29 November, 2006

ACV1 Clinical Trials Update

Metabolic's neuropathic pain drug, ACV1 - additional clinical studies reveal:

• First patients have been treated with ACV1 in the Phase 2A sciatica trial
• A separate trial to test safety of a higher dose level in healthy volunteers has commenced – important information for regulatory authorities and potential licensing partners
• The three clinical trials for ACV1 in progress or final planning are progressing on time

24 November, 2006

Excitatory short kappaA-conotoxins from C. purpurascens

Teichert RW, Jacobsen R, Terlau H, Yoshikami D, Olivera BM.(2006) Discovery and characterization of the short kappaA-conotoxins: A novel subfamily of excitatory conotoxins. Toxicon. 2006 Oct 14; [Epub ahead of print]
Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112, USA.

Abstract: We have characterized the defining members of a novel subfamily of excitatory conotoxins, the short kappaA-conotoxins (kappaA(S)-conotoxins). kappaA-conotoxins PIVE and PIVF (kappaA-PIVE and kappaA-PIVF) were purified from Conus purpurascens venom. Both peptides elicited excitatory activity upon injection into fish. kappaA-PIVE was synthesized for further characterization. The excitatory effects of kappaA-PIVE in vivo were dose dependent, causing hyperactivity at low doses and rapid immobilization at high doses, symptomatic of a type of excitotoxic shock. Consistent with these observations, kappaA-PIVE caused repetitive action potentials in frog motor axons in vitro. Similar results have been reported for other structurally distinct conotoxin families; such peptides appear to be required by most fish-hunting cone snails for the rapid immobilization of prey. Unexpected structure-function relationships were revealed between these peptides and two families of homologous conotoxins: the alphaA-conotoxins (muscle nAChR antagonists) and kappaA-conotoxins (excitotoxins), which all share a common arrangement of cysteine residues (CC-C-C-C-C). Biochemically, the kappaA(S)-conotoxins more closely resemble the alphaA(S)-conotoxins than the other kappaA-conotoxin subfamily, the long kappaA-conotoxins (kappaA(L)-conotoxins); however, kappaA(S)- and alphaA(S)-conotoxins produce different physiological effects. In contrast, the kappaA(S)-and kappaA(L)-conotoxins that diverge in several biochemical characteristics are clearly more similar in their physiological effects.

23 November, 2006

ACV1 PreClinical Update

Metabolic's neuropathic pain drug ACV1 - additional preclinical studies reveal greater potential This ASX Announcement by Metabolic Pharmaceuticals reports that the:


• Latest oral version of ACV1 works as well as the injected version in new animal studies
• Provides proof-of-concept for Metabolic's Oral Peptide Delivery Platform
• An independent study provides new knowledge about how ACV1 works and reveal the likely biochemical target for ACV1

I-conotoxins revisisted

Mondal S, Bhavna R, Mohan Babu R, Ramakumar S. (2006) Pseudo amino acid composition and multi-class support vector machines approach for conotoxin superfamily classification. J Theor Biol. 243:252-260.
Department of Physics, Indian Institute of Science, Bangalore 560 012, India.

Abstract: Conotoxins are disulfide rich small peptides that target a broad spectrum of ion-channels and neuronal receptors. They offer promising avenues in the treatment of chronic pain, epilepsy and cardiovascular diseases. Assignment of newly sequenced mature conotoxins into appropriate superfamilies using a computational approach could provide valuable preliminary information on the biological and pharmacological functions of the toxins. However, creation of protein sequence patterns for the reliable identification and classification of new conotoxin sequences may not be effective due to the hypervariability of mature toxins. With the aim of formulating an in silico approach for the classification of conotoxins into superfamilies, we have incorporated the concept of pseudo-amino acid composition to represent a peptide in a mathematical framework that includes the sequence-order effect along with conventional amino acid composition. The polarity index attribute, which encodes information such as residue surface buriability, polarity, and hydropathy, was used to store the sequence-order effect. Several methods like BLAST, ISort (Intimate Sorting) predictor, least Hamming distance algorithm, least Euclidean distance algorithm and multi-class support vector machines (SVMs), were explored for superfamily identification. The SVMs outperform other methods providing an overall accuracy of 88.1% for all correct predictions with generalized squared correlation of 0.75 using jackknife cross-validation test for A, M, O and T superfamilies and a negative set consisting of short cysteine rich sequences from different eukaryotes having diverse functions. The computed sensitivity and specificity for the superfamilies were found to be in the range of 84.0-94.1% and 80.0-95.5%, respectively, attesting to the efficacy of multi-class SVMs for the successful in silico classification of the conotoxins into their superfamilies.

Mondal S, Babu RM, Bhavna R, Ramakumar S. (2006) I-conotoxin superfamily revisited. J Pept Sci. 12:679-685.
Department of Physics, Indian Institute of Science, Bangalore 560012, India.

Abstract: The I-conotoxin superfamily (I-Ctx) is known to have four disulfide bonds with the cysteine arrangement C-C-CC-CC-C-C, and the members inhibit or modify ion channels of nerve cells. Recently, Olivera and co-workers (FEBS J. 2005; 272: 4178-4188) have suggested that the previously described I-Ctx should now be divided into two different gene superfamilies, namely, I(1) and I(2), in view of their having two different types of signal peptides and exhibiting distinct functions. We have revisited the 28 entries presently grouped as I-Ctx in UniProt Swiss-Prot knowledgebase, and on the basis of in silico analysis have divided them into I(1) and I(2) superfamilies. The sequence analysis has provided a framework for in silico annotation enabling us to carry out computer-based functional characterization of the UniProtKB/TrEMBL entry Q59AA4 from Conus miles and to predict it as a member of the I(2) superfamily. Furthermore, we have predicted the mature toxin of this entry and have proposed that it may be an inhibitor of voltage-gated potassium channels. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

Mondal S, Vijayan R, Shichina K, Babu RM, Ramakumar S. (2005) I-superfamily conotoxins: sequence and structure analysis. In Silico Biol. 5:557-571.
Department of Physics, Indian Institute of Science, Bangalore 560 012, India.

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.

15 November, 2006

alpha-conotoxin RgIA from Conus regius interacts with alpha9/alpha10 nAChRs and suppresses pain

Snail toxin may help nerve pain relief: Associated Press, November 14 - Toxins from cone snail venom may help point the way to better relief of severe nerve pain in people, researchers report. Results were promising in tests on rats with a type of nerve pain similar to sciatica, according to researchers.

Michelle Vincler, Shannon Wittenauer, Renee Parker, Michael Ellison, Baldomero M. Olivera, and J. Michael McIntosh (2006) Molecular mechanism for analgesia involving specific antagonism of alpha9alpha10 nicotinic acetylcholine receptors. PNAS 103: 17880-17884
*Department of Anesthesiology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157; and Departments of Psychiatry, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112

Abstract: alpha10 nicotinic acetylcholine receptors (nAChRs) have been identified in a variety of tissues including lymphocytes and dorsal root ganglia; except in the case of the auditory system, the function of alpha10 nAChRs is not known. Here we show that selective block (rather than stimulation) of alpha10 nAChRs is analgesic in an animal model of nerve injury pain. In addition, blockade of this nAChR subtype reduces the number of choline acetyltransferase-positive cells, macrophages, and lymphocytes at the site of injury. Chronic neuropathic pain is estimated to affect up to 8% of the world's population; the numerous analgesic compounds currently available are largely ineffective and act through a small number of pharmacological mechanisms. Our findings not only suggest a molecular mechanism for the treatment of neuropathic pain but also demonstrate the involvement of alpha10 nAChRs in the pathophysiology of peripheral nerve injury.

Opoiorphin, a natural painkiller in saliva

Natural chemical, "beats morphine": BBC (UK), November 14 - The human body produces a natural painkiller several times more potent than morphine, research suggests. When given to rats, the chemical, called opiorphin, was able to curb pain at much lower concentration than the powerful painkiller morphine.

Anne Wisner, Evelyne Dufour, Michaël Messaoudi, Amine Nejdi, Audrey Marcel, Marie-Noelle Ungeheuer, and Catherine Rougeot (2006) Human Opiorphin, a natural antinociceptive modulator of opioid-dependent pathways. PNAS 103: 17979-17984

Abstract: Mammalian zinc ectopeptidases play important roles in turning off neural and hormonal peptide signals at the cell surface, notably those processing sensory information. We report here the discovery of a previously uncharacterized physiological inhibitor of enkephalin-inactivating zinc ectopeptidases in humans, which we have named Opiorphin. It is a QRFSR peptide that inhibits two enkephalin-catabolizing ectoenzymes, human neutral ecto-endopeptidase, hNEP (EC 3.4.24.11), and human ecto-aminopeptidase, hAP-N (EC 3.4.11.2). Opiorphin displays potent analgesic activity in chemical and mechanical pain models by activating endogenous opioid-dependent transmission. Its function is closely related to the rat sialorphin peptide, which is an inhibitor of pain perception and acts by potentiating endogenous µ- and delta-opioid receptor-dependent enkephalinergic pathways. Here we demonstrate the functional specificity in vivo of human Opiorphin. The pain-suppressive potency of Opiorphin is as effective as morphine in the behavioral rat model of acute mechanical pain, the pin-pain test. Thus, our discovery of Opiorphin is extremely exciting from a physiological point of view in the context of endogenous opioidergic pathways, notably in modulating mood-related states and pain sensation. Furthermore, because of its in vivo properties, Opiorphin may have therapeutic implications.

6 November, 2006

alpha3/alpha6beta2* nAChRs identified using (125)I-alpha-conotoxinMII

Bordia, T., Parameswaran, N., Fan, H., Langston, J.W., McIntosh, J.M. and Quik, M. (2006) Partial recovery of striatal nicotinic receptors in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys with chronic oral nicotine. J Pharmacol Exp Ther. 2006 Oct;319(1):285-92. Epub 2006 Jul 12.
The Parkinson's Institute, 1170 Morse Ave, Sunnyvale, CA 94089-1605, USA.

Abstract: Recent studies in nonhuman primates show that chronic nicotine treatment protects against nigrostriatal degeneration, with a partial restoration of neurochemical and functional measures in the striatum. The present studies were done to determine whether long-term nicotine treatment also protected against striatal nicotinic receptor (nAChR) losses after nigrostriatal damage. Monkeys were administered nicotine in the drinking water for 6 months and subsequently lesioned with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) over several months while nicotine was continued. (125)I-Epibatidine, [(125)I]5-[(125)I]iodo-3(2(S)-azetidinylmethoxy)-pyridine (A85380), and (125)I-alpha-conotoxinMII autoradiography was performed to evaluate changes in alpha4beta2* and alpha3/alpha6beta2* nAChRs, the major striatal subtypes. Nicotine treatment increased alpha4beta2* nAChRs by > or =50% in striatum of both unlesioned and lesioned animals. This increase in alpha4beta2* nAChRs was significantly greater in lesioned compared with unlesioned monkey striatum. Chronic nicotine treatment led to a small decrease in alpha3/alpha6beta2* nAChR subtypes. The decline in alpha3/alpha6beta2* subtypes, defined using alpha-conotoxinMII-sensitive (125)I-epibatidine or [(125)I]A85380 binding, was significantly smaller in striatum of nicotine-treated lesioned monkeys compared with unlesioned monkeys. This difference was not observed for alpha3/alpha6beta2* nAChRs identified using (125)I-alpha-conotoxinMII. These data suggest that there are at least two striatal alpha3/alpha6beta2* subtypes that are differentially affected by chronic nicotine treatment in lesioned animals. In addition, the results showing an improvement in striatal alpha4beta2* and select alpha3/alpha6beta2* nAChR subtypes, combined with previous work, demonstrate that chronic nicotine treatment restores and/or protects against the loss of multiple molecular markers after nigrostriatal damage. Such findings suggest that nicotine or nicotinic agonists may be of therapeutic value in Parkinson's disease.

29 October, 2006

muConotoxin MrVIB from Conus marmoreus selectively blocks sodium channels on sensory neurons and chronic pain behavior

Ekberg, J., Jayamanne, A., Vaughan, C.W., Aslan, S., Thomas, L.,, Mould, J., Drinkwater, R., Baker, M.D., Abrahamsen, B., Wood, J.N., Adams, D.J., Christie, M.J., Lewis RJ.(2006) {micro}O-conotoxin MrVIB selectively blocks Nav1.8 sensory neuron specific sodium channels and chronic pain behavior without motor deficits. Proc Natl Acad Sci U S A. 103: 17030-17035
Institute for Molecular Bioscience and School of Biomedical Sciences, University of Queensland, QLD 4072, Australia; Pain Management Research Institute and Kolling Institute, Northern Clinical School, University of Sydney at Royal North Shore Hospital, St Leonards, NSW 2065, Australia.

Abstract: The tetrodotoxin-resistant voltage-gated sodium channel (VGSC) Nav1.8 is expressed predominantly by damage-sensing primary afferent nerves and is important for the development and maintenance of persistent pain states. Here we demonstrate that microO-conotoxin MrVIB from Conus marmoreus displays substantial selectivity for Nav1.8 and inhibits pain behavior in models of persistent pain. In rat sensory neurons, submicromolar concentrations of MrVIB blocked tetrodotoxin-resistant current characteristic of Nav1.8 but not Nav1.9 or tetrodotoxin-sensitive VGSC currents. MrVIB blocked human Nav1.8 expressed in Xenopus oocytes with selectivity at least 10-fold greater than other VGSCs. In neuropathic and chronic inflammatory pain models, allodynia and hyperalgesia were both reduced by intrathecal infusion of MrVIB (0.03-3 nmol), whereas motor side effects occurred only at 30-fold higher doses. In contrast, the nonselective VGSC blocker lignocaine displayed no selectivity for allodynia and hyperalgesia versus motor side effects. The actions of MrVIB reveal that VGSC antagonists displaying selectivity toward Nav1.8 can alleviate chronic pain behavior with a greater therapeutic index than nonselective antagonists.

Novel muO-conotoxin lt7a, from Conus litteratus

Pi, C., Liu, J., Wang, L., Jiang, X., Liu, Y., Peng, C., Chen, S. and Xu, A. (2006) Soluble expression, purification and functional identification of a disulfide-rich conotoxin derived from Conus litteratus. J Biotechnol. 2006 Sep 27; [Epub ahead of print]
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, Guangzhou 510275, People's Republic of China.

Abstract: Conotoxins are a diverse array of small peptides mostly with multiple disulfide bridges. These peptides become an increasing significant source of neuro-pharmacological probes and drugs as a result of the high selectivity for ion channels and receptors. Usually, the analogue of natural conotoxins is produced by means of chemical synthesis. Here, we present a simple and fast strategy of producing disulfide-rich conotoxins via recombinant expression. By fused with thioredoxin and His tag, a novel O-superfamily conotoxin lt7a was successfully expressed in Escherichia coli and purified, resulting in a high yield of recombinant lt7a about 6mg/l. The purity of target protein is up to 95% as identified by HPLC results. Whole cell patch-clamp recording revealed that the new conotoxin blocked voltage-sensitive sodium channels in rat dorsal root ganglion neurons, indicating it might be a novel muO-conotoxin.

Disulfide bond rearrangement during synthesis of alpha-Conotoxin GI

Szabo, I., Schlosser, G., Hudecz, F., Mezo, G. (2006). Disulfide bond rearrangement during regioselective oxidation in PhS(O)Ph/CH(3)SiCl(3) mixture for the synthesis of alpha-conotoxin GI. Biopolymers. 2006 Oct 24.; [Epub ahead of print]
Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eotvos L. University, Budapest, Hungary.

Abstract: Rearrangement of disulfide bonds during the synthesis of alpha-conotoxin GI using PhS(O)Ph/CH(3)SiCl(3) oxidation procedure was observed. We have demonstrated that the protecting scheme (order of Acm and (t)Bu protecting groups) of the Cys residues as well as the reaction time influenced the ratio of the native and the mispaired compounds, while the temperature of the reaction mixture had no significant effect. However, in all cases the nonnative derivative was produced in high amount. The structure of the isomers was identified by the combination of enzymatic digestion and mass spectrometry measurements. We had to conclude that the air oxidation followed by the application of Tl(tfa)(3) for the regioselective formation of disulfide bonds lead up to the appropriate compound in case of the synthesis of alpha-conotoxin GI, while the oxidation procedure using PhS(O)Ph/CH(3)SiCl(3) system resulted in the non-native disulfide isomer. (c) 2006 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2006.

Omega conotoxins for pain treatment: Review

Schroeder CI, Doering CJ, Zamponi GW, Lewis RJ. N-type calcium channel blockers: novel therapeutics for the treatment of pain. Med Chem. 2006 Sep;2(5):535-43.
Institute for Molecular Bioscience, University of Queensland, St Lucia, QLD 4072, Australia.

Abstract: Highly selective Ca(v)2.2 voltage-gated calcium channel (VGCC) inhibitors have emerged as a new class of therapeutics for the treatment of chronic and neuropathic pain. Cone snail venoms provided the first drug in class with FDA approval granted in 2005 to Prialt (omega-conotoxin MVIIA, Elan) for the treatment of neuropathic pain. Since this pioneering work, major efforts underway to develop alternative small molecule inhibitors of Ca(v)2.2 calcium channel have met with varied success. This review focuses on the properties of the Ca(v)2.2 calcium channel in different pain states, the action of omega-conotoxins GVIA, MVIIA and CVID, describing their structure-activity relationships and potential as leads for the design of improved Ca(v)2.2 calcium channel therapeutics, and finally the development of small molecules for the treatment of chronic pain.

28 October, 2006

Bromotryptophan, a post-translational modification of conotoxins, is a feature of many marine species

Bittner, S., Scherzer, R. and Harlev, E. (2006) The five bromotryptophans. [Review] Amino Acids. 2006 Oct 12; [Epub ahead of print]
Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel.

Abstract: The five regioisomeric bromotryptophans (BrTrps) play an important role in the life of sponges and lower marine invertebrates. These bromo-amino acids, which are formed by post-translational modifications, are not found in nature in their free state, but rather are involved in more complex structures. Any of the BrTrps can be part of a peptide, a cyclic peptide, an indole alkaloid, an ergot alkaloid, a macrocycle and others. The present review covers the synthesis, physical and spectroscopic properties of the five BrTrps. It also describes the many exiting pharmacological and biological activities played by the BrTrps and by various secondary metabolites containing brominated tryptophan moieties. Of special interest are cyclic peptides containing the 2-BrTrp unit, which were isolated from marine sponges e.g. konbamide, orbiculamide A, the various keramamides, jaspamide eusynstyelamide and more. Important families of non-cyclic peptides containing the 6-BrTrp, include the styelins, the conotoxins, the cathelicidins and several constrained macrocyclic peptides. Many marine secondary BrTrp-containing, non-peptidic metabolites also display a remarkable spectrum of bioactivities, which can be harnessed for therapeutic and other purposes. Examples are: barettin, bromotryptanthrin, tetraacetyl clionamide, cyclocinamide A, clavicipitic acid, various brominated beta-carbolines. In this review we have presented the various synthetic routes leading to the preparation of the five BrTrps and many of its derivatives. Also, we have introduced the reader to many synthetic routes leading to BrTrp-containing non-peptidic natural products. Although the functional role of the various compounds in the human body is only poorly understood, its effects were extensively studied. Almost all of these compounds exhibit important therapeutic properties e.g. antifungal, antimicrobial, antihelmintic, insecticidal ichthyotoxic and anticancer activity. In the present review attempts have been made to provide synopsis, synthesis and symbiosis of chemical and biological actions, which may provide future guidance and facilitate further research in this area.

Development of omega-conotoxins GVIA, MVIIA, CVID for the treatment of chronic pain

Schroeder, C.I., Doering, C.J., Zamponi, G.W. and Lewis, R.J. (2006) N-type calcium channel blockers: novel therapeutics for the treatment of pain. Med Chem. 2: 535-543.
Institute for Molecular Bioscience, University of Queensland, St Lucia, QLD 4072, Australia.

Abstract: Highly selective Ca(v)2.2 voltage-gated calcium channel (VGCC) inhibitors have emerged as a new class of therapeutics for the treatment of chronic and neuropathic pain. Cone snail venoms provided the first drug in class with FDA approval granted in 2005 to Prialt (omega-conotoxin MVIIA, Elan) for the treatment of neuropathic pain. Since this pioneering work, major efforts underway to develop alternative small molecule inhibitors of Ca(v)2.2 calcium channel have met with varied success. This review focuses on the properties of the Ca(v)2.2 calcium channel in different pain states, the action of omega-conotoxins GVIA, MVIIA and CVID, describing their structure-activity relationships and potential as leads for the design of improved Ca(v)2.2 calcium channel therapeutics, and finally the development of small molecules for the treatment of chronic pain.

28 October, 2006

Redox-active agents influence oxidative folding of conotoxins GI, PVIIA and r11a

Buczek, O., Green, B.R. and Bulaj, G. (2006) Albumin is a redox-active crowding agent that promotes oxidative folding of cysteine-rich peptides. Biopolymers. 2006 Oct 23; [Epub ahead of print]
Department of Biology, University of Utah, Salt Lake City, Utah 84112.

Abstract: Oxidative folding that occurs in a crowded cellular milieu is characterized by multifaceted interactions that occur among nascent polypeptides and resident components of the endoplasmic reticulum (ER) lumen. Macromolecular crowding has been considered an essential factor in the folding of polypeptides, but the excluded volume effect has not been evaluated for small, disulfide-rich peptides. In the research presented, we examined how macromolecular crowding agents, such as albumin, ovalbumin and polysaccharides influenced the kinetics and thermodynamics of forming disulfide bonds in four model peptides of varying molecular size from 13-residues (1.4 kDa) to 58-residues (6.5 kDa): conotoxins: GI, PVIIA, r11a, and bovine pancreatic trypsin inhibitor. Our results indicate that the excluded volume effect does not significantly alter the folding rates nor equilibria for these peptides. In stark contrast, folding reactions were dramatically accelerated, when protein-based crowding agents were present at concentrations lower than those predicted to provide the excluded volume effect. Submillimolar albumin alone was as effective as glutathione in promoting the oxidative folding of GI conotoxin at concentrations typically found in the ER. To best of our knowledge, this is the first report and quantitative characterization of oxidative folding of peptides mediated by other than thioredoxinbased protein disulfide bonds. Our work raises a possibility that concurrent secretory and ER resident proteins may influence the oxidative folding of small, cysteine-rich peptides not as crowding agents, but as redox-active factors. (c) 2006 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2006.

Ziconotide a treatment option for neuraxial anaesthesia.

Schug, S.A., Saunders, D., Kurowski, I. and Paech, M.J. (2006) Neuraxial drug administration : a review of treatment options for anaesthesia and analgesia. CNS Drugs. 20: 917-933.
Pharmacology Unit, School of Medicine and Pharmacology, UWA Anaesthesia, University of Western Australia, Perth, Western Australia, AustraliaDepartment of Anaesthesia and Pain Medicine, Royal Perth Hospital, Perth, Western Australia, Australia.

Abstract: Neuraxial drug administration describes techniques that deliver drugs in close proximity to the spinal cord, i.e. intrathecally into the CSF or epidurally into the fatty tissues surrounding the dura, by injection or infusion. This approach was initially developed in the form of spinal anaesthesia over 100 years ago. Since then, neuraxial drug administration has evolved and now includes a wide range of techniques to administer a large number of different drugs to provide anaesthesia, but also analgesia and treatment of spasticity in a variety of acute and chronic settings.This review concentrates on the pharmacological agents used and the clinical basis behind currently utilised approaches to neuraxial drug administration. With regard to local anaesthetics, the main focus is on the development of the enantiomer-specific compounds ropivacaine and levobupivacaine, which provide similar efficacy to bupivacaine with a reduced risk of severe cardiotoxicity. Opioids are the other group of drugs widely used neuraxially, in particular to provide analgesia alone or more commonly in combination with other agents. The physicochemical properties of the various opioids explain the main differences in efficacy and safety between these drugs when used intrathecally, of which morphine, fentanyl and sufentanil are most commonly used. Another group of drugs including clonidine, dexmedetomidine and epinephrine (adrenaline) provide neuraxial analgesia via alpha-adrenergic receptors and are used mainly as adjuvants to local anaesthetics and opioids. Furthermore, intrathecal baclofen is in routine clinical use to treat spasticity in a number of neurological conditions.Beside these established approaches, a wide range of other drugs have been assessed for neuraxial administration to provide analgesia; however, most are in various early stages of investigation and are not used routinely. These drugs include neostigmine, ketamine, midazolam and adenosine, and the conotoxin ziconotide. The latter is possibly the most unusual compound here; it has recently gained registration for intrathecal use in specific chronic pain conditions.

27 October, 2006

A synthetic cyclic version of Conotoxin MrIA, from Conus marmoreus, retains biological activity

Lovelace, E.S., Armishaw, C.J., Colgrave, M.L., Wahlstrom, M.E., Alewood, P.F., Daly, N.L. and Craik, D.J. (2006) Cyclic MrIA: A Stable and Potent Cyclic Conotoxin with a Novel Topological Fold that Targets the Norepinephrine Transporter. J Med Chem. 2006 Nov 2;49(22):6561-8.
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia.

Abstract: Conotoxins, disulfide-rich peptides from the venom of cone snails, have created much excitement over recent years due to their potency and specificity for ion channels and their therapeutic potential. One recently identified conotoxin, MrIA, a 13-residue member of the chi-conotoxin family, inhibits the human norepinephrine transporter (NET) and has potential applications in the treatment of pain. In the current study, we show that the beta-hairpin structure of native MrIA is retained in a synthetic cyclic version, as is biological activity at the NET. Furthermore, the cyclic version has increased resistance to trypsin digestion relative to the native peptide, an intriguing result because the cleavage site for the trypsin is not close to the cyclization site. The use of peptides as drugs is generally hampered by susceptibility to proteolysis, and so, the increase in enzymatic stability against trypsin observed in the current study may be useful in improving the therapeutic potential of MrIA. Furthermore, the structure reported here for cyclic MrIA represents a new topology among a growing number of circular disulfide-rich peptides.

Craik, D. (2006) Animal, plant and microbial toxins - 15th world congress. (Meeting Report). IDrugs. 2006 Oct;9(10):679-81.
Institute for Molecular Bioscience, Australian Research Council Special Research Center for Functional and Applied Genomics, University of Queensland, Brisbane, QLD 4072, Australia. d.craik@imb.uq.edu.au.

No abstract available; an edited extract follows.

Extract: Update on the development of the analgesic conotoxin ACV1.
Several other presentations at the meeting described preclinical and clinical studies on conopeptides, revealing the apparent importance of these promising disufide-rich molecules as drug leads. Bruce Livett [Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Australia] presented two posters that updated progress on the analgesic peptide ACV1 (Metabolic Pharmaceuticals Ltd) and related conotoxins derived from the venom of Conus victoriae. ACV1 is a synthetic peptide corresponding to the non-post-translationally modified venom peptide alpha-conotoxin Vc1.1. The conotoxin compound is currently undergoing Phase II clinical trials for neuropathic pain. In the first poster, Dr. Livett and colleagues described a chemical analysis of the C. victoriae venom in combination with the specific chemical synthesis of some post-translationally modified analogs of ACV1. In several animal models of human neuropathic pain, previous research had established that the 16-amino acid peptide Vc1.1 is active, but that the major post-translational form of Vc1.1 present in the venom, the peptide vc1a, which contains a hydroxyproline at position 6 and a gamma-carboxyglutamic acid at position 14, is inactive. The new research detected the presence of a previously unreported dimeric version of the post-translationally modified peptide, 6-Hyp Vc1.1. This dimeric form of the 6-Hyp peptide exhibited 36% of the activity of the monomeric alpha-conotoxin Vc1.1 (ACV1) in a neuronal nicotinic receptor assay. The second poster provided the first report that ACV1 is effective at alleviating neuropathic pain in an animal model of diabetic neuropathy. In the streptozotocin-induced diabetic rat model of peripheral neuropathy, an antiallodynic effect was observed at doses of 30 and 300 ug/kg within 1 h of dosing. ACV1 attenuated allodynia for up to 1 week following cessation of treatment, and also reduced oxidative stress markers such as lipid hydroperoxidases and nitrotyrosine. These findings broaden the indications for the development of ACV1 to treat neuropathic pain and diabetic neuropathy.

26 October, 2006

Rearrangement of disulfide bonds during synthesis of alpha-conotoxin GI from Conus geographus

Szabo, I., Schlosser, G., Hudecz, F. and Mezo, G.(2006). Disulfide bond rearrangement during regioselective oxidation in PhS(O)Ph/CH(3)SiCl(3) mixture for the synthesis of alpha-conotoxin GI. Biopolymers. 2006 Oct 24; [Epub ahead of print]
Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eotvos L. University, Budapest, Hungary.

Abstract: Rearrangement of disulfide bonds during the synthesis of alpha-conotoxin GI using PhS(O)Ph/CH(3)SiCl(3) oxidation procedure was observed. We have demonstrated that the protecting scheme (order of Acm and (t)Bu protecting groups) of the Cys residues as well as the reaction time influenced the ratio of the native and the mispaired compounds, while the temperature of the reaction mixture had no significant effect. However, in all cases the nonnative derivative was produced in high amount. The structure of the isomers was identified by the combination of enzymatic digestion and mass spectrometry measurements. We had to conclude that the air oxidation followed by the application of Tl(tfa)(3) for the regioselective formation of disulfide bonds lead up to the appropriate compound in case of the synthesis of alpha-conotoxin GI, while the oxidation procedure using PhS(O)Ph/CH(3)SiCl(3) system resulted in the non-native disulfide isomer. (c) 2006 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2006.

24 October, 2006

O-conotoxin from Conus marmoreus from Hainan

Luo, S., Zhangsun, D., Lin, Q., Xie, L., Wu, Y. and Zhu, X. (2006) Sequence diversity of O-superfamily conopetides from Conus marmoreus native to Hainan. Peptides. 2006 Sep 29; [Epub ahead of print]
Key Laboratory for Tropical Biology Resources, Ministry of Education, Ocean College, Center for Experimental Biotechnology, Hainan University, Haikou, Hainan 570228, China.

Abstract: The full-length cDNAs of six new O-superfamily conotoxins (CTX) were cloned and sequenced from Conus marmoreus native to Hainan in China South Sea using RT-PCR and 3'-RACE. Six novel conotoxin precursors encoded by these cDNAs consist of three typical regions of signal, pro-peptide and mature peptide. All the six toxin regions share a common O-superfamily cysteine pattern (C-C-CC-C-C, with three disulfide bridges). The predicted precursors are composed of 73-88 amino acids, and the predicted mature peptides consist of 26-34 amino acids. Phylogenetic analysis of new conotoxins from C. marmoreus from the present study and published homologue T-superfamily sequences from other Conus species was performed systematically. Patterns of sequence divergence for three regions of signal, pro-region and mature peptides, as well as Cys codon usage define the major O-superfamily branches and suggest how these separate branches arose. Percent identities of the amino acid sequences of the signal region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical to highly divergent between inter- and intra-species. Notably, the diversity of the pro-region was also high with intermediate divergence between that observed in signal and toxin regions. Amino acid sequences and their mode of action (target) of previously identified conotoxins from molluscivorous C. marmoreus for the known conotoxins classes are discussed in detail. The data presented are new and should pave the way for chemical synthesis of these unique conotoxins for to allow determination of the molecular targets of these peptides, and also to provide clues for a better understanding of the phylogeny of these peptides.

T-conotoxins from Conus lividus and Conus litteratus

Luo, S., Zhangsun, D., Wu, Y., Zhu, X., Xie, L., Hu, Y., Zhang, J. and Zhao, X. (2006) Identification and Molecular Diversity of T-superfamily Conotoxins from Conus lividus and Conus litteratus.Chem Biol Drug Des. 2006 Aug;68(2):97-106.
Key Laboratory for Tropical Biological Resources (MOE), Ocean College, Center for Experimental Biotechnology, Hainan University, Haikou, Hainan 570228 China.

Abstract: The T-superfamily conotoxins comprise a large and diverse group of biologically active peptides and are widely distributed in venom ducts of all major feeding types of Conus. Six novel T-superfamily peptides from the two worm-hunting cone snail species of Conus lividus and Conus litteratus native to Hainan were identified and determined to share a common signal sequence as well as a conserved arrangement of cysteine residues (CC-CC). The predicted mature peptides consist of 11-15 amino acids only. Phylogenetic analyses of new conotoxins from C. lividus and C. litteratus in present study and published homologue T-superfamily sequences from the other Conus species was systematically performed. Phylogenetic trees, residue substitutions to view evolutionary relationships of the precursors' signal, propeptide, and mature toxin regions were explored, as well as residue frequency component and cystine codon usage. Percent divergence of the amino acid sequences of the signal-region exhibited high conservation, whereas the sequences of the mature peptides ranged from high similarity to high divergence between inter- and intro-species. Notably, diversity of pro-peptide region was also high with intermediate percent divergence between that observed in signal and toxin-regions. Consensus hydrophobic residues Leu, Val, Ala, Ile and Pro of signal regions were abundant, whereas among propeptides, basic residues Arg and Lys and acidic residue Asp, addition of hydrophilic residues Thr and Ser were abundant. Residue frequency components were hypervariable in mature toxin region except for highly conservative cystine frame residues. The T-superfamily conotoxins have been previously found mainly in piscivorous and molluscivorous cone snails. The newly identified six T-superfamily peptides described in this investigation exemplify the first to be found from vermivorous C. lividus and C. litteratus. The elucidated cDNAs of the six toxins will facilitate a better understanding of the relationship between structure and function as well as provide a framework for their further research and development.

alpha-conotoxins from Cone shells native to Hainan

Luo, S., Zhangsun, D., Zhang, B., Quan, Y. and Wu, Y. (2006) Novel alpha-conotoxins identified by gene sequencing from cone snails native to Hainan, and their sequence diversity. J Pept Sci. 2006 Nov;12(11):693-704.
Key Laboratory for Tropical Aquatic and Terrestrial Biological Resources (MOE), Ocean College, Center for Experimental Biotechnology, Hainan University, Haikou Hainan, 570228 China.

Abstract: Conotoxins (CTX) from the venom of marine cone snails (genus Conus) represent large families of proteins, which show a similar precursor organization with surprisingly conserved signal sequence of the precursor peptides, but highly diverse pharmacological activities. By using the conserved sequences found within the genes that encode the alpha-conotoxin precursors, a technique based on RT-PCR was used to identify, respectively, two novel peptides (LiC22, LeD2) from the two worm-hunting Conus species Conus lividus, and Conus litteratus, and one novel peptide (TeA21) from the snail-hunting Conus species Conus textile, all native to Hainan in China. The three peptides share an alpha4/7 subfamily alpha-conotoxins common cysteine pattern (CCX(4)CX(7)C, two disulfide bonds), which are competitive antagonists of nicotinic acetylcholine receptor (nAChRs). The cDNA of LiC22N encodes a precursor of 40 residues, including a propeptide of 19 residues and a mature peptide of 21 residues. The cDNA of LeD2N encodes a precursor of 41 residues, including a propeptide of 21 residues and a mature peptide of 16 residues with three additional Gly residues. The cDNA of TeA21N encodes a precursor of 38 residues, including a propeptide of 20 residues and a mature peptide of 17 residues with an additional residue Gly. The additional residue Gly of LeD2N and TeA21N is a prerequisite for the amidation of the preceding C-terminal Cys. All three sequences are processed at the common signal site -X-Arg- immediately before the mature peptide sequences. The properties of the alpha4/7 conotoxins known so far were discussed in detail. Phylogenetic analysis of the new conotoxins in the present study and the published homologue of alpha4/7 conotoxins from the other Conus species were performed systematically. Patterns of sequence divergence for the three regions of signal, proregion, and mature peptides, both nucleotide acids and residue substitutions in DNA and peptide levels, as well as Cys codon usage were analyzed, which suggest how these separate branches originated. Percent identities of the DNA and amino acid sequences of the signal region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical to highly divergent between inter- and intra-species. Notably, the diversity of the proregion was also high, with an intermediate percentage of divergence between that observed in the signal and in the toxin regions. The data presented are new and are of importance, and should attract the interest of researchers in this field. The elucidated cDNAs of these toxins will facilitate a better understanding of the relationship of their structure and function, as well as the process of their evolutionary relationships. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

Discovery and development of alpha-conotoxins as novel analgesics

Livett, B.G., Sandall, D.W., Keays, D., Down, J., Gayler, K.R., Satkunanathan, N. and Khalil Z. (2006) Therapeutic applications of conotoxins that target the neuronal nicotinic acetylcholine receptor. Toxicon. 2006 Jul 15; [Epub ahead of print]
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria 3010, Australia.

Abstract: Pain therapeutics discovered by molecular mining of the expressed genome of Australian predatory cone snails are providing lead compounds for the treatment of neurological diseases such as multiple sclerosis, shingles, diabetic neuropathy and other painful neurological conditions. The high specificity exhibited by these novel compounds for neuronal receptors and ion channels in the brain and nervous system indicates the high degree of selectivity that this class of neuropeptides can be expected to show when used therapeutically in humans. A lead compound, ACV1 (conotoxin Vc1.1 from Conus victoriae), has entered Phase II clinical trials and is being developed for the treatment for neuropathic pain. ACV1 will be targeted initially for the treatment of sciatica, shingles and diabetic neuropathy. The compound is a 16 amino acid peptide [Sandall et al., 2003. A novel alpha-conotoxin identified by gene sequencing is active in suppressing the vascular response to selective stimulation of sensory nerves in vivo. Biochemistry 42, 6904-6911], an antagonist of neuronal nicotinic acetylcholine receptors. It has potent analgesic activity following subcutaneous or intramuscular administration in several preclinical animal models of human neuropathic pain [Satkunanathan et al., 2005. Alpha conotoxin Vc1.1 alleviates neuropathic pain and accelerates functional recovery of injured neurons. Brain. Res. 1059, 149-158]. ACV1 may act as an analgesic by decreasing ectopic excitation in sensory nerves. In addition ACV1 appears to accelerate the recovery of injured nerves and tissues.

Unusual structure of alpha-conotoxin BuIA from Conus bullatus

Chi SW, Kim DH, Olivera BM, McIntosh JM, Han KH. (2006) NMR structure determination of alpha-conotoxin BuIA, a novel neuronal nicotinic acetylcholine receptor antagonist with an unusual 4/4 disulfide scaffold. Biochem Biophys Res Commun. 349:1228-1234.
Molecular Cancer Research Center, Division of Molecular Therapeutics, KRIBB, Daejeon 305-806, Republic of Korea.

Abstract: We have determined a high-resolution three-dimensional structure of alpha-conotoxin BuIA, a 13-residue peptide toxin isolated from Conus bullatus. Despite its unusual 4/4 disulfide bond layout alpha-conotoxin BuIA exhibits strong antagonistic activity at alpha6/alpha3beta2beta3, alpha3beta2, and alpha3beta4 nAChR subtypes like some alpha4/7 conotoxins. alpha-Conotoxin BuIA lacks the C-terminal beta-turn present within the second disulfide loop of alpha4/7 conotoxins, having only a "pseudo omega-shaped" molecular topology. Nevertheless, it contains a functionally critical two-turn helix motif, a feature ubiquitously found in alpha4/7 conotoxins. Such an aspect seems mainly responsible for similarities in the receptor recognition profile of alpha-conotoxin BuIA to alpha4/7 conotoxins. Structural comparison of alpha-conotoxin BuIA with alpha4/7 conotoxins and alpha4/3 conotoxin ImI suggests that presence of the second helical turn portion of the two-turn helix motif in alpha4/7 and alpha4/4 conotoxins may be important for binding to the alpha3 and/or alpha6 subunit of nAChR.

23 October, 2006

Novel M-superfamily conotoxins from Conus marmoreus, C.textile, C. leopardus and C. quercinus

Han YH, Wang Q, Jiang H, Liu L, Xiao C, Yuan DD, Shao XX, Dai QY, Cheng JS, Chi CW. (2006) Characterization of novel M-superfamily conotoxins with new disulfide linkage. FEBS J. 273: 4972-4982
Key laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai, China.

Abstract: The M-superfamily with the typical Cys framework (-CC-C-C-CC-) is one of the seven major superfamilies of conotoxins found in the venom of cone snails. Based on the number of residues in the last Cys loop (between C4 and C5), M-superfamily conotoxins can be provisionally categorized into four branches (M-1, M-2, M-3, M-4) [Corpuz GP, Jacobsen RB, Jimenez EC, Watkins M, Walker C, Colledge C, Garrett JE, McDougal O, Li W, Gray WR, et al. (2005) Biochemistry44, 8176-8186]. Here we report the purification of seven M-superfamily conotoxins from Conus marmoreus (five are novel and two are known as mr3a and mr3b) and one known M-1 toxin tx3a from Conus textile. In addition, six novel cDNA sequences of M-superfamily conotoxins have been identified from C. marmoreus, Conus leopardus and Conus quercinus. Most of the above novel conotoxins belong to M-1 and M-2 and only one to M-3. The disulfide analyses of two M-1 conotoxins, mr3e and tx3a, revealed that they possess a new disulfide bond arrangement (C1-C5, C2-C4, C3-C6) which is different from those of the M-4 branch (C1-C4, C2-C5, C3-C6) and M-2 branch (C1-C6, C2-C4, C3-C5). This newly characterized disulfide connectivity was confirmed by comparing the HPLC profiles of native mr3e and its two regioselectively folded isoforms. This is the first report of three different patterns of disulfide connectivity in conotoxins with the same cysteine framework.

22 October, 2006

3-D Model of alpha-Conotoxin Vc1.1




The PDB coordinates of alpha-conotoxin Vc1.1 have been released and can be found under ID code # 2H8S following the link:

http://www.rcsb.org/pdb/explore.do?structureId=2H8S

Clark, R.J., Fischer, H., Nevin, S.T., Adams, D.J. and Craik, D.J. (2006) The synthesis, structural chracterisation and receptor specificity of the alpha-conotoxin Vc1.1. J Biol Chem. 281: 23254-23263.





16 October, 2006

Roman Coligula spawns modern counterpart

'An image and a thousand words' is a regular column at the Toronto Star written by Peter Calamai, National Science Editor, Canada. This article about Bruce Livett's cone shell research appeared the Toronto Star on October 16 (Bruce Livett's 30th wedding anniversary as it happens !).

To read the article in the Toronto Star Click here

16 October, 2006

Recombinant conotoxin MVIIA expressed in E. coli is 800 times stronger than morphine

Xia, Z., Chen, Y., Zhu, Y., Wang, F., Xu, X. and Zhan, J. (2006) Recombinant omega-Conotoxin MVIIA possesses strong analgesic activity.BioDrugs 20:275-281.
Department of Biochemistry and Molecular Biology, Zhejiang University Medical School, Hangzhou, People's Republic of China.

Abstract: omega-Conotoxin (CTX) MVIIA is a specific antagonist of N-type voltage-sensitive calcium channels. A synthetic peptide version of CTX MVIIA (ziconotide) has been approved by the US FDA for severe and chronic pain. Given the high cost and complexity of the synthetic process of the disulfide-rich peptide, the genetic recombinant approach may simplify the development of this potent therapeutic agent. AIM: In this study, we report a new method for production of the recombinant CTX MVIIA. METHOD: A novel DNA fragment encoding CTX MVIIA was designed using Escherichia coli-preferred codons, and the fragment was cloned into the expression vector pGEX(2T). The fusion protein, CTX MVIIA and glutathione-S-transferase (GST) [GST-CTX MVIIA], was expressed in E. coli and purified by affinity chromatography on a glutathione-agarose column. After digestion with thrombin, the CTX MVIIA fragment was purified on a Sephacryltrade mark S-100 HR column and identified by mass spectrometry. The bioactivity of the peptide was evaluated by the hot tail-flick assay, in which the CTX MVIIA was intracerebroventricularly administered into Sprague-Dawley rats and its antinociceptive effect measured. RESULTS: The analgesic activity of the conotoxin was about 800 times stronger than that of morphine. CONCLUSION: The recombinant CTX MVIIA expressed in E. coli has shown marked analgesic activity, which may have potential in clinical application.




15 October, 2006

Conotoxins provide protection to the rapidly diversifying species of Conus. REVIEW

Olivera BM.(2006) Conus peptides, biodiversity-based discovery and exogenomics..J Biol Chem. 2006 Aug 11; [Epub ahead of print] .
Dept. of Biology, University of Utah, Salt Lake City, UT 84112-0840

Abstract: The 700 different predatory cone snails (genus Conus) have evolved complex venoms, each species with it's own distinct set of 100 to 200 biologically active venom peptides. These are specified by a small number of gene superfamilies that rapidly diversify as speciation occurs. Most conopeptides are multiply disulfide cross-linked, with peptides in a superfamily having a characteristic disulfide framework, resulting in generally similar structures, but diverging function. Conopeptides have direct therapeutic applications - at least five are in clinical trials, and one (Prialt) has been approved as a drug for severe pain. Many conopeptides have exquisite targeting selectivity, and have the potential to be diagnostic ligands for a particular molecular isoform of an ion channel or receptor (the majority of conopeptide targets fall into these categories). The rapid diversification observed in conopeptide genes may be a general feature of all genes used by organisms to interact with other organisms in their environment. Across megadiverse taxa, each individual species has it's own complement of predators, prey and competitors; those genes whose products are used to mediate the biotic interactions of that particular species should thus be rapidly diversifying, if compared to homologous genes of other species. For conopeptides, an integrated approach to discovery that incorporates phylogenetics has been highly successful for identifying peptides with differing subtype-specificity.

3-D Structure of alpha-conotoxin BuIA from Conus bullatus

Chi, S.W., Kim, D.H., Olivera, B.M., McIntosh, J.M. and Han, K.H.(2006). NMR structure determination of alpha-conotoxin BuIA, a novel neuronal nicotinic acetylcholine receptor antagonist with an unusual 4/4 disulfide scaffold. Biochem Biophys Res Commun. 349:1228-1234.
Molecular Cancer Research Center, Division of Molecular Therapeutics, KRIBB, Daejeon 305-806, Republic of Korea.

Abstract: We have determined a high-resolution three-dimensional structure of alpha-conotoxin BuIA, a 13-residue peptide toxin isolated from Conus bullatus. Despite its unusual 4/4 disulfide bond layout alpha-conotoxin BuIA exhibits strong antagonistic activity at alpha6/alpha3beta2beta3, alpha3beta2, and alpha3beta4 nAChR subtypes like some alpha4/7 conotoxins. alpha-Conotoxin BuIA lacks the C-terminal beta-turn present within the second disulfide loop of alpha4/7 conotoxins, having only a "pseudo omega-shaped" molecular topology. Nevertheless, it contains a functionally critical two-turn helix motif, a feature ubiquitously found in alpha4/7 conotoxins. Such an aspect seems mainly responsible for similarities in the receptor recognition profile of alpha-conotoxin BuIA to alpha4/7 conotoxins. Structural comparison of alpha-conotoxin BuIA with alpha4/7 conotoxins and alpha4/3 conotoxin ImI suggests that presence of the second helical turn portion of the two-turn helix motif in alpha4/7 and alpha4/4 conotoxins may be important for binding to the alpha3 and/or alpha6 subunit of nAChR.

6 September, 2006

Selectivity of alpha-conotoxin BuIA from Conus bullatus

Shiembob, D.L., Roberts, R.L., Luetje, C.W., McIntosh, J.M. (2006) Determinants of alpha-conotoxin BuIA selectivity on the nicotinic acetylcholine receptor beta subunit. Biochemistry. 45:11200-11207.

Department of Biology, University of Utah, Salt Lake City, Utah, Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami, Miami, Florida, and Department of Psychiatry, University of Utah, Salt Lake City, Utah.

Abstract: Neuronal nicotinic acetylcholine receptors (nAChRs) are pentamers composed of alpha and beta subunits. Different molecular compositions of these subunits constitute various receptor subtypes that are implicated in the pathophysiology and/or treatment of several disease states but are difficult to distinguish pharmacologically. alpha-Conotoxins are a group of small, structurally defined peptides that may be used to molecularly dissect the nAChR-binding site. Heteromeric nAChRs generally contain either a beta2 or beta4 subunit in addition to an alpha subunit at the ligand-binding interface. alpha-Conotoxin BuIA kinetically distinguishes between beta2- and beta4-containing nAChRs, with long off times for the latter. Mutational studies were used to assess the influence of residues that line the putative acetylcholine-binding pocket but differ between beta2 and beta4 subunits. Residues Thr/Lys59, Val/Ile111, and Phe/Gln119 of the respective beta2 and beta4 subunits are critical to off-rate differences. Among these residues, Thr59 of nAChR beta2 may interfere with effective access to the binding site, whereas Lys59 may facilitate this binding.

Kasheverov, I.E., Zhmak, M.N., Vulfius, C.A., Gorbacheva, E.V., Mordvintsev, D.Y., Utkin, Y.N., van Elk, R., Smit, A.B., Tsetlin, V.I. (2006) alpha-Conotoxin analogs with additional positive charge show increased selectivity towards Torpedo californica and some ne