospecting for Drugs from the Sea
      Gayler, K., Sandall, D., Greening, D., Keays, D., Polidano, M., Livett B., Down J., Satkunanathan N. and Khalil, Z. (2005) Molecular Prospecting for Drugs from the Sea. IEEE Engineering in Medicine and Biology Magazine. 24 (2) 80-84.
      Department of Biochemistry and Molecular Biology, University of Melbourne, Bio21 Molecular Science & Biotechnology Institute, 30 Flemington Rd., Parkville, 3010 Australia.

      In this article, the Melbourne-based researchers describe why Conus are valuable animals for medical research and the alternative approaches they have taken to realize the therapeutic potential of their venom.

    31 March, 2005

      Sequence information determines folding of contoxins
      Fuller, E., Green, B.R., Catlin, P., Buczek, O., Nielsen, J.S., Olivera, B.M. and Bulaj, G. (2005) Oxidative folding of conotoxins sharing an identical disulfide bridging framework. FEBS J. 272 : 1727-38.
      Cognetix Inc, Salt Lake City, UT, USA.

      Abstract: Conotoxins are short, disulfide-rich peptide neurotoxins produced in the venom of predatory marine cone snails. It is generally accepted that an estimated 100 000 unique conotoxins fall into only a handful of structural groups, based on their disulfide bridging frameworks. This unique molecular diversity poses a protein folding problem of relationships between hypervariability of amino acid sequences and mechanism(s) of oxidative folding. In this study, we present a comparative analysis of the folding properties of four conotoxins sharing an identical pattern of cysteine residues forming three disulfide bridges, but otherwise differing significantly in their primary amino acid sequence. Oxidative folding properties of M-superfamily conotoxins GIIIA, PIIIA, SmIIIA and RIIIK varied with respect to kinetics and thermodynamics. Based on rates for establishing the steady-state distribution of the folding species, two distinct folding mechanisms could be distinguished: first, rapid-collapse folding characterized by very fast, but low-yield accumulation of the correctly folded form; and second, slow-rearrangement folding resulting in higher accumulation of the properly folded form via the reshuffling of disulfide bonds within folding intermediates. Effects of changing the folding conditions indicated that the rapid-collapse and the slow-rearrangement mechanisms were mainly determined by either repulsive electrostatic or productive noncovalent interactions, respectively. The differences in folding kinetics for these two mechanisms were minimized in the presence of protein disulfide isomerase. Taken together, folding properties of conotoxins from the M-superfamily presented in this work and from the O-superfamily published previously suggest that conotoxin sequence diversity is also reflected in their folding properties, and that sequence information rather than a cysteine pattern determines the in vitro folding mechanisms of conotoxins.

    29 March, 2005

      Frequently Asked Questions
      Updated the Frequently Asked Questions in the More Info section of this web page, in response to more questions. Thanks to those of you who asked. Some of the answers even amazed me !

    28 March, 2005

    27 March, 2005

    26 March, 2005

      Prialt, (Ziconotide, SNX111, omega-conotoxin MVIIA) and other conopeptides under development for treament of chronic pain
      Stix, G. (2005) A Toxin against Pain Scientific American. (April 2005), 70-75.

      Abstract: The past 18 months were very good ones for hypnotists, yoga teachers and acupuncturists. For many chronic pain sufferers, promises of relief from various forms of alternative medicine seemed like rational options amid the unending stream of negative reports about Vioxx, Celebrex, Aleve and Rush Limbaugh's addiction to painkillers.

      Not all was lost for patients who prefer medicine to meditation. With little fanfare, the Food and Drug Administration approved in late December two new drugs intended to treat a form of pain that often proves resistant to anti-inflammatories and opiates--the two predominant classes of pharmaceuticals for analgesia. Medical specialists welcomed their arrival. "It's an embarrassment that we're treating pain with opiates and aspirinlike compounds," notes Edwin McCleskey of the Oregon Health and Sciences University. "Opiates are more than 2,000 years old, and aspirin is nearly 200 years old."

      Further reading:

    22 March, 2005

      Use of conotoxins as analgesics
      Radbruch, L. and Elsner, F.(2005) Emerging analgesics in cancer pain management. Expert Opin Emerg Drugs. 10: 151-171.
      University of Aachen, Department of Palliative Medicine, Pauwelsstrasse 30, 52074 Aachen, Germany.

      Abstract: Cancer pain is one of the most frequent symptoms in malignant disease, severely impairing the patients' quality of life. The recommendations of the World Health Organization will provide adequate pain relief for the vast majority of cancer patients. However, some patients will suffer from inadequate analgesia or intolerable side effects. Cyclooxygenase-2 (COX-2)-selective non-steroidal anti-inflammatory drugs (NSAIDs), new anticonvulsants, cannabinoids and NMDA receptor antagonists are being developed for these patients. NSAIDs with nitric oxide-releasing moieties are an interesting addition, as this new class of analgesics combines improved analgesic efficacy with higher tolerability. Conotoxins and other drugs such as nicotinic acetylcholinergic receptor agonists will be advantageous only for a few patients in the near future, as side-effect profile and risk of complications, as well as the burden on the patient, often are not worth the additional analgesic benefit.

    18 March, 2005

      Conotoxin Vc1.1 from Conus victoriae inhibits human sensory nerve activation
      Lang PM, Burgstahler R, Haberberger RV, Sippel W, Grafe P. (2005) A conus peptide blocks nicotinic receptors of unmyelinated axons in human nerves. Neuroreport. 16: 479-483.
      (1)Departments of Anesthesiology, (2) Physiology, Ludwig-Maximilians University, 80336 Munich, Germany, (3) Institute for Anatomy and Cell Biology, Justus-Liebig-University, 35385 Giessen, Germany.

      Abstract: The novel alpha-conotoxin Vc1.1 is a potential analgesic for the treatment of painful neuropathic conditions. In the present study, the effects of Vc1.1 were tested on the nicotine-induced increase in excitability of unmyelinated C-fiber axons in isolated segments of peripheral human nerves. Vc1.1 in concentrations above 0.1 muM antagonized the increase in axonal excitability produced by nicotine; the maximal inhibition was observed with 10 muM. We also demonstrate immunoreactivity for alpha3 and alpha5 subunits of neuronal nicotinic receptors on unmyelinated peripheral human axons. Blockade of nicotinic receptors on unmyelinated peripheral nerve fibers may be helpful in painful neuropathies affecting unmyelinated sympathetic and/or sensory axons.

    17 March, 2005

      The ctl-2 gene in a multicapsid nucleopolyhedrovirus found in a poplar tree moth lavae, encodes a conotoxin-like protein.
      Jakubowska A, van Oers MM, Cory JS, Ziemnicka J, Vlak JM. (2005) European Leucoma salicis NPV is closely related to North American Orgyia pseudotsugata MNPV. J Invertebr Pathol. 2005 Feb;88(2):100-107.
      Laboratory of Virology, Wageningen University, Binnenhaven 11, Wageningen 6709 PD, The Netherlands; Department of Biological Control and Quarantine, Institute of Plant Protection, Miczurina 20, Poznan 60-318, Poland.

      Abstract: The satin moth Leucoma salicis L. (Lepidoptera, Lymantriidae) is a frequent defoliator of poplar trees (Populus spp.) in Europe and Asia (China, Japan). Around 1920 the insect was introduced into the USA and Canada. In this paper, a multicapsid nucleopolyhedrovirus isolated from L. salicis larvae in Poland (LesaNPV) was characterized and appeared to be a variant of Orgyia pseudotsugata (Op) MNPV. O. pseudotsugata, the Douglas fir tussock moth (Lepidoptera, Lymantriidae), occurs exclusively in North America. Sequences of three conserved baculovirus genes, polyhedrin, lef-8, and pif-2, were amplified in polymerase chain reactions using degenerate primer sets, and revealed a high degree of homology to OpMNPV. Restriction enzyme analysis confirmed the close relationship between LesaNPV and OpMNPV, although a number of restriction fragment length polymorphisms were observed. The lef-7 gene, encoding late expression factor 7, and the ctl-2 gene, encoding a conotoxin-like protein, were chosen as putative molecular determinants of the respective viruses. The ctl-2 region appeared suitable for unequivocal identification of either virus as LesaNPV lacked a dUTPase gene in this region. Our observations may suggest that LesaNPV, along with L. salicis, was introduced into O. pseudotsugata after introduction of the former insect into North America in the 1920s.

    9 March, 2005

    8 March, 2005

      Radiation of Cape Verde Conus
      Duda, T.F. Jr, Rolan, E. (2005) Explosive radiation of Cape Verde Conus, a marine species flock. Mol Ecol.14:267-272.
      Naos Marine Laboratory, Smithsonian Tropical Research Institute, Apartado 2072, Balboa Ancon, Republic of Panama. tfduda@umich.edu

      Abstract: Nearly 50 species of the marine gastropod genus Conus are restricted to the Cape Verde archipelago. This unusual concentration of endemics within a single set of oceanic islands is extremely uncharacteristic of marine taxa. Here we used phylogenetic analyses of 90 Conus species, including 30 endemics from Cape Verde, to reveal the relationships and origins of the endemic Cape Verde Conus. Results show that these species group in two distinct clades and represent a marine species flock that is restricted to a very narrowly confined geographical area. Species' originations occurred in exceptionally limited parts of the archipelago and in some cases radiations took place solely within single islands. Finally, comparison of levels of divergence between Cape Verde endemics and other Conus species suggests that the radiation of Conus in Cape Verde occurred during the last few million years.

      Conus clades and their migrations
      Duda, T.F. Jr, Kohn, A.J. (2005) Species-level phylogeography and evolutionary history of the hyperdiverse marine gastropod genus Conus. Mol Phylogenet Evol. 34: 257-272.
      Naos Marine Laboratory, Smithsonian Tropical Research Institute, Apartado 2072, Balboa, Ancon, Panama. tfduda@umich.edu

      Abstract: Phylogenetic and paleontological analyses are combined to reveal patterns of species origination and divergence and to define the significance of potential and actual barriers to dispersal in Conus, a species-rich genus of predatory gastropods distributed throughout the world's tropical oceans. Species-level phylogenetic hypotheses are based on nucleotide sequences from the nuclear calmodulin and mitochondrial 16S rRNA genes of 138 Conus species from the Indo-Pacific, eastern Pacific, and Atlantic Ocean regions. Results indicate that extant species descend from two major lineages that diverged at least 33 mya. Their geographic distributions suggest that one clade originated in the Indo-Pacific and the other in the eastern Pacific + western Atlantic. Impediments to dispersal between the western Atlantic and Indian Oceans and the central and eastern Pacific Ocean may have promoted this early separation of Indo-Pacific and eastern Pacific + western Atlantic lineages of Conus. However, because both clades contain both Indo-Pacific and eastern Pacific + western Atlantic species, migrations must have occurred between these regions; at least four migration events took place between regions at different times. In at least three cases, incursions between regions appear to have crossed the East Pacific Barrier. The paleontological record illustrates that distinct sets of Conus species inhabited the Indo-Pacific, eastern Pacific + western Atlantic, and eastern Atlantic + former Tethys Realm in the Tertiary, as is the case today. The ranges of <1% of fossil species (N=841) spanned more than one of these regions throughout the evolutionary history of this group.

      Gene deletion effects on alpha6 nicotinic receptor
      Gotti,C.*(1), Moretti, M.(1), Clementi, F. (1), Riganti, L.(1), McIntosh, JM.(2), Collins, A.C.(3), Marks, M.J.(3) and Whiteaker, P.(3) (2005) Expression of nigrostriatal {alpha}6-containing nicotinic acetylcholine receptors is selectively reduced, but not eliminated, by {beta}3 subunit gene deletion. Mol. Pharmacol. 67: 2007-2015.
      (1) CNR, Institute of Neuroscience, Milan, Italy. (2) University of Utah (3) University of Colorado, USA.
      * Address correspondence to: E-mail: c.gotti@in.cnr.it

      Abstract: mRNAs for the neuronal nicotinic receptor (nAChR) {alpha}6 and {beta}3 subunits are abundantly expressed and colocalized in dopaminergic cells of the substantia nigra and ventral tegmental area. Studies using subunit-null mutant mice have shown that {alpha}6- or {beta}3-dependent nAChRs bind alpha-conotoxin MII (alpha-CtxMII) with high affinity and modulate striatal dopamine release. This study explores the effects of {beta}3 subunit-null mutation on striatal and midbrain nAChR expression, composition, and pharmacology. Ligand binding and immunoprecipitation experiments using subunit-specific antibodies indicated that {beta}3-null mutation selectively reduced striatal alpha* nAChR expression by 76% vs. beta3+/+ control. Parallel experiments showed a smaller reduction in both midbrain {beta}3* and {alpha}6* nAChRs (34% and 42% vs. {beta}3+/+ control, respectively). Sedimentation coefficient determinations indicated that residual {alpha}6* nAChRs in {beta}3-/- striatum were pentameric, like their wild-type counterparts. Immunoprecipitation experiments on immunopurified {beta}3* nAChRs demonstrated that almost all wild-type striatal {beta}3* nAChRs also contain {alpha4, {alpha}6, and {beta}2 subunits, although a small population of non-{beta}3 {alpha}6* nAChRs is also expressed. {beta}3 subunit incorporation appeared to increase {alpha}4 participation in {alpha}6{beta}2* complexes. 125I-Epibatidine competition binding studies showed that the alpha-CtxMII affinity of {alpha}6* nAChRs from the striata of {beta}-/- mice was similar to those isolated from {beta}3+/+ animals. Taken together, the results of these experiments show that the {beta}3 subunit is important for the correct assembly, stability and/or transport of {alpha}6* nAChRs in dopaminergic neurons, and influences their subunit composition. However, {beta}3 subunit expression is not essential for the expression of {alpha}6*, high affinity alpha-CtxMII-binding nAChRs.

    4 March, 2005

      Venom peptides as potential drug leads
      Oren Bogin (2005) Venom peptides and their mimetics as potential drugs.Modulator, Issue 9, No. 19. Spring 2005, pp. 14-20.
      Recombinant Protein Group, Alomone Labs Ltd., Harhotzvim Hi-Tech Park, PO Box 4287, Jerusalem 91042, Israel

      Abstract:Venomous creatures have a sophisticated mechanism for prey capture which includes a vast array of biologically-active compounds, such as enzymes, proteins, peptides and small molecular weight compounds. These substances target an immense number of receptors and membrane proteins with high affinity, selectivity and potency, and can serve as potential drugs or scaffolds for drug design.

      Ion Channels in Pain Sensation
      Ofra Gohar (2005) Contribution of Ion Channels in Pain Sensation.Modulator, Issue 9, No. 19. Spring 2005, pp. 9-13.
      Antibody Group, Alomone Labs Ltd., Harhotzvim Hi-Tech Park, PO Box 4287, Jerusalem 91042, Israel

      Abstract:Many ion channels are located at the nociceptor peripheral terminal, affecting neuron excitability after injury and as a result affecting pain sensation.11 Voltage gated Na+ and Ca2+ channels, TRP, ASIC, ligand gated ion channels, P2X, NMDA, AMPA and Kainate receptors are some of the ion channels involved in the pathogenesis of pain,10 several of which will be reviewed here.

    3 March, 2005

      Conus marmoreus T-superfamily conotoxins
      Han YH, Wang Q, Jiang H, Miao XW, Chen JS, Chi CW. (2005) Sequence diversity of T-superfamily conotoxins from Conus marmoreus. Toxicon. 45: 481-487.
      Institute of Biochemistry and Cell Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 200031, China; Institute of Protein Research, TongJi University, Shanghai 200092, China.

      Abstract: Remarkable sequence diversity of T-superfamily conotoxins was found in a mollusk-hunting cone snail Conus marmoreus. The sequence of mr5a purified from the snail venom was determined, while six other sequences of Mr5.1a, Mr5.1b, Mr5.2, Mr5.3, Mr5.4a, and Mr5.4b were deduced from their corresponding cDNA cloned by RACE approach. mr5a of 10 amino acid residues is one of the shortest T-superfamily conotoxins ever found. They all share a typical (-CC-CC-) Cys pattern, a conserved signal peptide and a long 3'-untranslated region. A consensus Glu residue is preceded by the second two adjacent cysteines in all these toxins except in mr5a, whereas Mr5.1a, Mr5.1b, Mr5.4a and Mr5.4b are abundant in Trp residues. The identification of these highly divergent T-superfamily conotoxins will facilitate the understanding the relationship of their structure and function.

    28 February, 2005

      Prialt - Clinical Trial currently recruiting
      Ziconotide (Prialt) Effectiveness and Safety Trial in Patients with Chronic Severe Pain
      This study is currently recruiting patients.

      The purpose of this open-label study is to give chronic severe pain patients with existing intrathecal pump systems access to ziconotide. Study start is February 2004. Ziconotide/Prialt™ is the synthetic form of conotoxin MVIIA, a potent analgesic derived from the venom of the cone snail Conus magus.

    • For eligibility, location and contact information click here and click on box by "recruiting" and then click on "Display Selected Studies".

    27 February, 2005

    22 February, 2005

      European Commission grants Elan permission to market PRIALT (synthetic conotoxin MVIIA from Conus magus)

      Elan Receives Approval from the European Commission to Market PRIALT -100 Micrograms/ML Solution for Infusion- for Severe Chronic Pain
      DUBLIN, Ireland--(BUSINESS WIRE)--Feb. 22, 2005--
      New Non-Narcotic Treatment Based on Marine Snail Peptide Blocks Pain Signals; Approval Based on Results From Three Pivotal Studies

      Elan Corporation, plc today announced that the European Commission (EC) has granted marketing approval for PRIALT(TM) (ziconotide) for the treatment of severe, chronic pain in patients who require intrathecal (IT) analgesia.

      Abstract:"The EC approval of PRIALT, along with the US approval in December 2004, signal the successful completion of years of research, development, and commitment on the part of Elan to bring this innovative therapy to market," said Lars Ekman, MD, PhD, executive vice president and president, Research and Development, Elan. "PRIALT can offer new hope for patients in Europe as the first new IT analgesic approved in more than two decades. We are deeply gratified today that physicians and patients now have a significant option for the treatment of severe chronic pain, an area in which there is tremendous unmet medical need."
      This approval follows a positive opinion in November 2004 by the European Committee for Medicinal Products for Human Use (CHMP), the human medicines scientific body of the European Medicines Agency. Marketing approval of PRIALT was based on the treatment of more than 1,000 patients, including three pivotal clinical studies, which evaluated the efficacy and safety of IT PRIALT in patients with severe chronic pain that was not adequately managed despite a regimen of systemic and/or IT analgesics.

      • PRIALT has been awarded orphan drug status in the European Union, which designates it as a product used for the diagnosis, prevention or treatment of life-threatening or very serious rare disorders or conditions.
      • PRIALT, developed by scientists at Elan, is in a class of non-opioid analgesics known as N-type calcium channel blockers. PRIALT is the synthetic equivalent of a naturally occurring conopeptide found in a marine snail known as Conus magus. Research suggests that the mechanism of action of PRIALT works by targeting and blocking N-type calcium channels on nerves that transmit pain signals.
      • "PRIALT is the first new IT analgesic for severe chronic pain in many years and in many cases represents an effective alternative to currently available opioids, with no evidence of the development of tolerance to treatment over time, drug addiction or respiratory depression," said Ann Ver Donck, MD, one of the clinical investigators for PRIALT and pain therapy specialist, Multidisciplinary Pain Centre, Brugge, Belgium. "Patients and physicians alike should be encouraged by the body of research supporting PRIALT, which now includes one of the largest and most comprehensive safety databases available for any IT therapy."
      • The approval of PRIALT was based on three independent pivotal studies, each of which demonstrated significant improvement on the Visual Analog Scale of Pain Intensity, a well-accepted pain outcome measure.

      Results of the PRIALT Pivotal Studies
      The results of the PRIALT pivotal studies were previously announced upon U.S. Food and Drug Administration approval of PRIALT. The data from the most recent Phase III study will be presented this week at the American Academy of Pain Medicine meeting in Palm Springs, California. In two other Phase III clinical studies, IT-administered PRIALT was found to significantly reduce severe chronic pain in a variety of opioid-resistant patient populations with neuropathic pain and pain related to cancer and AIDS. The results of the first fast-titration study were published last year in the Journal of the American Medical Association (January 7, 2004, Vol. 291, No. 1). The results of the second fast-titration study are currently in preparation for publication.

      PRIALT Safety
      PRIALT has been evaluated as an IT infusion in more that 1,000 patients participating in chronic pain studies. The longest treatment duration to date has been more than six years.
      The four most commonly reported adverse drug reactions (ADRs) in long-term clinical studies were dizziness, nausea, nystagmus and confusion. Most ADRs were mild to moderate in severity and resolved over time.

      • Information about PRIALT is available through a toll-free number in EUROPE, 00-800-2683-4736.

      About Severe Chronic Pain
      Severe chronic pain is defined as pain lasting longer than six months and has multiple causes, such as failed back surgery, injury, accident, cancer, AIDS, and other nervous system disorders.

    15 February, 2005

      Disulfide connectivity of alpha-conotoxin SII from Conus striatus

      Bingham, J.P., Broxton, N.M., Livett, B.G., Down, J.G., Jones, A. and Moczydlowski, E.G. (2005) Optimizing the connectivity in disulfide-rich peptides: alpha-conotoxin SII as a case study. Anal Biochem. 338: 48-61.
      Department of Biology, Clarkson University, Potsdam, NY 13699, USA; Department of Biochemistry and Molecular Biology, University of Melbourne, Victoria, 3010, Australia, and the Centre for Drug Design and Development, University of Queensland, St. Lucia, Qld. 4072, Australia.

      Abstract: We describe a strategy for the efficient, unambiguous assignment of disulfide connectivities in alpha-conotoxin SII, of which approximately 30% of its mass is cysteine, as an example of a generalizable technique for investigation of cysteine-rich peptides. alpha-Conotoxin SII was shown to possess 3-8, 2-18, and 4-14 disulfide bond connectivity. Sequential disulfide bond connectivity analysis was performed by partial reduction with Tris(2-carboxyethyl)phosphine and real-time mass monitoring by direct-infusion electrospray mass spectrometry (ESMS). This method achieved high yields of the differentially reduced disulfide bonded intermediates and economic use of reduced peptide. Intermediates were alkylated with either N-phenylmaleimide or 4-vinylpyridine. The resulting alkyl products were assigned by ESMS and their alkyl positions sequentially identified via conventional Edman degradation. The methodology described allows a more efficient, rapid, and reliable assignment of disulfide bond connectivity in synthetic and native cysteine-rich peptides.

      Current Medicinal Chemistry: Special Focus Issue

      Jones, RM and Semple, G. (editors) (2004) "New Clinical Applications for Naturally Occuring Peptide Toxins" Current Medicinal Chemistry 11 (23), Dec. 2004 / Arena Pharm. San Diego USA

      Preface: We are proud to present readers with this thematic issue of Current Medicinal Chemistry devoted to monographs discussing frontier research and development of natural peptide toxins. These manuscripts are authored by leading scientists in the field and their content truly epitomizes the current status of peptide toxin research at therapeutically relevant targets. Where relevant, particular emphasis has been placed on the potential for pharmaceutical development.
      Alongside the huge molecular and functional diversity of ion channels, a no less impressive structural diversity of animal toxins have been revealed through the discovery of an increasing number of distinct polypeptide folds. Indeed, it appears such peptides have evolved distinct architectural motifs to adapt to different ion channel modulating strategies. Toxins acting at ion channels are principally highly potent, compact peptides that have been isolated from a wide variety of unrelated species including the platypus, scorpions, snakes, sea anemones, jellyfish, marine cone snails, worms and bacteria. The means by which various peptide toxins bind to their respective targets have been mapped topologically, revealing two generally accepted molecular signatures. The first is a generic conserved scaffold that facilitates recognition at most, if not all members of the target family, whilst the second component affords high affinity and selectivity for specific subtypes.
      The toxins present in the venoms of carnivorous marine Conidae, are for prey capture and defense and exhibit rich structural diversity. Individual conotoxins have activities at a broad spectrum of pharmaceutically interesting targets making them an excellent resource for delineating the biology of normal and diseased states. A growing number of these peptides have shown efficacy in vivo, with several having entered pre-clinical or clinical development in recent years.
      Three separate monographs in this issue of “CMC” exemplify the current development status of conotoxins as bone fide therapeutic solutions for certain intractable CNS disorders. The most advanced of these, as George Miljanich describes, is the spinally delivered N-type (Cav2.2) channel pore occluder, ziconotide (Prialt®). It is currently in late stage development for the treatment of pain arising from both visceral and somatic disease states and contingent on regulatory approval it will represent the first in a new class of neurological drugs.
      Rik Layer, John Wagstaff and Steve White from Cognetix describe SARs for the conantokin family, in particular conantokin-G, a novel NR2B subunit selective NMDA receptor antagonist isolated from the venom of the piscivorous cone snail, Conus geographus. Spinal injection of conantokins ameliorates experimental brain injury in rodents and also produce profound efficacy in animal models of epilepsy and analgesia.
      In the third article, Bob French and Heinz Terlau examine the molecular determinants that permit mu-conotoxins to exhibit Na+ channel selectivities and explore their potential in aiding future drug design. Selective sodium channel blockade with toxins might have considerable therapeutic value, given the compelling evidence that Nav1.8, a TTX resistant channel expressed specifically in sensory neurons, is intimately involved in neuropathic pain states.
      Voltage-dependent potassium ion (Kv) channels and their accessory subunits have been implicated in several diseases of genetic or autoimmune origin. Studies seeking direct correlations between identified channel subtypes and physiological roles (e.g., neurotransmitter release) are still in their infancy. Nevertheless, selective inhibition could have marked consequences for therapeutic intervention. In the wake of encouraging clinical data generated for 4-aminopyridine, for improving both sensory and motor function, Ray Norton, Mike Pennington and Heike Wulff have provided a monograph that relays the discovery and characterization of ShK toxin, a novel Kv1.3 channel blocker isolated from the sea anemone Stichodactyla helianthus, that is showing promise for treatment of multiple sclerosis. The potassium channel modulation theme is continued by Alan Harvey and Brian Robertson who present a monograph on the dendrotoxins that were first isolated some 20 years ago from mamba (Dendroaspis) snake venoms.
      In the final review article, Roger Aoki from Allergan, traces the successful development history of serotypes of Botulinum neurotoxin, a presynaptic neuromuscular blocking agent, isolated from the anaerobic bacterium Clostrtidium botulinium. Botox® induces a selective and reversible muscle weakness of up to several months when injected intra muscularly in minute quantities. Treatment of neurogenic detrusor overactivity and detrusor sphincter dyssynergia with botulinum-A toxin is a clinically proven alternative to conservative medication or surgery.
      We are both grateful to the Editors and staff of “CMC” for the publication of this special thematic issue, and to the contributors for their excellent reviews in their respective fields and for their commitment and enthusiasm during the preparation of this issue. We hope scientists involved in this field, as well as newcomers, will find the topics covered beneficial and informative.

    • Miljanich, GP (2004)Ziconotide: neuronal calcium channel blocker for treating severe chronic pain. Curr Med Chem. 11:3029-3040.
    • Layer, R.T., Wagstaff, J.D. and White, H.S. (2004) Conantokins: peptide antagonists of NMDA receptors. Curr Med Chem. 11: 3073-3084.
    • French, R.J. and Terlau, H. (2004) Sodium channel toxins--receptor targeting and therapeutic potential. Curr Med Chem. 11:3053-3064.
    • Norton, R.S., Pennington, M.W. and Wulff, H. (2004) Potassium channel blockade by the sea anemone toxin ShK for the treatment of multiple sclerosis and other autoimmune diseases. Curr Med Chem. 11:3041-3052.
    • Harvey, A.L. and Robertson, B. (2004) Dendrotoxins: structure-activity relationships and effects on potassium ion channels. Curr Med Chem. 11: 3065-3072.
    • Aoki, K.R. (2004) Botulinum toxin: a successful therapeutic protein. Curr Med Chem.11: 3085-3092.

    11 February, 2005

      Conus Biodiversity Website updated

      Alan Kohn and Trevor Anderson announced some additions and updates to the Conus Biodiversity Website.

      Color photographic images of primary type specimens of 176 species-group taxa of Conus described between 1758 and 1840 have been added. These represent about 75% of the species whose types are known to exist. Most of those remaining to be posted are of species described as Cenozoic fossils.

      Navigating the site is now easier. The images of types may be accessed directly via the Type Gallery, and single mouse clicks from there lead to the taxonomic entries and original citations.

      The Catalogue has been updated and navigation within it made more user-friendly. Some nominal species-group taxa introduced in 2004 are included, and the authors would like to be advised of any they have missed. Hard copies of original descriptions are particularly appreciated, and they are willing to post photographs of primary type specimens of new nominal taxa.

    8 February, 2005

      Selective actions of rho-conotoxin TIA from Conus tulipa

      Lima, V., Mueller, A., Kamikihara, S.Y., Raymundi, V., Alewood, D., Lewis, R.J., Chen, Z., Minneman K.P. and Pupo, A.S. (2005) Differential antagonism by conotoxin rho-TIA of contractions mediated by distinct alpha(1)-adrenoceptor subtypes in rat vas deferens, spleen and aorta. Eur J Pharmacol. 2005 Jan 31;508(1-3):183-92. Epub 2005 Jan 12. PMID: 15680270 [PubMed - in process]
      Department of Pharmacology, Instituto de Biociencias, UNESP, Botucatu, SP 18618-000, Brazil.

      Abstract: The ability of the conotoxin rho-TIA, a 19-amino acid peptide isolated from the marine snail Conus tulipa, to antagonize contractions induced by noradrenaline through activation of alpha(1A)-adrenoceptors in rat vas deferens, alpha(1B)-adrenoceptors in rat spleen and alpha(1D)-adrenoceptors in rat aorta, and to inhibit the binding of [(125)I]HEAT (2-[[beta-(4-hydroxyphenyl)ethyl]aminomethyl]-1-tetralone) to membranes of human embryonic kidney (HEK) 293 cells expressing each of the recombinant rat alpha(1)-adrenoceptors was investigated. rho-TIA (100 nM to 1 muM) antagonized the contractions of vas deferens and aorta in response to noradrenaline without affecting maximal effects and with similar potencies (pA(2) approximately 7.2, n=4). This suggests that rho-TIA is a competitive antagonist of alpha(1A)- and alpha(1D)-adrenoceptors with no selectivity between these subtypes. Incubation of rho-TIA (30 to 300 nM) with rat spleen caused a significant reduction of the maximal response to noradrenaline, suggesting that rho-TIA is a non-competitive antagonist at alpha(1B)-adrenoceptors. After receptor inactivation with phenoxybenzamine, the potency of rho-TIA in inhibiting contractions was examined with similar occupancies ( approximately 25%) at each subtype. Its potency (pIC(50)) was 12 times higher in spleen (8.3+/-0.1, n=4) than in vas deferens (7.2+/-0.1, n=4) or aorta (7.2+/-0.1, n=4). In radioligand binding assays, rho-TIA decreased the number of binding sites (B(max)) in membranes from HEK293 cells expressing the rat alpha(1B)-adrenoceptors without affecting affinity (K(D)). In contrast, in HEK293 cells expressing rat alpha(1A)- or alpha(1D)-adrenoceptors, rho-TIA decreased the K(D) without affecting the B(max). It is concluded that rho-TIA will be useful for distinguishing the role of particular alpha(1)-adrenoceptor subtypes in native tissues.

    30 January, 2005

    28 January, 2005

      Conus textile: Specialized expression, processing and secretion of conotoxins in different regions of the venom duct

      Conus textile shell & dissected venom apparatus (Fig 1 Garrett et al 2005)

      Garrett, J.E., Buczek, O., Watkins, M., Olivera, B.M., Bulaj, G. (2005) Biochemical and gene expression analyses of conotoxins in Conus textile venom ducts. Biochem Biophys Res Commun. 2005 Mar 4;328(1):362-367. PMID: 15670792 [PubMed - in process]
      Cognetix, Inc., 421 Wakara Way, Suite 201, Salt Lake City, UT 84108, USA.

      Abstract: Each Conus snail species produces 50-200 unique peptide-based conotoxins, derived from a number of different gene superfamilies. Conotoxins are synthesized and secreted in a long venom duct, but biochemical and molecular aspects of their biosynthesis remain poorly understood. Here, we analyzed expression patterns of conotoxin genes belonging to different superfamilies in Conus textile venom ducts. The results demonstrate that specific gene families are expressed in particular regions of the venom duct. Biochemical analysis using liquid chromatography and mass spectrometry revealed an even more localized accumulation of individual conotoxins. This study demonstrates for the first time that specialization of gene expression, processing, and secretion of conotoxins occurs in different regions of the venom duct.

      Conantokin-G interaction with the NMDA glutamate receptor

      Tsai, V.W., Dodd, P.R. and Lewis, R.J. (2005) The effects of alanine-substituted conantokin-G and ifenprodil on the human spermine-activated N-methyl-d-aspartate receptor. Neuroscience. 2005;130(2):457-464.
      School of Molecular and Microbial Sciences, University of Queensland, Brisbane 4072, Australia.

      Abstract: We evaluated the effects of Ala-7-conantokin-G (Con-G(A7)) and ifenprodil on the modulation by spermine of [(3)H]MK801 binding to human cortical membranes. Human cortical tissue was obtained at autopsy and stored at -80 degrees C until assay. Both Con-G(A7) and ifenprodil inhibited [(3)H]MK801 binding, but spermine affected these inhibitions differently. Con-G(A7) IC(50) changed little with spermine concentration, indicative of a non-competitive interaction, whereas the rightward shift in ifenprodil IC(50) with increasing spermine concentration suggested partial competition. When the two agents were tested against the biphasic activation of [(3)H]MK801 binding by spermine, they again differed in their effects. In the activation phase Con-G(A7) was a non-competitive inhibitor of spermine activation, and may even enhance the spermine EC(50), while the ifenprodil data indicated a partially competitive interaction. Both agents were non-competitive in the inhibitory phase. Overall, the data suggest that Con-G(A7) and ifenprodil interact differently with the polyamine modulation of the glutamate-N-methyl-d-aspartate receptor

    24 January, 2005

      alphaA Conotoxin-OIVB from Conus obscurus selectively inhibits fetal neuromuscular cholinergic receptor

      Teichert, R.W., Rivier, J., Torres, J., Dykert, J., Miller, C. and Olivera, B.M. (2005) A Uniquely Selective Inhibitor of the Mammalian Fetal Neuromuscular Nicotinic Acetylcholine Receptor. J Neurosci. 25: 732-736.
      Department of Biology, University of Utah, Salt lake City, Utah 84112, and Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037.

      Abstract: We have purified and characterized a novel conotoxin from the venom of Conus obscurus, which has the unique property of selectively and potently inhibiting the fetal form of the mammalian neuromuscular nicotinic acetylcholine receptor (nAChR) (alpha1beta1gammadelta-subunits). Although this conotoxin, alphaA-conotoxin OIVB (alphaA-OIVB), is a high-affinity antagonist (IC(50) of 56 nm) of the fetal muscle nAChR, it has >1800-fold lower affinity for the adult muscle nAChR (alpha1beta1epsilondelta-subunits) and virtually no inhibitory activity at a high concentration on various neuronal nAChRs (IC(50) > 100 mum in all cases). The peptide (amino acid sequence, CCGVONAACPOCVCNKTCG), with three disulfide bonds, has been chemically synthesized in a biologically active form. Although the neuromuscular nAChRs are perhaps the most extensively characterized of the receptors/ion channels of the nervous system, the precise physiological roles of the fetal form of the muscle nAChR are essentially unknown.alphaA-OIVB is a potentially important tool for delineating the functional roles ofalpha1beta1gammadelta receptors in normal development, as well as in various adult tissues and in pathological states. In addition to its potential as a research tool, alphaA-OIVB may have some direct biomedical applications.

      The chi-Conotoxin MrIB from Conus marmoreus (Xen2174) acts as an alpha2-adrenoceptor antagonist to prevent pain

      Obata, H., Conklin, D. and Eisenach, J.C. (2005) Spinal noradrenaline transporter inhibition by reboxetine and Xen2174 reduces tactile hypersensitivity after surgery in rats. Pain 113: 271-276.
      Department of Anesthesiology and Center for the Pharmacologic Plasticity in the Presence of Pain, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157; Department of Anesthesiology, Gunma University Graduate School of Medicine, 3-39-22 Showa, Maebashi, Gunma 371-8511, Japan.

      Spinal noradrenaline (NA) released in response to noxious stimuli may play an important role in suppression of nociceptive transmission. Here, we investigated the efficacy of a competitive NA transporter inhibitor (reboxetine) and a noncompetitive NA transporter inhibitor peptide, Xen2174, isolated from the Pacific cone snail, to treat tactile hypersensitivity following paw incisional surgery. Male Sprague-Dawley rats were anesthetized, an incision of the plantar aspect of the hind paw was performed, and withdrawal threshold to von Frey filaments near the surgical site determined. Reboxetine (0.5-5mug) and Xen2174 (0.3-100mug) increased withdrawal threshold when injected 24h after paw incision, with a peak effect at 15-60min, for Xen2174, an ED50 value of 0.64mug. Administration of Xen2174 (3-30mug) 15min before incision also reduced hypersensitivity in a dose-dependent manner. Withdrawal threshold after the single 30mug dose was greater than vehicle control even at 2, 3, and 5 days after incision. Doses < = 30mug did not alter spontaneous behavior. The anti-hypersensitivity effect of 10mug of Xen2174 was totally blocked by the alpha2-adrenoceptor antagonist, idazoxan, and partially blocked by the muscarinic antagonist, atropine. These data suggest that selective NA transporter inhibition suppresses post-incisional hypersensitivity through a different mechanism from that of neuropathic pain, since we previously reported that reversal of hypersensitivity by intrathecal clonidine, an alpha2-adrenoceptor agonist, following spinal nerve ligation is completely blocked by intrathecal atropine. Finally, these data suggest that intrathecal administration of Xen2174 at the time of spinal anesthesia might produce postoperative analgesia in humans.

    17 January, 2005

      O-superfamily contoxins from Conus capitaneus, imperialis, striatus, vexillum and virgo

      Kaufersteinm, S., Melaun, C. and Mebs, D. (2005) Direct cDNA cloning of novel conopeptide precursors of the O-superfamily. Peptides, 26(3):361-367.
      Zentrum der Rechtsmedizin, University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt, Germany.

      Abstract: Conotoxins from the venom of marine cone snails (genus Conus) represent large families of proteins exhibiting a similar precursor organization, but highly diverse pharmacological activities. A directed PCR-based approach using primers according to the conserved signal sequence was applied to investigate the diversity of conotoxins from the O-superfamily. Using 3' RACE, cDNA sequences encoding precursor peptides were identified in five Conus species (Conus capitaneus, Conus imperialis, Conus striatus, Conus vexillum and Conus virgo). In all cases, the sequence of the signal region exhibited high conservancy, whereas the sequence of the mature peptides was either almost identical or highly divergent among the five species. These findings demonstrate that beside a common genetic pattern divergent evolution of toxins occurred in a highly mutating peptide family.

    11 January, 2005

      Dr. J. Michael McIntosh's compound which "caused mice to shake", now approved as an analgesic

      A goldfish is enveloped by the "rostrum" (a type of mouth) of a Conus geographus (Al Hartmann/The Salt Lake Tribune).

      Twenty five years ago, University of Utah researchers Dr J. Michael McIntosh and Dr. Baldomero Olivera, discovered a compound (omega-conotoxin MVIIA) in the venom from the fish-hunting cone snail, Conus magus (the Cone of Maggi, or the Magician's Cone) which when injected caused mice to shake. This compound, a 25 amino acid peptide with three disulfide bridges, is now the basis of a new pain-fighting drug (Prialt = Ziconotide) developed initially by Dr. George Miljanich and colleagues at Neurex Corp. and more recently by the Irish pharmaceutical company, Elan Corp. The drug has recently been approved by the FDA for treatment of chronic pain.
      (See article "25-year-old University of Utah research helps turn snail venom into painkiller" by Greg Lavine in The Salt Lake City Tribune, 11 January 2005)

      • Information about PRIALT, including prescribing information and comprehensive support services, is available through a toll-free number, 1-888-PRIALT-1, and at www.PRIALT.com.
      • More information on the drug is available on the Resources page, and at http://www.elan.com.

      Synthesis and folding of delta-conotoxin PVIA from Conus purpurascens

      Buczek, P., Buczek, O., Bulaj, G. (2005) Total chemical synthesis and oxidative folding of delta-conotoxin PVIA containing an N-terminal propeptide. Biopolymers. 2005 Jan 7; [Epub ahead of print]
      Cognetix, Inc., 421 Wakara Way Suite 201, Salt Lake City, Utah 84108.

      Abstract: Small disulfide-rich peptides are translated as larger precursors typically containing an N-terminal prepro sequence. In this study, we investigated the role of a propeptide in the oxidative folding of an extremely hydrophobic delta-conotoxin, PVIA. delta-Conotoxin PVIA (delta-PVIA) is a 29-amino acid neurotoxin stabilized by three disulfide bridges. Previous folding studies on delta-conotoxins revealed that their poor folding properties resulted from their hydrophobicity. However, low folding yields of delta-PVIA could be improved by the presence of a nonionic detergent, which acted as a chemical chaperone. delta-PVIA provided an attractive model to investigate whether the hydrophilic propeptide region could function as an intramolecular chaperone. A 58-amino acid precursor for delta-PVIA (pro-PVIA), containing the N-terminal propeptide covalently attached to the mature conotoxin, was synthesized using native chemical ligation. Oxidative folding of pro-PVIA resulted in a very low accumulation of the correctly folded form, comparable to that for the mature conotoxin delta-PVIA. Our results are in accord with the relevant data previously observed for alpha- and omega-conotoxins, indicating that conotoxin prepro sequences are so-called class II propeptides, which are not directly involved in the oxidative folding. We hypothesize that these propeptide regions may be important for interactions with protein folding catalysts and sorting receptors during the secretory process.

      Image of Conus purpurascens

    9 January, 2005

      Analgesic actions of omega-conotoxin CVID from Conus catus

      Blake, D.W., Scott, D.A., Angus, J.A., Wright, C.E. (2005) Synergy between intrathecal omega-conotoxin CVID and dexmedetomidine to attenuate mechanical hypersensitivity in the rat. Eur J Pharmacol. 506: 221-227.
      Department of Pharmacology, University of Melbourne, Victoria 3010, Australia.

      Abstract: The analgesic effects of intrathecal (i.t.) omega-conotoxin CVID, an N-type Ca(2+) channel antagonist, and the alpha(2)-adrenoceptor agonist, dexmedetomidine, were tested alone and in combination following unilateral ligation of L (lumbar) 5/6 spinal nerves in rats. Mechanical allodynia was observed prior to insertion of an i.t. catheter. Effects and interactions of omega-conotoxin CVID (0.01-10 mug/kg) and dexmedetomidine (0.1-10 mug/kg) were tested on allodynic and tail flick (thermal stimulus) responses. Only dexmedetomidine increased the latency of the tail flick response. Both dexmedetomidine and omega-conotoxin CVID completely inhibited allodynia (ED(50) 0.78+/-0.02 and 0.35+/-0.08 mug/kg, respectively; n=63, 41). Dexmedetomidine and omega-conotoxin CVID combined in dose ratios 0.7 and 1.3 (adjusted for ED(50)) were synergistic in decreasing mechanical hypersensitivity; interaction index (gamma) 0.39 (confidence interval [CI] 0.33, 0.46) and 0.3 (CI 0.23, 0.38). Despite the necessity for i.t. administration, these data suggest that the synergistic combination confers enhanced potency (lower doses) of both drugs that may avoid clinical toxicity of single drug therapy.

    3 January, 2005

      Prialt (Ziconotide): a Review

      Miljanich, G.P. (2004) Ziconotide: neuronal calcium channel blocker for treating severe chronic pain. Curr Med Chem. 2004 Dec;11(23):3029-3040.
      Elan Pharmaceuticals, Inc., 7475 Lusk Boulevard, San Diego, CA 92121, USA. george.miljanich@elan.com

      Abstract: Ziconotide (PRIALT) is a neuroactive peptide in the final stages of clinical development as a novel non-opioid treatment for severe chronic pain. It is the synthetic equivalent of omega-conotoxin MVIIA, a component of the venom of the marine snail, Conus magus. The mechanism of action underlying ziconotide's therapeutic profile derives from its potent and selective blockade of neuronal N-type voltage-sensitive calcium channels (N-VSCCs). Direct blockade of N-VSCCs inhibits the activity of a subset of neurons, including pain-sensing primary nociceptors. This mechanism of action distinguishes ziconotide from all other analgesics, including opioid analgesics. In fact, ziconotide is potently anti-nociceptive in animal models of pain in which morphine exhibits poor anti-nociceptive activity. Moreover, in contrast to opiates, tolerance to ziconotide is not observed. Clinical studies of ziconotide in more than 2,000 patients reveal important correlations to ziconotide's non-clinical pharmacology. For example, ziconotide provides significant pain relief to severe chronic pain sufferers who have failed to obtain relief from opiate therapy and no evidence of tolerance to ziconotide is seen in these patients. Contingent on regulatory approval*, ziconotide will be the first in a new class of neurological drugs: the N-type calcium channel blockers, or NCCBs. Its novel mechanism of action as a non-opioid analgesic suggests ziconotide has the potential to play a valuable role in treatment regimens for severe chronic pain. If approved* for clinical use, ziconotide will further validate the neuroactive venom peptides as a source of new and useful medicines.

      * Note: Prialt(R) (Ziconotide intrathecal infusion) was approved by the FDA on December 28 (2004) "for the management of severe chronic pain in patients for whom intrathecal (IT) therapy is warranted, and who are intolerant of or refractory to other treatment, such as systemic analgesics, adjunctive therapies or IT morphine."
      • Detailed product information about Prialt is available for download.
      • (see also entry for December 28 in What's New for 2004)

    2 January, 2005

      Conotoxins from Conus delessertii from Mexico

      Aguilar, M.B., Lopez-Vera, E., Imperial, J.S., Falcon, A., Olivera, B.M., de la Cotera E.P. (2005) Putative gamma-conotoxins in vermivorous cone snails: the case of Conus delessertii. Peptides 26:23-27.
      Laboratory of Marine Neuropharmacology, Institute of Neurobiology, Universidad Nacional Autonoma de Mexico, Juriquilla, Qro. 76230, Mexico.

      Abstract: Peptide de7a was purified from the venom of Conus delessertii, a vermivorous cone snail collected in the Yucatan Channel, Mexico. Its amino acid sequence was determined by automatic Edman degradation after reduction and alkylation. The sequence shows six Cys residues arranged in the pattern that defines the O-superfamily of conotoxins, and several post-translationally modified residues. The determination of its molecular mass by means of laser desorption ionization time-of-flight mass spectrometry (average mass, 3170.0Da) confirmed the chemical data and suggested amidation of the C-terminus. The primary structure


      [O, hydroxyproline; gamma, gamma-carboxyglutamate; *, amidated C-terminus]; calculated average mass, 3169.66Da) of de7a contains a motif (gammaCCS) that has previously only been found in two other toxins, both from molluscivorous cone snails: TxVIIA from Conus textile and gamma-PnVIIA from Conus pennaceus. These toxins cause depolarization and increased firing of action potentials in molluscan neuronal systems, and toxin gamma-PnVIIA has been shown to act as an agonist of neuronal pacemaker cation currents. The similarities to toxins TxVIIA and gamma-PnVIIA suggest that peptide de7a might also affect voltage-gated nonspecific cation pacemaker channels.

      Conotoxins used to distinguish 3 nicotinic receptor subtypes in Lymnaea

      Vulfius, C.A., Tumina, O.B., Kasheverov, I.E., Utkin, Y.N., Tsetlin, VI. (2005) Diversity of nicotinic receptors mediating Cl(-) current in Lymnaea neurons distinguished with specific agonists and antagonist.Neurosci Lett. 373:232-236
      Laboratory of Cellular Neurobiology, Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.

      Abstract: Diversity of nicotinic acetylcholine receptors (nAChRs) mediating Cl(-) current in voltage-clamped identifiable Lymnaea stagnalis neurons was studied using acetylcholine (ACh), three agonists and alpha-conotoxin ImI (ImI). Cytisine, nicotine, and choline, full agonists at alpha7 subunit-containing nAChRs of vertebrates, were found to evoke at saturating concentration 84-92% of the maximal current elicited by ACh. ImI, known to block selectively alpha7 and alpha9 nAChRs, markedly diminished the responses to ACh. The average maximal ImI-induced block was 80%, leaving a residual current which had very slow kinetics. The choline-, cytisine-, and nicotine-induced currents were blocked by ImI almost completely, suggesting that they activate only ImI-sensitive receptors. Two groups of cells which differ in desensitization kinetics and in sensitivity to ImI were revealed. IC(50) values for ImI against ACh were 10.3 and 288nM, respectively, with the rapidly desensitizing current being the more sensitive to ImI. The data obtained suggest the existence of at least three pharmacologically distinct subtypes of nicotinic receptors in Lymnaea neurons. Two of the subtypes are similar to alpha7 nAChRs of vertebrates, but differ from each other in their affinity for ImI and in their desensitization kinetics. The third subtype is quite distinct, in that it is resistant to ImI, is not activated by nicotine, cytisine or choline, and mediates a very slowly developing current.

    1 January, 2005

      Ziconotide and other calcium-channel blockers for chronic pain

      McGivern, J.G. and McDonough, S.I. (2004) Voltage-gated calcium channels as targets for the treatment of chronic pain. Curr Drug Targets CNS Neurol Disord. 3:457-478.
      Department of HTS-Molecular Pharmacology, Amgen Inc., Thousand Oaks, California 91320, USA. mcgivern@amgen.com.

      Abstract: This review focuses on the importance of voltage-gated calcium channels in modulating and controlling the function of peripheral and central neurons involved in nociceptive processing. We describe the different families of voltage-gated calcium channels that are expressed in pain pathway neurons, how the expression levels of calcium channel currents change in chronic pain conditions, and the validation of N-type, T-type, and P-type calcium channels as targets for the treatment of pain. The molecular mechanism of action is reviewed for the most prominent calcium channel-targeted drugs including gabapentin and ziconotide as well as antiepileptics administered off-label for the treatment of pain. We discuss how the major genetic, functional, and pharmacological differences between subtypes of neuronal calcium channels can be leveraged to identify new molecular targets and to discover and develop new therapeutic agents for the treatment of chronic pain syndromes.

      Bonicalzi, V., Canavero, S. (2004) Intrathecal ziconotide for chronic pain. JAMA. 292(14):1681-1682; author reply 1682. No abstract available.
      Comment on: JAMA. 2004 Jan 7;291(1):63-70. Intrathecal ziconotide for chronic pain. Bonicalzi V, Canavero S. Publication Types: Comment Letter

      Bayes, M., Rabasseda, X., Prous, J.R. (2004) Gateways to clinical trials. Methods Find Exp Clin Pharmacol. 26 :473-503. Abstract: Gateways to Clinical Trials is a guide to the most recent clinical trials in current literature and congresses. The data in the following tables has been retrieved from the Clinical Trials Know- ledge Area of Prous Science Integrity, the drug discovery and development portal, http://integrity.prous.com. This issue focuses on the following selection of drugs: ABI-007, Ad.Egr.TNF.11D, adefovir dipivoxil, AdPEDF.11, AES-14, albumex, alefacept, alemtuzumab, aliskiren fumarate, alvimopan hydrate, aAminolevulinic acid hydrochloride, aminolevulinic acid methyl ester, anakinra, anti-IL-12 MAb, aprepitant, atazanavir sulfate, atrasentan, avanafil; Banoxantrone, BG-12, bimatoprost, bortezomib, bosentan; Calcipotriol/betamethasone dipropionate, caspofungin acetate, CBT-1, ciclesonide, clofarabine, conivaptan hydrochloride, CpG-7909, C-Vax, Cypher; DA-8159, DAC:GLP-1, darbepoetin alfa, darifenacin, duloxetine hydrochloride; Eculizumab, efalizumab, efaproxiral sodium, EGF vaccine, eletriptan, epratuzumab, erlotinib hydrochloride, escitalopram oxalate, ETC-642, etoricoxib, everolimus, exenatide; Gefitinib, IV gamma-globulin; Human insulin, gamma-hydroxybutyrate sodium; IDN-6556, iguratimod, imatinib mesylate, indiplon, ixabepilone; Laquinimod, LB-80380, lidocaine/prilocaineliraglutide, lopinavir, lopinavir/ritonavir, lucinactant; MAb-14.18, melatonin, MLN-591-DM1; NC-531, neridronic acid, nesiritide, neutrophil-inhibitory factor, niacin/lovastatin; Oblimersen sodium, olcegepant, oral Insulin, ORV-105; Palonosetron hydrochloride, PAmAb, pegaptanib sodium, peginterferon alfa-2a, pegvisomant, perifosine, pexelizumab, phenoxodiol, phenserine tartrate, pimecrolimus, pramlintide acetate, pregabalin, PRO-542, prostate cancer vaccine, PT-141; Ramelteon, rasagiline mesilate, rDNA insulin, reslizumab, rh-Lactoferrin, ribamidine hydrochloride, rosuvastatin calcium; S-8184l, SC-1, sorafenib, St. John's Wort extract, SU-11248; Taxus, telbivudine, tenofovir disoproxil fumarate, teriparatide, testosterone gel, tezosentan disodium, tipifarnib, tolvaptan, trabectedin, travoprost, travoprost/timolol, treprostinil sodium; Vardenafil hydrochloride hydrate; Xcellerated T cells, XR-5944; Yttrium 90 (90Y) ibritumomab tiuxetan; Ziconotide.

      Bayes, M., Rabasseda, X., Prous, J.R. (2004) Gateways to clinical trials. Methods Find Exp Clin Pharmacol.26:211-244.
      Prous Science, S.A., Barcelona, Spain. mbayes@prous.com

      Abstract: Gateways to Clinical Trials is a guide to the most recent clinical trials in current literature and congresses. The data in the following tables has been retrieved from the Clinical Studies Knowledge Area of Prous Science Integrity(R), the drug discovery and development portal, http://integrity.prous.com. This issue focuses on the following selection of drugs: ABI-007, adalimumab, adefovir dipivoxil, alefacept, alemtuzumab, 3-AP, AP-12009, APC-8015, L-Arginine hydrochloride, aripiprazole, arundic acid, avasimibe; Bevacizumab, bivatuzumab, BMS-181176, BMS-184476, BMS-188797, bortezomib, bosentan, botulinum toxin type B, BQ-123, BRL-55730, bryostatin 1; CEP-1347, cetuximab, cinacalcet hydrochloride, CP-461, CpG-7909; D-003, dabuzalgron hydrochloride, darbepoetin alfa, desloratadine, desoxyepothilone B, dexmethylphenidate hydrochloride, DHA-paclitaxel, diflomotecan, DN-101, DP-b99, drotrecogin alfa (activated), duloxetine hydrochloride, duramycin; Eculizumab, Efalizumab, EKB-569, elcometrine, enfuvirtide, eplerenone, erlotinib hydrochloride, ertapenem sodium, eszopiclone, everolimus, exatecan mesilate, ezetimibe; Fenretinide, fosamprenavir calcium, frovatriptan; GD2L-KLH conjugate vaccine, gefitinib, glufosfamide, GTI-2040; Hexyl insulin M2, human insulin, hydroquinone, gamma-Hydroxybutyrate sodium; IL-4(38-37)-PE38KDEL, imatinib mesylate, indisulam, inhaled insulin, ixabepilone; KRN-5500; LY-544344; MDX-210, melatonin, mepolizumab, motexafin gadolinium; Natalizumab, NSC-330507, NSC-683864; 1-Octanol, omalizumab, ortataxel; Pagoclone, peginterferon alfa-2a, peginterferon alfa-2b, pemetrexed disodium, phenoxodiol, pimecrolimus, plevitrexed, polyphenon E, pramlintide acetate, prasterone, pregabalin, PX-12; QS-21; Ragaglitazar, ranelic acid distrontium salt, RDP-58, recombinant glucagon-like peptide-1 (7-36) amide, repinotan hydrochloride, rhEndostatin, rh-Lactoferrin, (R)-roscovitine; S-8184, semaxanib, sitafloxacin hydrate, sitaxsentan sodium, sorafenib, synthadotin; Tadalafil, tesmilifene hydrochloride, theratope, tipifarnib, tirapazamine, topixantrone hydrochloride, trabectedin, traxoprodil, Tri-Luma; Valdecoxib, valganciclovir hydrochloride, vinflunine; Ximelagatran; Ziconotide. mbayes@prous.com

    Continued in What's new in 2004
    See also : What's new in 2006, 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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