What's New in 2008

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

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

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

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

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

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

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

    For a video simulation of cone shell envenomaton click here

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

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    31 December 2008

    Complications with Ziconotide

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

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

    30 November 2008

    Novel conopeptides from Conus delessertii

    Aguilar MB, Flores-Torres A, Batista CV, Falcón A, López-Vera E, de la Cotera EP (2010). Structural characterization of five post-translationally modified isomorphs of a novel putative delta-conotoxin from the vermivorous snail Conus delessertii from the Mexican Caribbean Sea. Peptides. 30: 458-466.

    Laboratorio de Neurofarmacología Marina, Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro 76230, Mexico. maguilar@servidor.unam.mx

    Abstract: A novel peptide, de7b, was isolated from the venom of Conus delessertii, a worm-hunting species collected in the Caribbean Sea off the Yucatan Peninsula. Its primary structure was determined by automated Edman degradation and confirmed by mass spectrometry: it contains 28 amino acids, including six Cys residues. Peptide de7b is the second, O-conotoxin-like peptide isolated from the venom of this species, and it exists in different post-translationally modified isomorphs, some of which contain gamma-carboxy-glutamate (gamma) and/or 4-hydroxy-proline (O) at positions 4, 7, and/or 14. Its primary structure is DCI(P/O)GG(E/gamma)NCDVFR(O/P)YRCCSGYCILLLCA, with molecular masses varying from 3078.6 to 3154.6Da, depending on the number and kind of modified amino acid residues. Peptide de7b shows significant sequence identity with several O-conotoxins purified and biologically characterized from molluscivorous and piscivorous cone snails of the Indo-Pacific region, the tropical Atlantic and Eastern Pacific Oceans, especially with the delta-conotoxins but also with the omega-conotoxins from molluscivorous species, which suggests that it might affect voltage-gated Na(+) or Ca(2+)channels. Peptide de7b has 32% sequence identity with putative gamma-conotoxin de7a, previously characterized from the same species; both peptides contain the same number of amino acid residues and of non-Cys residues between the pairs of consecutive Cys residues. However, these peptides have charge differences at seven positions within the N-terminal half indicating that they might have distinct molecular targets that remain to be identified.

    Alpha-conotoxin GID and analogues from Conus geographus

    Millard EL, Nevin ST, Loughnan ML, Nicke A, Clark RJ, Alewood PF, Lewis RJ, Adams DJ, Craik DJ, Daly NL (2008). Inhibition of neuronal nicotinic acetylcholine receptor subtypes by alpha-Conotoxin GID and analogues. J Biol Chem. 284: 4944-4951.

    Institute for Molecular Bioscience, University of Queensland, Brisbane QLD 4072, Australia. Abstract: alpha-Conotoxins are small disulfide-rich peptides from the venom of the Conus species that target the nicotinic acetylcholine receptor (nAChR). They are valuable pharmacological tools and also have potential therapeutic applications particularly for the treatment of chronic pain. alpha-Conotoxin GID is isolated from the venom of Conus geographus and has an unusual N-terminal tail sequence that has been shown to be important for binding to the alpha4beta2 subtype of the nAChR. To date, only four conotoxins that inhibit the alpha4beta2 subtype have been characterized, but they are of considerable interest as it is the most abundant nAChR subtype in the mammalian brain and has been implicated in a range of diseases. In this study, analysis of alanine-scan and truncation mutants of GID reveals that a conserved proline in alpha-conotoxins is important for activity at the alpha7, alpha3beta2, and alpha4beta2 subtypes. Although the proline residue was the most critical residue for activity at the alpha3beta2 subtype, Asp(3), Arg(12), and Asn(14) are also critical at the alpha7 subtype. Interestingly, very few of the mutations tested retained activity at the alpha4beta2 subtype indicating a tightly defined binding site. This lack of tolerance to sequence variation may explain the lack of selective ligands discovered for the alpha4beta2 subtype to date. Overall, our findings contribute to the understanding of the structure-activity relationships of alpha-conotoxins and may be beneficial for the ongoing attempts to exploit modulators of the neuronal nAChRs as therapeutic agents.

    Drugs from marine natural products

    Molinski TF, Dalisay DS, Lievens SL, Saludes JP (2008). Drug development from marine natural products. Nat Rev Drug Discov. 8: 69-85. Review.

    Department of Chemistry and Biochemistry and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive, MC 0358, La Jolla, California 92093, USA. tmolinski@ucsd.edu Abstract: Drug discovery from marine natural products has enjoyed a renaissance in the past few years. Ziconotide (Prialt; Elan Pharmaceuticals), a peptide originally discovered in a tropical cone snail, was the first marine-derived compound to be approved in the United States in December 2004 for the treatment of pain. Then, in October 2007, trabectedin (Yondelis; PharmaMar) became the first marine anticancer drug to be approved in the European Union. Here, we review the history of drug discovery from marine natural products, and by describing selected examples, we examine the factors that contribute to new discoveries and the difficulties associated with translating marine-derived compounds into clinical trials. Providing an outlook into the future, we also examine the advances that may further expand the promise of drugs from the sea.

    Intrathecal therapy with Ziconotide

    Vitale V, Battelli D, Gasperoni E, Monachese N (2008). Intrathecal therapy with ziconotide: clinical experience and considerations on its use. Minerva Anestesiol. 74: 727-733. Review.

    Anesthesiology, Intensive Care and Analgesic Unit, San Marino Republic Hospital, San Marino, Republic of San Marino.

    Abstract: Ziconotide is a synthetic peptide equivalent of an w-conotoxin, obtained from the marine snail Conus magus, which acts by blocking N-type calcium channels in the spinal cord, reducing the perception of pain. It is a newly marketed drug, exclusively for intrathecal use, indicated for severe chronic pain. Ziconotide came to the physicians' table with both doubts and promises; to determine its safety and efficacy, one of the largest and most well-designed randomized double-blind studies in the history of intrathecal therapy was undertaken, and this drug demonstrated efficacy in relieving chronic pain in patients coming from many years of different therapies and therapy failures. However, the pain relief came with some adverse effects, which are few compared with morphine's adverse effects but in some cases could undermine the course of therapy with this conotoxin. The experience described in this paper began in June 2007 and gave us the opportunity to analyze how the conotoxin works outside of the papers. We noted differences between the well-known activity of morphine on pain and mood, and the more focused action of ziconotide on pain. In addition, it is important to consider the lack of addiction, opioid-induced hyperalgesia and other systemic effects that are common with morphine. These are the reasons why the Polyanalgesic Consensus Conference of 2007 put ziconotide in the first line of intrathecal therapy management.

    Intrathecal Ziconotide

    Mitchell AA, Sapienza-Crawford AJ, Hanley KL, Lokey KJ, Wells L (2008). Using ziconotide for intrathecal infusions. Nursing 38: 19.

    Integrated Pain Solutions in Westerville, Ohio, USA.

    Integrated Pain Solutions in Westerville, Ohio, USA.

    No Abstract

    alpha4 and alpha6 nicotinic receptors in the VTA

    Pons S, Fattore L, Cossu G, Tolu S, Porcu E, McIntosh JM, Changeux JP, Maskos U, Fratta W (2008). Crucial role of alpha4 and alpha6 nicotinic acetylcholine receptor subunits from ventral tegmental area in systemic nicotine self-administration. J Neurosci. 28: 12318-122327.

    Unité Neurobiologie intégrative des systèmes cholinergiques, Institut Pasteur, 75724 Paris Cedex 15, France.

    Abstract: The identification of the molecular mechanisms involved in nicotine addiction and its cognitive consequences is a worldwide priority for public health. Novel in vivo paradigms were developed to match this aim. Although the beta2 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) has been shown to play a crucial role in mediating the reinforcement properties of nicotine, little is known about the contribution of the different alpha subunit partners of beta2 (i.e., alpha4 and alpha6), the homo-pentameric alpha7, and the brain areas other than the ventral tegmental area (VTA) involved in nicotine reinforcement. In this study, nicotine (8.7-52.6 microg free base/kg/inf) self-administration was investigated with drug-naive mice deleted (KO) for the beta2, alpha4, alpha6 and alpha7 subunit genes, their wild-type (WT) controls, and KO mice in which the corresponding nAChR subunit was selectively re-expressed using a lentiviral vector (VEC mice). We show that WT mice, beta2-VEC mice with the beta2 subunit re-expressed exclusively in the VTA, alpha4-VEC mice with selective alpha4 re-expression in the VTA, alpha6-VEC mice with selective alpha6 re-expression in the VTA, and alpha7-KO mice promptly self-administer nicotine intravenously, whereas beta2-KO, beta2-VEC in the substantia nigra, alpha4-KO and alpha6-KO mice do not respond to nicotine. We thus define the necessary and sufficient role of alpha4beta2- and alpha6beta2-subunit containing nicotinic receptors (alpha4beta2*- and alpha6beta2*-nAChRs), but not alpha7*-nAChRs, present in cell bodies of the VTA, and their axons, for systemic nicotine reinforcement in drug-naive mice.

    31 October 2008

    Determinants of potency and selectivity for receptor interaction with alpha-conotoxin RgIA

    Pérez EG, Cassels BK, Zapata-Torres G. (2009) Molecular modeling of the alpha9alpha10 nicotinic acetylcholine receptor subtype. Bioorg Med Chem Lett. 19: 251-254.

    Department of Chemistry, Faculty of Sciences, University of Chile, Casilla, Santiago, Chile.

    Abstract: This study reports the comparative molecular modeling, docking and dynamic simulations of human alpha9alpha10 nicotinic acetylcholine receptors complexed with acetylcholine, nicotine and alpha-conotoxin RgIA, using as templates the crystal structures of Aplysia californica and Lymnaea stagnalis acetylcholine binding proteins. The molecular dynamics simulations showed that Arg112 in the complementary alpha10(-) subunit, is a determinant for recognition in the site that binds small ligands. However, Glu195 in the principal alpha9(+), and Asp114 in the complementary alpha10(-) subunit, might confer the potency and selectivity to alpha-conotoxin RgIA when interacting with Arg7 and Arg9 of this ligand.

    conantokin-G and omega-conotoxin MVIIA are synergistic for antinociception

    Hama A, Sagen J. (2008) Antinociceptive effects of the marine snail peptides conantokin-G and conotoxin MVIIA alone and in combination in rat models of pain. Neuropharmacology 56: 556-563.

    The Miami Project to Cure Paralysis, University of Miami, Miller School of Medicine, 1095 NW 14th Terrace, R-48, Miami, FL 33136, USA. ahama@med.miami.edu

    Abstract: There are a number of neurologically active ion channel blocking peptides derived from cone snail venom, such as conantokin-G and omega-conotoxin MVIIA. Conantokin-G inhibits NMDA receptors containing the NR2B subunit whereas omega-conotoxin MVIIA blocks N-type Ca(2+) channels. Separately, these peptides induce antinociceptive effects in pre-clinical pain models following intrathecal injection. In the current study, the efficacies of these peptides were determined separately and in combination by intrathecal injection into rats with a spinal nerve ligation, in rats with a spinal cord compression injury and in the formalin test. Separately, both conantokin-G and omega-conotoxin MVIIA dose-dependently attenuated nociceptive responses in all of these models. However, at high antinociceptive doses for both formalin and nerve injury models, omega-conotoxin MVIIA evoked untoward side effects. Using isobolographic analysis, the combination of sub-antinociceptive doses of peptides demonstrated additive antinociception in rats with a nerve ligation and in the formalin test, without apparent adverse side effects. In a model of neuropathic spinal cord injury pain, which is clinically difficult to treat, the combination of conantokin-G and omega-conotoxin MVIIA resulted in robust synergistic antinociception. These data suggest that a combination of these peptides may be analgesic across diverse clinical pains with limited untoward side effects, and particularly potent for reducing spinal cord injury pain.

    The conantokin, con-G[S16Y], a potent analgesic

    Xiao C, Huang Y, Dong M, Hu J, Hou S, Castellino FJ, Prorok M, Dai Q (2008) NR2B-selective conantokin peptide inhibitors of the NMDA receptor display enhanced antinociceptive properties compared to non-selective conantokins. Neuropeptides 42: 601-609.

    Institute of Biotechnology, Beijing 100071, China.

    Abstract: NR2B-selective inhibitors show lower side-effects in preclinical pain models than non-selective NMDA receptor (NMDAR) antagonists, but it is unclear whether the improved safety of NR2B-selective inhibitors is due to their subtype selectivity or to a unique mode of inhibition of the receptor. In this study, the analgesic effects of intracerebral bolus injections of conantokin peptides with different NMDAR subunit selectivity were determined in mice by the standard hot-plate test, and following stimuli with acetic acid, formalin and complete Freund's adjuvant (CFA). In the standard hot-plate model, con-G[S16Y], a NR2B-selective inhibitor, showed the highest analgesic activity among conantokin peptides tested. In the acetic acid- and CFA-induced pain models, con-G[S16Y] and, to a lesser extent, con-G exhibited higher analgesic activity compared to non-selective inhibitors, such as con-R[1-17]. In the formalin test, while all conantokin peptides could partially suppress the first phase response, only con-G[S16Y] and con-G significantly inhibited the second phase response and suppressed paw edema. Our results suggest that the antinociceptive action of the conantokins may be related to their NR2B-selectivity and that these peptides may be useful as both neurobiological tools for probing mechanisms of nociception and as therapeutic agents for pain relief.

    Omega-conotoxin GVIA: mechanism of action

    Yarotskyy V, Elmslie KS (2009). omega-conotoxin GVIA alters gating charge movement of N-type (CaV2.2) calcium channels. J Neurophysiol. 101: 332-340

    Department of Anesthesiology, Penn State College of Medicine, Milton S. Hershey Medical Center, Hershey, PA 17033, USA.

    Abstract: Omega-conotoxin GVIA (omegaCTX) is a specific blocker of N-type calcium (CaV2.2) channels that inhibits neuropathic pain. While the toxin appears to be an open channel blocker, we show that N-channel gating charge movement is modulated. Gating currents were recorded from N-channels expressed along with beta2a and alpha2delta subunits in HEK293 cells in external solutions containing either lanthanum and magnesium (La-Mg) or 5 mM Ca2+ plus omegaCTX (omegaCTX-Ca). A comparison showed that omegaCTX induced a 10-mV right shift in the gating charge versus voltage (Q-V) relationship, smaller off-gating current time constant (tau Q(Off)), a lower tau Q(Off) voltage dependence, and smaller on-gating current (Q(On)) tau. We also examined gating current in La-Mg plus omegaCTX and found no significant difference from that in omegaCTX-Ca; this demonstrates that the modulation was induced by the toxin. A model with strongly reduced open-state occupancy reproduced the omegaCTX effect on gating current and showed that the gating modulation alone would inhibit N-current by 50%. This mechanism of N-channel inhibition could be exploited to develop novel analgesics that induce only a partial block of N-current, which may limit some of the side effects associated with the toxin analgesic currently approved for human use (i.e., Prialt).

    30 September 2008

    Intrathecal drug delivery for pain management

    Simpson KH, Jones I (2008). Intrathecal drug delivery for management of cancer and noncancer pain. J Opioid Manag. 4: 293-304.

    Pain Management Service, Seacroft Hospital, Leeds, United Kingdom.

    Abstract: Intrathecal drug delivery (ITDD) has been an option for the management of persistent pain since the 1980s. The discovery of opioid receptors in the central nervous system was the impetus for early attempts to deliver opioids intraspinally. Approximately, 10-20 percent patients with cancer pain get inadequate analgesia from conventional medical management; this group particularly may benefit from ITDD. However, there is also some evidence for the use of ITDD in those with noncancer pain. This review presents options for ITDD, available drugs, evidence for efficacy, principles of patient selection, and problems with the intrathecal route.

    Non-opioid treatments for management of pain: Review

    Fürst Z. (2008) [Central and peripheral mechanisms in antinociception: current and future perspectives]. Neuropsychopharmacol Hung. 10: 127-130. Review. Hungarian.

    Semmelweis Egyetem, Farmakológiai és Farmakoterápiás Intézet, Budapest, MTA-SE Neuropszichofarmakológiai Kutatócsoport. furzsu@pharma.sote.hu

    Abstract: As it is well known opioids are the most powerful drugs used for acute and chronic pain, although, their several serious side effects, such as respiratory depression, mental clouding, constipation, and tolerance dependence producing capacity, as well as large interpatient variability in responses limit their safe everyday use. Furthermore, the treatment of certain types of pain (e.g. neuropathic pain) is not very satisfactorily managed. Consequently, there is a continuous need to find analgesics efficient against chronic neuropathic pain and avoid these side actions and still retain opioid like potency. There are several possible way to find new targets for these purposes. Recently opioid receptors have been identified on peripheral processes of sensory neurons. These findings provide new insights into intrinsic mechanisms of pain control and suggest innovative strategies for developing drugs and alternative approaches to pain treatment. In the effort to discover better analgesic drugs for chronic pain, attention is being paid to specific ion channels at the periphery, include members of transient receptor potential family (TRPV1, capsaicin receptors), as well as P2x receptors, sensitive to purines released from tissue injury. A special tetradotoxin-resistant, voltage dependent type of sodium channel is associated with dorsal root ganglia neurons is blocked by mexiletine, used in chronic pain. A synthetic peptide analogue of marine snail toxin ziconitine blocks N-type calcium channels. GABA and NMDA receptors are also involved in the antinociceptive actions of gabapentin and ketamine, respectively. Furthermore nicotine and analogues (epibatidine) induce analgesia through nicotinic ACh receptors. We studied mostly the peripheral targets of hydrophilic heterocyclic opioids in antinociceptive processes.

    Novel M-superfamily of conotoxins from Conus bullatus

    Holford M, Zhang MM, Gowd KH, Azam L, Green BR, Watkins M, Ownby JP, Yoshikami D, Bulaj G, Olivera BM. (2009) Pruning nature: Biodiversity-derived discovery of novel sodium channel blocking conotoxins from Conus bullatus. Toxicon.53: 90-98.

    Department of Biology, University of Utah, 257 S. 1400 E. Rm. 114, Salt Lake City, UT 84112, USA. holford@biology.utah.edu

    Abstract: Described herein is a general approach to identify novel compounds using the biodiversity of a megadiverse group of animals; specifically, the phylogenetic lineage of the venomous gastropods that belong to the genus Conus ("cone snails"). Cone snail biodiversity was exploited to identify three new mu-conotoxins, BuIIIA, BuIIIB and BuIIIC, encoded by the fish-hunting species Conus bullatus. BuIIIA, BuIIIB and BuIIIC are strikingly divergent in their amino acid composition compared to previous mu-conotoxins known to target the voltage-gated Na channel skeletal muscle subtype Na(v)1.4. Our preliminary results indicate that BuIIIB and BuIIIC are potent inhibitors of Na(v)1.4 (average block approximately 96%, at a 1muM concentration of peptide), displaying a very slow off-rate not seen in previously characterized mu-conotoxins that block Na(v)1.4. In addition, the three new C. bullatus mu-conopeptides help to define a new branch of the M-superfamily of conotoxins, namely M-5. The exogene strategy used to discover these Na channel-inhibiting peptides was based on both understanding the phylogeny of Conus, as well as the molecular genetics of venom mu-conotoxin peptides previously shown to generally target voltage-gated Na channels. The discovery of BuIIIA, BuIIIB and BuIIIC Na channel blockers expands the diversity of ligands useful in determining the structure-activity relationship of voltage-gated sodium channels.

    P-conotoxin-like peptide, pa19a, from the venom duct of a Turrid snail

    Aguilar MB, de la Rosa RA, Falcón A, Olivera BM, Heimer de la Cotera EP (2009). Peptide pal9a from the venom of the turrid snail Polystira albida from the Gulf of Mexico: purification, characterization, and comparison with P-conotoxin-like (framework IX) conoidean peptides. Peptides. 30: 467-476.

    Laboratorio de Neurofarmacología Marina, Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de Mexico, Campus Juriquilla, Querétaro 76230, Mexico. maguilar@servidor.unam.mx

    Abstract: A novel peptide, pal9a, was purified from the venom duct extract of the turrid snail, Polystira albida (superfamily Conoidea, family Turridae), collected in the Gulf of Mexico. Its primary structure was determined by automated Edman degradation and confirmed by mass spectrometry. Turritoxin pal9a contains 34 amino acid residues, including 6 Cys residues arranged in the pattern C-C-C-C-C-C (framework IX, where "-" represents one or more non-Cys amino acids), which characterizes the P-conotoxins. Peptide pal9a is the first P-conotoxin-like turritoxin characterized from a member of family Turridae of the Western Atlantic. The primary structure of turritoxin pal9a, NVCDGDACPDGVCRSGCTCDFNVAQRKDTCFYPQ-nh(2) (-nh(2), amidated C-terminus; calculated monoisotopic mass, 3679.48Da; experimental monoisotopic mass, 3678.84Da), shows variable degrees of low sequence similarity with framework IX-toxins from turrid (three species of Lophiotoma, and four species of Gemmula), terebrid (Hastula hectica), and Conus species of the Indo-Pacific (C. textile, C. gloriamaris, C. amadis, and C. litteratus) and of the Western Atlantic (C. regius). During the comparison of peptide pal9a with the other framework IX-toxins known to date, we realized that, in general, these peptides are hydrophilic, acidic compounds that have not been found in the fish-hunting Conus species studied thus far; we also found support for the notion that they may belong to several distinct gene superfamilies, even those from the same species. Given the broad distribution of framework IX-toxins within superfamily Conoidea, it will be interesting to identify the still-unknown molecular targets of P-conotoxins, P-conotoxin-like turritoxins, and P-conotoxin-like augertoxins.

    NMR-based mapping of mu-conotoxin SxIIIA from Conus striatus

    Walewska A, Skalicky JJ, Davis DR, Zhang MM, Lopez-Vera E, Watkins M, Han TS, Yoshikami D, Olivera BM, Bulaj G. (2008))NMR-based mapping of disulfide bridges in cysteine-rich peptides: application to the mu-conotoxin SxIIIA. J Am Chem Soc. 130: 14280-14286.

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

    Abstract: Disulfide-rich peptides represent a megadiverse group of natural products with very promising therapeutic potential. To accelerate their functional characterization, high-throughput chemical synthesis and folding methods are required, including efficient mapping of multiple disulfide bridges. Here, we describe a novel approach for such mapping and apply it to a three-disulfide-bridged conotoxin, mu-SxIIIA (from the venom of Conus striolatus), whose discovery is also reported here for the first time. Mu-SxIIIA was chemically synthesized with three cysteine residues labeled 100% with (15)N/(13)C, while the remaining three cysteine residues were incorporated using a mixture of 70%/30% unlabeled/labeled Fmoc-protected residues. After oxidative folding, the major product was analyzed by NMR spectroscopy. Sequence-specific resonance assignments for the isotope-enriched Cys residues were determined with 2D versions of standard triple-resonance ((1)H, (13)C, (15)N) NMR experiments and 2D [(13)C, (1)H] HSQC. Disulfide patterns were directly determined with cross-disulfide NOEs confirming that the oxidation product had the disulfide connectivities characteristic of mu-conotoxins. Mu-SxIIIA was found to be a potent blocker of the sodium channel subtype Na(V)1.4 (IC50 = 7 nM). These results suggest that differential incorporation of isotope-labeled cysteine residues is an efficient strategy to map disulfides and should facilitate the discovery and structure-function studies of many bioactive peptides.

    mu-SIIIA: An analgesic mu-conotoxin from Conus striatus

    Yao S, Zhang MM, Yoshikami D, Azam L, Olivera BM, Bulaj G, Norton RS (2008). Structure, dynamics, and selectivity of the sodium channel blocker mu-conotoxin SIIIA. Biochemistry. 47: 10940-10949.

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

    Abstract: mu-SIIIA, a novel mu-conotoxin from Conus striatus, appeared to be a selective blocker of tetrodotoxin-resistant sodium channels in frog preparations. It also exhibited potent analgesic activity in mice, although its selectivity profile against mammalian sodium channels remains unknown. We have determined the structure of mu-SIIIA in aqueous solution and characterized its backbone dynamics by NMR and its functional properties electrophysiologically. Consistent with the absence of hydroxyprolines, mu-SIIIA adopts a single conformation with all peptide bonds in the trans conformation. The C-terminal region contains a well-defined helix encompassing residues 11-16, while residues 3-5 in the N-terminal region form a helix-like turn resembling 3 10-helix. The Trp12 and His16 side chains are close together, as in the related conotoxin mu-SmIIIA, but Asn2 is more distant. Dynamics measurements show that the N-terminus and Ser9 have larger-magnitude motions on the subnanosecond time scale, while the C-terminus is more rigid. Cys4, Trp12, and Cys13 undergo significant conformational exchange on microsecond to millisecond time scales. mu-SIIIA is a potent, nearly irreversible blocker of Na V1.2 but also blocks Na V1.4 and Na V1.6 with submicromolar potency. The selectivity profile of mu-SIIIA, including poor activity against the cardiac sodium channel, Na V1.5, is similar to that of the closely related mu-KIIIA, suggesting that the C-terminal regions of both are critical for blocking neuronal Na V1.2. The structural and functional characterization described in this paper of an analgesic mu-conotoxin that targets neuronal subtypes of mammalian sodium channels provides a basis for the design of novel analogues with an improved selectivity profile.

    31 August 2008

    Analgesic effects of Ph alpha 1beta, a calcium channel blocker from spider venom

    Souza AH, Ferreira J, Cordeiro Mdo N, Vieira LB, De Castro CJ, Trevisan G, Reis H, Souza IA, Richardson M, Prado MA, Prado VF, Gomez MV (2008). Analgesic effect in rodents of native and recombinant Ph alpha 1beta toxin, a high-voltage-activated calcium channel blocker isolated from armed spider venom. Pain 140: 115-126.

    Departamento de Farmacologia, ICB, UFMG, Belo Horizonte, MG, Brazil.

    Abstract: Calcium influx through neuronal voltage-sensitive calcium channels (VSCC S) mediates nociceptive information in the spinal dorsal horn. In fact, spinally administered VSCC S blockers, such as omega-conotoxin MVIIA, have analgesic effect apart of their low therapeutic index and many side effects. Here we study the analgesic potential of Ph alpha 1beta, a calcium channel blocker, in rodent models of acute and persistent pain. Spinally administered Ph alpha 1beta showed higher efficacy and long-lasting analgesia in a thermal model of pain, when compared with omega-conotoxin MVIIA. Moreover, Ph alpha 1beta was more effective and potent than omega-conotoxin MVIIA not only to prevent, but especially to reverse, previously installed persistent chemical and neuropathic pain. Furthermore, the analgesic action of both toxins are related with the inhibition of Ca2+-evoked release of pro-nociceptive neurotransmitter, glutamate, from rat spinal cord synaptosomes and decrease of glutamate overflow in cerebrospinal fluid. When side effects were assessed, we found that Ph alpha 1beta had a therapeutic index wider than omega- conotoxin MVIIA. Finally, recombinant Ph alpha 1beta expressed in Escherichia coli showed marked analgesic activity similar to the native toxin. Taken together, the present study demonstrates that native and recombinant Ph alpha 1beta have analgesic effects in rodent models of pain, suggesting that this toxin may have potential to be used as a drug in the control of persistent pathological pain.

    Omega conotoxin GVIA inhibits nicotinic response from deer chromaffin cells

    Douglas SA, Stevenson KE, Knowles PJ, Bunn SJ (2008). Characterization of catecholamine release from deer adrenal medullary chromaffin cells. Neurosci Lett. 445: 126-129.

    Department of Anatomy and Structural Biology, The University of Otago, P.O. Box 913, Dunedin, New Zealand.

    Abstract: Isolated adrenal medullary chromaffin cells maintained in culture have been widely used to study neurosecretory events. Many of these studies have been conducted using cells obtained from the bovine adrenal. In this study we have cultured chromaffin cells from an alternative large animal model, the deer, and have conducted the first characterization of secretion from this preparation. Cervine chromaffin cells, preloaded with [3H]noradrenalin, displayed a strong secretory response to the cholinergic agonist carbachol, with a maximal secretion of approximately 28% cell content over 15 min. This response was reproduced by nicotinic but not muscarinic agonists and was similarly inhibited by nicotinic but not muscarinic antagonists. Nicotine-evoked secretion measured over a 15 min time period was inhibited approximately 50% by the L-type Ca2+-channel antagonist nifedipine and approximately 20% by N-type (omega-conotoxin GVIA) or N, P/Q-type (omega-conotoxin MVIIC) antagonists. In contrast the response was unaffected by omega-agatoxin IVA, a P/Q-type antagonist. In addition to nicotinic receptor stimulation, activation of PACAP or histamine H1 receptors resulted in a concentration-dependent increase in secretion. PACAP was approximately two-fold more effective than histamine although both were weaker secretagogues than nicotine. In contrast, cervine chromaffin cells did not respond to angiotensin II or bradykinin, two agents known to stimulate secretion from bovine chromaffin cells. These data provide an initial characterization of the secretory response from cervine adrenal medullary chromaffin cells indicating that there are marked similarities but also potentially significant differences between them and their far more extensively described bovine counterparts.

    Discovery of new members of a Conus species complex

    Duda TF Jr, Bolin MB, Meyer CP, Kohn AJ (2008). Hidden diversity in a hyperdiverse gastropod genus: discovery of previously unidentified members of a Conus species complex. Mol Phylogenet Evol. 49: 867-876.

    Department of Ecology and Evolutionary Biology, Museum of Zoology, University of Michigan, 1109 Geddes Avenue, Ann Arbor, MI 48109, USA. tfduda@umich.edu

    Abstract: Molecular sequence data are a powerful tool for delimiting species, particularly in cases where morphological differences are obscure. Distinguishing species in the Conus sponsalis complex of tropical marine gastropods has long been difficult, because descriptions and identification has relied exclusively on shell characters, primarily color patterns, and these often appear to intergrade among putative species. Here we use molecular sequence data from two mitochondrial gene regions (16S rRNA and cytochrome oxidase subunit I) and one nuclear locus (a four-loop conotoxin gene) to characterize the genetic discontinuity of the nominal species of this group currently accepted as valid: the Indo-West Pacific C. sponsalis, C. nanus, C. ceylanensis, C. musicus and C. parvatus, and the eastern Pacific C. nux. In these analyses C. nanus and C. sponsalis resolve quite well and appear to represent distinct evolutionary units that are mostly congruent with morphology-based distinctions. We also identified several cryptic entities whose genetic uniqueness suggests species-level distinctions. Two of these fit the original description of C. sponsalis; three forms appear to represent C. nanus but differ in adult shell size or possess a unique shell color pattern.

    Flexibility of alpha conotoxin GI and analogues

    Jiang N, Ma J (2008). Conformational simulations of aqueous solvated alpha-conotoxin GI and its single disulfide analogues using a polarizable force field model. J Phys Chem A. 112: 9854-9867.

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

    Abstract: The solution conformation of alpha-conotoxin GI and its two single disulfide analogues are simulated using a polarizable force field in combination with the molecular fragmentation quantum chemical calculation. The polarizability is explicitly described by allowing the partial charges and fragment dipole moments to be variables, with values coming from the linear-scaling energy-based molecular fragmentation calculations at the B3LYP/6-31G(d) level. In comparison with the full quantum chemical calculations, the fragmentation approaches can yield precise ground-state energies, dipole moments, and static polarizabilities for peptides. The B3LYP/6-31G(d) charges and fragment-centered dipole moments are introduced in calculations of electrostatic terms in both AmberFF03 and OPLS force fields. Our test calculations on the gas-phase glucagon (PDB code: 1gcn) and solvated alpha-conotoxin GI (PDB code: 1not) demonstrate that the present polarization model is capable of describing the structural properties (such as the relative conformational energies, intramolecular hydrogen bonds, and disulfide bonds) with accuracy comparable to some other polarizable force fields (ABEEM/MM and OPLS-PFF) and the quantum mechanics/molecular mechanics (QM/MM) hybrid model. The employment of fragment-centered dipole moments in calculations of dipole-dipole interactions can save computational time in comparison with those polarization models using atom-centered dipole moments without much loss of accuracy. The molecular dynamics simulations using the polarizable force field demonstrate that two single disulfide GI analogues are more flexible and less structured than the native alpha-conotoxin GI, in agreement with NMR experiments. The polarization effect is important in simulations of the folding/unfolding process of solvated proteins.

    Alpha-conotoxin Arenatus IB[V11L,V16D] selectively inhibits alpha7 nicotinic receptors

    Innocent N, Livingstone PD, Hone A, Kimura A, Young T, Whiteaker P, McIntosh JM, Wonnacott S (2008). Alpha-conotoxin Arenatus IB[V11L,V16D] [corrected] is a potent and selective antagonist at rat and human native alpha7 nicotinic acetylcholine receptors. J Pharmacol Exp Ther. 327: 529-537. [Erratum in: J Pharmacol Exp Ther. 2008 Dec;327(3):1001]

    Department of Biology and Biochemistry, University of Bath, Bath BA27AY, UK. Erratum in: J Pharmacol Exp Ther. 2008 Dec;327(3):1001.

    Abstract: A recently developed alpha-conotoxin, alpha-conotoxin Arenatus IB-[V11L,V16D] (alpha-CtxArIB[V11L,V16D]) [corrected], is a potent and selective competitive antagonist at rat recombinant alpha7 nicotinic acetylcholine receptors (nAChRs), making it an attractive probe for this receptor subtype. alpha7 nAChRs are potential therapeutic targets that are widely expressed in both neuronal and non-neuronal tissues, where they are implicated in a variety of functions. In this study, we evaluate this toxin at rat and human native nAChRs. Functional alpha7 nAChR responses were evoked by choline plus the allosteric potentiator PNU-120596 [1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea] in rat PC12 cells and human SH-SY5Y cells loaded with calcium indicators. alpha-CtxArIB[V11L,V16D] specifically inhibited alpha7 nAChR-mediated increases in Ca2+ in PC12 cells. Responses to other stimuli, 5-I-A-85380 [5-iodo-3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride], nicotine, or KCl, that did not activate alpha7 nAChRs were unaffected. Human alpha7 nAChRs were also sensitive to alpha-CtxArIB[V11L, V16D]; acetylcholine-evoked currents in Xenopus laevis oocytes expressing human alpha7 nAChRs were inhibited by alpha-CtxArIB[V11L,V16D] (IC(50), 3.4 nM) in a slowly reversible manner, with full recovery taking 15 min. This is consistent with the time course of recovery from blockade of rat alpha7 nAChRs in PC12 cells. alpha-CtxArIB[V11L,V16D] inhibited human native alpha7 nAChRs in SHSY5Y cells, activated by either choline or AR-R17779 [(2)-spiro[1-azabicyclo[2.2.2]octane-3,59-oxazolidin]-29-one] plus PNU-120596. Rat brain alpha7 nAChRs contribute to dopamine release from striatal minces; alpha-CtxArIB[V11L,V16D] (300 nM) selectively inhibited choline-evoked dopamine release without affecting responses evoked by nicotine that activates heteromeric nAChRs. This study establishes that alpha-CtxArIB[V11L,V16D] selectively inhibits human and rat native alpha7 nAChRs with comparable potency, making this a potentially useful antagonist for investigating alpha7 nAChR functions.

    Asymmetric evolution of conotoxin loci in C. abbrevieatus and C. miliaris

    Duda TF Jr. (2008) Differentiation of venoms of predatory marine gastropods: divergence of orthologous toxin genes of closely related Conus species with different dietary specializations. J Mol Evol. 67: 315-321.

    University of Michigan Museum of Zoology, 1109 Geddes Avenue, Ann Arbor, MI 48109, USA. tfduda@umich.edu

    Abstract: Venoms of Conus are remarkably diverse among species and the genes that encode conotoxins show high rates of evolution. Yet no prior studies have specifically explored how conotoxin gene evolution contributes to the differentiation of venoms of closely related Conus species. Previous investigations of four-loop conotoxin expression patterns of six closely related Conus species identified 12 sets of putative orthologous loci from these species, including eight pairs of loci that are coexpressed by two of these six species, C. abbreviatus and C. miliaris. Here I analyze the molecular evolution of orthologous conotoxin loci of these species and specifically examine the divergence of the eight orthologous counterparts of C. abbreviatus and C. miliaris. Tree and maximum likelihood-based analyses of these sequences reveal that positive selection promotes the divergence of orthologous genes among species and that the evolution of orthologues of C. abbreviatus and C. miliaris is asymmetric among species. The asymmetric evolution of conotoxin loci among species may result from lineage-specific dietary shifts or interspecific differences in the impact of selection from predator-prey interactions on conotoxin loci.

    Effects of loop size on structure and function of alpha-conotoxins

    Jin AH, Daly NL, Nevin ST, Wang CI, Dutertre S, Lewis RJ, Adams DJ, Craik DJ, Alewood PF (2008). Molecular engineering of conotoxins: the importance of loop size to alpha-conotoxin structure and function. J Med Chem. 51: 5575-5584.

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

    Abstract: Alpha-conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). The majority of currently characterized alpha-conotoxins have a 4/7 loop size, and the major features of neuronal alpha-conotoxins include a globular disulfide connectivity and a helical structure centered around the third of their four cysteine residues. In this study, a novel "molecular pruning" approach was undertaken to define the relationship between loop size, structure, and function of alpha-conotoxins. This involved the systematic truncation of the second loop in the alpha-conotoxin [A10L]PnIA [4/7], a potent antagonist of the alpha7 nAChR. The penalty for truncation was found to be decreased conformational stability and increased susceptibility to disulfide bond scrambling. Truncation down to 4/4[A10L]PnIA maintained helicity and did not significantly reduce electrophysiological activity at alpha7 nAChRs, whereas 4/3[A10L]PnIA lost both alpha7 nAChR activity and helicity. In contrast, all truncated analogues lost approximately 100-fold affinity at the AChBP, a model protein for the extracellular domain of the nAChR. Docking simulations identified several hydrogen bonds lost upon truncation that provide an explanation for the reduced affinities observed at the alpha7 nAChR and AChBP.

    Salivary gland specific alpha-conotoxins in Conus pulicarius

    31 July 2008

    Biggs JS, Olivera BM, Kantor YI (2008). Alpha-conopeptides specifically expressed in the salivary gland of Conus pulicarius. Toxicon. 52: 101-105.

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

    Abstract: To date, studies conducted on cone snail venoms have attributed the origins of this complex mixture of neuroactive peptides entirely to gene expression by the secretory cells lining the lumen of the venom duct. However, specialized tissues such as the salivary glands also secrete their contents into the anterior gut and could potentially contribute some venom components injected into target animals; evidence supporting this possibility is reported here. Sequence analysis of a cDNA library created from a salivary gland of Conus pulicarius revealed the expression of two transcripts whose predicted gene products, after post-translational processing, strikingly resemble mature conopeptides belonging to the alpha-conotoxin family. These two transcripts, like alpha-conotoxin transcripts, putatively encode mature peptides containing the conserved A-superfamily cysteine pattern (CC-C-C) but the highly conserved A-superfamily signal sequences were not present. Analysis of A-superfamily members expressed in the venom duct of the same C. pulicarius specimens revealed three putative alpha-conotoxin sequences; the salivary gland transcripts were not found in the venom duct cDNA library, suggesting that these alpha-conotoxins are salivary gland specific. Therefore, expression of conotoxin-like gene products by the salivary gland could potentially add to the complexity of Conus venoms.

    omega-Conotoxin CVIB, a reversible channel inhibitor

    Motin L, Adams DJ (2008). omega-Conotoxin inhibition of excitatory synaptic transmission evoked by dorsal root stimulation in rat superficial dorsal horn. Neuropharmacology 55: 860-864. Queensland Brain Institute, University of Queensland, Brisbane, Queensland 4072, Australia.

    Abstract: A number of omega-conotoxins are potent and selective antagonists of N-type voltage-gated calcium channels (VGCCs) and are potentially effective as analgesic agents. omega-Conotoxins CVID and CVIB, venom peptides from Conus catus, inhibit N-type and N/P/Q-type VGCCs, respectively, in rat dorsal root ganglion sensory neurons. In the present study, we tested the effects of five different omega-conotoxins, CVID, CVIB, MVIIA, MVIIC and GVIA, on excitatory synaptic transmission between primary afferents and dorsal horn superficial lamina neurons of rat spinal cord. The N-type VGCC antagonists CVID (200nM) and MVIIA (500nM) completely and irreversibly inhibited excitatory postsynaptic currents (EPSCs) in the dorsal horn superficial lamina. The N- and P/Q-type VGCC antagonist CVIB (200nM) reversibly reduced evoked EPSC amplitude an average of 34+/-8%, whereas MVIIC (200nM) had no effect on excitatory synaptic transmission. In neurons receiving polysynaptic input, CVIB reduced both the EPSC amplitude and the "success rate" calculated as the relative number of primary afferent stimulations that resulted in postsynaptic responses. These results indicate that (i) the analgesic action of omega-conotoxins that antagonise N-type VGCCs may be attributed to inhibition of neurotransmission between primary afferents and superficial dorsal horn neurons, (ii) nociceptive synaptic transmission between primary afferents and superficial lamina neurons is mediated predominantly by N-type VGCCs, and (iii) in contrast to the irreversible inhibition by CVID, MVIIA and GVIA, the inhibition of excitatory monosynaptic transmission by CVIB is reversible.

    Actions of local anaesthetics at sodium channels

    Bruhova I, Tikhonov DB, Zhorov BS (2008). Access and binding of local anesthetics in the closed sodium channel. Mol Pharmacol. 74: 1033-1045.

    Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.

    Abstract: Local anesthetics (LAs) are known to bind Na+ channels in the closed, open, and inactivated states and reach their binding sites via extracellular and intracellular access pathways. Despite intensive studies, no atomic-scale theory is available to explain the diverse experimental data on the LA actions. Here we attempt to contribute to this theory by simulating access and binding of LAs in the KcsA-based homology model of the closed Na+ channel. We used Monte Carlo minimizations to model the channel with representative local anesthetics N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium (QX-314), cocaine, and tetracaine. We found the nucleophilic central cavity to be a common binding region for the ammonium group of LAs, whose aromatic group can extend either along the pore axis (vertical binding mode) or to the III/IV domain interface (horizontal binding mode). The vertical mode was earlier predicted for the open channel, but only the horizontal mode is consistent with mutational data on the closed-channel block. To explore hypothetical access pathways of the permanently charged QX-314, we pulled the ligand via the selectivity filter, the closed activation gate, and the III/IV domain interface. Only the last pathway, which leads to the horizontal binding mode, did not impose steric obstacles. The LA ammonium group mobility within the central cavity was more restricted in the vertical mode than in the horizontal mode. Therefore, occupation of the selectivity-filter DEKA locus by a Na+ ion destabilizes the vertical mode, thus favoring the horizontal mode. LA binding in the closed channel requires the resident Na+ ion to leave the nucleophilic central cavity through the selectivity filter, whereas the LA egress should be coupled with reoccupation of the cavity by Na+. This hypothesis on the coupled movement of Na+ and LA in the closed channel explains seemingly contradictory data on how the outer-pore mutations as well as tetrodotoxin and micro-conotoxin binding affect the ingress and egress of LAs.

    Interactions between mu-conotoxins and internal amine blockers at VGSCs

    Pavlov E, Britvina T, McArthur JR, Ma Q, Sierralta I, Zamponi GW, French RJ (2008). Trans-channel interactions in batrachotoxin-modified skeletal muscle sodium channels: voltage-dependent block by cytoplasmic amines, and the influence of mu-conotoxin GIIIA derivatives and permeant ions. Biophys J. 95: 4277-4288.

    Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada.

    Abstract: External mu-conotoxins and internal amine blockers inhibit each other's block of voltage-gated sodium channels. We explore the basis of this interaction by measuring the shifts in voltage-dependence of channel inhibition by internal amines induced by two mu-conotoxin derivatives with different charge distributions and net charges. Charge changes on the toxin were made at residue 13, which is thought to penetrate most deeply into the channel, making it likely to have the strongest individual interaction with an internal charged ligand. When an R13Q or R13E molecule was bound to the channel, the voltage dependence of diethylammonium (DEA)-block shifted toward more depolarized potentials (23 mV for R13Q, and 16 mV for R13E). An electrostatic model of the repulsion between DEA and the toxin simulated these data, with a distance between residue 13 of the mu-conotoxin and the DEA-binding site of approximately 15 A. Surprisingly, for tetrapropylammonium, the shifts were only 9 mV for R13Q, and 7 mV for R13E. The smaller shifts associated with R13E, the toxin with a smaller net charge, are generally consistent with an electrostatic interaction. However, the smaller shifts observed for tetrapropylammonium than for DEA suggest that other factors must be involved. Two observations indicate that the coupling of permeant ion occupancy of the channel to blocker binding may contribute to the overall amine-toxin interaction: 1), R13Q binding decreases the apparent affinity of sodium for the conducting pore by approximately 4-fold; and 2), increasing external [Na(+)] decreases block by DEA at constant voltage. Thus, even though a number of studies suggest that sodium channels are occupied by no more than one ion most of the time, measurable coupling occurs between permeant ions and toxin or amine blockers. Such interactions likely determine, in part, the strength of trans-channel, amine-conotoxin interactions.

    Mu-conotoxin GIIIA and sodium channel amine blockers interact

    Ma Q, Pavlov E, Britvina T, Zamponi GW, French RJ (2008). Trans-channel interactions in batrachotoxin-modified rat skeletal muscle sodium channels: kinetic analysis of mutual inhibition between mu-conotoxin GIIIA derivatives and amine blockers. Biophys J. 95: 4266-4276.

    Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada.

    Abstract: R13X derivatives of mu-conotoxin GIIIA bind externally to single sodium channels and block current incompletely with mean "blocked" durations of several seconds. We studied interactions between two classes of blockers (mu-conotoxins and amines) by steady state, kinetic analysis of block of BTX-modified Na channels in planar bilayers. The amines cause all-or-none block at a site internal to the selectivity filter. TPrA and DEA block single Na channels with very different kinetics. TPrA induces discrete, all-or-none, blocked events (mean blocked durations, approximately 100 ms), whereas DEA produces a concentration-dependent reduction of the apparent single channel amplitude ("fast" block). These distinct modes of action allow simultaneous evaluation of block by TPrA and DEA, showing a classical, competitive interaction between them. The apparent affinity of TPrA decreases with increasing [DEA], based on a decrease in the association rate for TPrA. When an R13X mu-conotoxin derivative and one of the amines are applied simultaneously on opposite sides of the membrane, a mutually inhibitory interaction is observed. Dissociation constants, at +50 mV, for TPrA ( approximately 4 mM) and DEA ( approximately 30 mM) increase by approximately 20%-50% when R13E (nominal net charge, +4) or R13Q (+5) is bound. Analysis of the slow blocking kinetics for the two toxin derivatives showed comparable decreases in affinity of the mu-conotoxins in the presence of an amine. Although this mutual inhibition seems to be qualitatively consistent with an electrostatic interaction across the selectivity filter, quantitative considerations raise questions about the mechanistic details of the interaction.

    30 June 2008

    Novel S-Superfamily conotoxins from Conus caracteristicus

    Liu L, Wu X, Yuan D, Chi C, Wang C. (2008) Identification of a novel S-superfamily conotoxin from vermivorous Conus caracteristicus.<>i Toxicon. 51: 1331-1337.

    Institute of Protein Research, College of Life Sciences and Technology, Tongji University, 1239 Siping Road, Shanghai 200092, PR China.

    Abstract: A new conotoxin, ca16a, containing 8 cysteine residues was purified, sequenced, and cloned from a worm-hunting snail, Conus caracteristicus. This conotoxin is an extremely hydrophilic peptide comprising 34 residues, with 4 acidic and 4 basic residues. It is rich in polar Gly, Ser, and Thr residues and includes a hydroxylated Pro residue. The cysteine arrangement pattern of ca16a (-C-C-CC-C-CC-C-, designated as framework #16) is distinct from that of other known conotoxins. Furthermore, the signal peptide sequence of this conotoxin does not share any homology with those of other conotoxins. Leu residues account for almost 50% of its 20-residue signal peptide. The unique cysteine framework and signal peptide sequence of ca16a suggest that it belongs to a new conotoxin superfamily.

    alpha-conotoxin MII[E11A], an alpha6-selective antagonist

    Meyer EL, Yoshikami D, McIntosh JM (2008). The neuronal nicotinic acetylcholine receptors alpha 4* and alpha 6* differentially modulate dopamine release in mouse striatal slices. J Neurochem. 105: 1761-1769

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

    Abstract: Striatal dopamine (DA) plays a major role in the regulation of motor coordination and in the processing of salient information. We used voltammetry to monitor DA-release evoked by electrical stimulation in striatal slices, where interneurons continuously release acetylcholine. Use of the alpha6-selective antagonist alpha-conotoxin MII[E11A] and alpha4 knockout mice enabled identification of two populations of DA-ergic fibers. The first population had a low action potential threshold, and action potential-evoked DA-release from these fibers was modulated by alpha6. The second population had a higher action potential threshold, and only alpha4(non-alpha6) modulated action potential-evoked DA-release. Striatal DA-ergic neurons fire in both tonic and phasic patterns. When stimuli were applied in a train to mimic phasic firing, more DA-release was observed in alpha4 knockout versus wild-type mice. Furthermore, block of alpha4(non-alpha6), but not of alpha6, increased DA release evoked by a train. These results indicate that there are different classes of striatal DA-ergic fibers that express different subtypes of nicotinic receptors.

    delta- and omega-conotoxins from Conus achatinus

    Gowd KH, Dewan KK, Iengar P, Krishnan KS, Balaram P (2008). Probing peptide libraries from Conus achatinus using mass spectrometry and cDNA sequencing: identification of delta and omega-conotoxins. J Mass Spectrom. 43: 791-805.

    Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India.

    Abstract: The peptide library present in the venom of the piscivorous marine snail Conus achatinus has been probed using a combination of mass spectrometry and cDNA sequencing methods. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis, before and following global reduction/alkylation of peptide mixtures, permits the rapid classification of individual components on the basis of the number of disulfide bonds. Mass fingerprinting and the reverse phase HPLC retention times permit a further deconvolution of the library in terms of peptide size and hydrophobicity. Sequencing of cDNA derived using O-superfamily specific primers yielded five complete conotoxin precursor sequences, ranging in polypeptide length from 75-87 residues containing six Cys residues at the C-terminus. Sequence analysis permits classification of the five putative mature peptides (Ac 6.1 to Ac 6.5) as delta, omega, and omega-like conotoxins. The presence of these predicted peptides in crude venom was established by direct matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) sequencing following trypsin digestion of the peptide mixture after global reduction/alkylation. The determination of partial peptide sequences and comparison with the predicted sequences resulted in the identification of four of the five predicted conotoxins. The characterization of posttranslationally modified analogs, which are hydroxylated at proline or amidated at the C-terminus is also demonstrated. Crude venom analysis should prove powerful in studying both inter- and intra-species variation in peptide libraries.

    V-superfamily of conotoxins from Conus virgo

    Peng C, Liu L, Shao X, Chi C, Wang C (2008). Identification of a novel class of conotoxins defined as V-conotoxins with a unique cysteine pattern and signal peptide sequence. Peptides 29: 985-991.

    Institute of Protein Research, Tongji University, 1239 Siping Road, Shanghai 200092, China.

    Abstract: Cone snails are predatory gastropod mollusks distributed in all tropical marine habitats with a highly sophisticated defense strategy using small peptides in their venoms. Here, we report the discovery and initial characterization of the V-superfamily conotoxins. A novel conotoxin vi15a was purified from the venom of a worm-hunting species Conus virgo. The sequence of vi15a was determined to have a unique arrangement of cysteine residues (C-C-CC-C-C-C-C), which defines the new V-superfamily conotoxins. The cDNA of vi15a was cloned with RACE method. Its unique signal peptide sequence led to the cloning of another V-superfamily conotoxin, Vt15.1, from Conus vitulinus. These results, as well as the existence of Lt15.1 from Conus litteratus and ca15a from Conus caracteristicus with the same cysteine pattern, suggest that V-superfamily might be a large and diverse group of peptides widely distributed in different Conus species. Like other eight Cys-containing toxins, V-superfamily conotoxins might also adopt an "ICK+1" disulfide bond connectivity. The identification of this novel class of conotoxins will certainly improve our understanding of the structure diversity of disulfide rich toxins.

    alpha-conotoxin Ar Ib[V11L;V16A] selectively inbibits alpha-7 nAChRs

    Whiteaker P, Marks MJ, Christensen S, Dowell C, Collins AC, McIntosh JM (2008). Synthesis and characterization of 125I-alpha-conotoxin ArIB[V11L;V16A], a selective alpha7 nicotinic acetylcholine receptor antagonist. J Pharmacol Exp Ther. 325: 910-919

    Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona, USA.

    Abstract: The alpha7 nicotinic acetylcholine receptors (nAChRs) are widely expressed both in the central nervous system (CNS) and periphery. In the CNS, 125I-alpha-bungarotoxin is commonly used to identify alpha7 nAChRs specifically. However, alpha-bungarotoxin also interacts potently with alpha1* and alpha9alpha10 nAChRs, two receptor subtypes in peripheral tissues that are colocalized with the alpha7 subtype. [3H]Methyllycaconitine is also frequently used as an alpha7-selective antagonist, but it has significant affinity for alpha6* and alpha9alpha10 nAChR subtypes. In this study, we have developed a highly alpha7-selective alpha-conotoxin radioligand by iodination of a naturally occurring histidine. Both mono- and diiodo derivatives were generated and purified (specific activities were 2200 and 4400 Ci mmol(-1), respectively). The properties of the mono- and diiodo derivatives were very similar to each other, but the diiodo was less stable. For monoidodo peptide, saturation binding to mouse hippocampal membranes demonstrated a K(d) value of 1.15 +/- 0.13 nM, similar to that of 125I-alpha-bungarotoxin in the same preparations (0.52 +/- 0.16 nM). Association and dissociation kinetics were relatively rapid (k(obs) for association at 1 nM was 0.027 +/- 0.007 min(-1); k(off) = 0.020 +/- 0.001 min(-1)). Selectivity was confirmed with autoradiography using alpha7-null mutant tissue: specific binding was abolished in all regions of alpha7(-/-) brains, whereas wild-type mice expressed high levels of labeling and low nonspecific binding. 125I-alpha-conotoxin ArIB[V11L; V16A] should prove useful where alpha7 nAChRs are coexpressed with other subtypes that are also labeled by existing ligands. Furthermore, true equilibrium binding experiments could be performed on alpha7 nAChRs, something that is impossible with 125I-alpha-bungarotoxin.

    Alpha-conotoxin MII modifies addictive behaviours

    Jerlhag E, Egecioglu E, Dickson SL, Svensson L, Engel JA (2008). Alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors are involved in mediating the ghrelin-induced locomotor stimulation and dopamine overflow in nucleus accumbens. Eur Neuropsychopharmacol. 18: 508-518

    Abstract: Section for Pharmacology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at Göteborg University, SE-405 30 Göteborg, Sweden. elisabet.jerlhag@pharm.gu.se

    Previously, we have reported that the orexigenic peptide ghrelin activates the cholinergic-dopaminergic reward link, involving nicotinic acetylcholine receptors (nAChR). The alpha(3)-alpha(7) and beta(2)-beta(4) subunits of the nAChR can be combined into pentameric nAChRs, with different functional roles. The present experiments show that the locomotor stimulatory effects of ghrelin, either into laterodorsal tegmental area (LDTg) or ventral tegmental area (VTA), are mediated via ventral tegmental nAChR, but neither the alpha(4)beta(2) (using dihydro-beta-erythroidine) nor the alpha(7) (using methyllycaconitine) subtypes appears to be involved. On the other hand, the alpha(3)beta(2), beta(3) and/or alpha(6) (using alpha-conotoxin MII) subtypes in the VTA mediate the stimulatory and DA-enhancing effects of ghrelin, a pattern that ghrelin shares with ethanol (n=5-8). Radioligand-binding experiments shown that ghrelin does not interfere directly with nAChRs (n=26). We therefore suggest that the alpha(3)beta(2), beta(3) and/or alpha(6) subtypes might be pharmacological targets for treatment of addictive behaviours including compulsive overeating and alcoholism.

    10 June 2008

    alpha-Conotoxin Lp1.1 from Conus leopardus

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

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

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

    Conotoxins Vc1.1 and RgIA: agonists at GABA(B) receptors

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

    Queensland Brain Institute and School of Biomedical Sciences, and Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.

    Abstract: alpha-Conotoxins Vc1.1 and Rg1A are peptides from the venom of marine Conus snails that are currently in development as a treatment for neuropathic pain. Here we report that the alpha9alpha10 nicotinic acetylcholine receptor-selective conotoxins Vc1.1 and Rg1A potently and selectively inhibit high-voltage-activated (HVA) calcium channel currents in dissociated DRG neurons in a concentration-dependent manner. The post-translationally modified peptides vc1a and [P6O]Vc1.1 were inactive, as were all other alpha-conotoxins tested. Vc1.1 inhibited the omega-conotoxin-sensitive HVA currents in DRG neurons but not those recorded from Xenopus oocytes expressing Ca(V)2.2, Ca(V)2.1, Ca(V)2.3, or Ca(V)1.2 channels. Inhibition of HVA currents by Vc1.1 was not reversed by depolarizing prepulses but was abolished by pertussis toxin (PTX), intracellular GDPbetaS, or a selective inhibitor of pp60c-src tyrosine kinase. These data indicate that Vc1.1 does not interact with N-type calcium channels directly but inhibits them via a voltage-independent mechanism involving a PTX-sensitive, G-protein-coupled receptor. Preincubation with a variety of selective receptor antagonists demonstrated that only the GABA(B) receptor antagonists, [S-(R*,R*)][-3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxy propyl]([3,4]-cyclohexylmethyl) phosphinic acid hydrochloride (2S)-3[[(1S)-1-(3,4-dichlorophenyl)-ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid and phaclofen, blocked the effect of Vc1.1 and Rg1A on Ca2+ channel currents. Together, the results identify Ca(V)2.2 as a target of Vc1.1 and Rg1A, potentially mediating their analgesic actions. We propose a novel mechanism by which alpha-conotoxins Vc1.1 and Rg1A modulate native N-type (Ca(V)2.2) Ca2+ channel currents, namely acting as agonists via G-protein-coupled GABA(B) receptors.

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

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

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

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

    Novel conotoxin family from Conus distans

    Chen P, Garrett JE, Watkins M, Olivera BM (2008) Purification and characterization of a novel excitatory peptide from Conus distans venom that defines a novel gene superfamily of conotoxins. Toxicon. 52: 139-145

    Department of Biology, University of Utah, 257 South, 1400 East, Salt Lake City, UT 84112, USA. pc15@utah.edu

    Abstract: An excitatory peptide, di16a, with 49 amino acids and 10 cysteine residues was purified and characterized from the venom of Conus distans. Five AA residues were modified: one gamma-carboxyglutamate (Gla), and four hydroxyproline (Hyp) residues. A cDNA clone encoding the precursor for the peptide was characterized; the peptide has a novel cysteine framework and a distinctive signal sequence that differs from any other conotoxin superfamily. The peptide was chemically synthesized and folded, and synthetic and native materials were shown to co-elute. Injection of the synthetic peptide causes a hyperexcitable phenotype in mice greater than 3 weeks of age at lower doses, and lethargy at higher doses. The peptide defines both a previously uncharacterized gene superfamily of conopeptides, and a new Cys pattern with three vicinal Cys residues.

    Two novel conotoxins from Conus caracteristicus

    Yuan DD, Liu L, Shao XX, Peng C, Chi CW, Guo ZY. (2009) New conotoxins define the novel I3-superfamily. Peptides. 30:861-865..

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

    Abstract: We purified two novel conotoxins, designated as ca11a and ca11b, from the venom of Conus caracteristicus. Based on the amino acid sequence of mature ca11a, we cloned its full-length cDNA. Based on the signal peptide of ca11a, several ca11a-like conotoxins were cloned from C. caracteristicus and C. pulicarius. These novel conotoxins have an I-superfamily cysteine pattern but with a novel signal peptide sequence, suggesting they belong to a new branch of I-superfamily, designated as I(3)-superfamily. Additionally, two O-superfamily conotoxins were also cloned based on the signal peptide of ca11a, suggesting a possible evolutionary relationship between O- and I-superfamilies.

    Novel conotoxin family from Conus caracteristicus

    Yuan DD, Liu L, Shao XX, Peng C, Chi CW, Guo ZY (2008) Isolation and cloning of a conotoxin with a novel cysteine pattern from Conus caracteristicus. Peptides. 29:1521-1525.

    Institute of Protein Research, College of Life Sciences and Technology, Tongji University, 1239 Siping Road, Shanghai 200092, PR China.

    Abstract: A new conotoxin, ca16a, containing 8 cysteine residues was purified, sequenced, and cloned from a worm-hunting snail, Conus caracteristicus. This conotoxin is an extremely hydrophilic peptide comprising 34 residues, with 4 acidic and 4 basic residues. It is rich in polar Gly, Ser, and Thr residues and includes a hydroxylated Pro residue. The cysteine arrangement pattern of ca16a (-C-C-CC-C-CC-C-, designated as framework #16) is distinct from that of other known conotoxins. Furthermore, the signal peptide sequence of this conotoxin does not share any homology with those of other conotoxins. Leu residues account for almost 50% of its 20-residue signal peptide. The unique cysteine framework and signal peptide sequence of ca16a suggest that it belongs to a new conotoxin superfamily.

    alphaA(S)-conotoxin analog, alphaA-OIVA[K15N], a selective high-affinity inhibitor of fetal muscle nAChRs

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

    Department of Biology, University of Utah, Salt Lake City, Utah, USA. russ_teichert@yahoo.com

    Abstract: Although the neuromuscular nicotinic acetylcholine receptor (nAChR) is one of the most intensively studied ion channels in the nervous system, the differential roles of fetal and adult subtypes of the nAChR under normal and pathological conditions are still incompletely defined. Until recently, no pharmacological tools distinguished between fetal and adult subtypes. Waglerin toxins (from snake venom) and alphaA(S)-conotoxins (from cone-snail venom) have provided such tools. Because these peptides were characterized by different research groups using different methods, we have: 1) more extensively tested their subtype selectivity, and 2) begun to explore how these peptides may be used in concert to elucidate expression patterns and functions of fetal and adult nAChRs. In heterologous expression systems and native tissues, Waglerin-1 and an alphaA(S)-conotoxin analog, alphaA-OIVA[K15N], are high-affinity, highly selective inhibitors of the adult and fetal muscle nAChRs, respectively. We have used the peptides and their fluorescent derivatives to explore the expression and function of the fetal and adult nAChR subtypes. While fluorescent derivatives of these peptides indicated a gradual transition from fetal to adult muscle nAChRs in mice during the first 2 weeks postnatal, we unexpectedly observed a steeper transition in functional expression in the mouse diaphragm muscle using electrophysiology. As a toolkit of pharmacological agents with complementary specificity, alphaA-OIVA[K15N] and Waglerin-1 should have further utility in determining the roles of fetal and adult nAChR subtypes in development, in mature tissues, and under pathological conditions.

    A novel conotoxin lt6c from Conus litteratus blocks Na channels

    Wang L, Pi C, Liu J, Chen S, Peng C, Sun D, Zhou M, Xiang H, Ren Z, Xu A (2008) Identification and characterization of a novel O-superfamily conotoxin from Conus litteratus. J Pept Sci. 14:1077-1083.

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

    Abstract: A novel conotoxin named lt6c, an O-superfamily conotoxin, was identified from the cDNA library of venom duct of Conus litteratus. The full-length cDNA contains an open reading frame encoding a predicted 22-residue signal peptide, a 22-residue proregion and a mature peptide of 28 amino acids. The signal peptide sequence of lt6c is highly conserved in O-superfamily conotoxins and the mature peptide consists of six cysteines arranged in the pattern of C-C-CC-C-C that is defined the O-superfamily of conotoxins. The mature peptide fused with thioredoxin, 6-His tag, and a Factor Xa cleavage site was successfully expressed in Escherichia coli. About 12 mg lt6c was purified from 1L culture. Under whole-cell patch-clamp mode, lt6c inhibited sodium currents on adult rat dorsal root ganglion neurons. Therefore, lt6c is a novel O-superfamily conotoxin that is able to block sodium channels.

    mu-conotoxins SIIIA and SIIIB from Conus striatus blocks Na channels

    Schroeder CI, Ekberg J, Nielsen KJ, Adams D, Loughnan ML, Thomas L, Adams DJ, Alewood PF, Lewis RJ (2008) Neuronally selective mu -conotoxins from Conus striatus utlise an alpha-helical motif to target mammalian sodium channels. J Biol Chem. 283:21621-21628.

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

    Abstract: Mu-conotoxins are small peptide inhibitors of muscle and neuronal tetrodotoxin (TTX)-sensitive voltage-gated sodium channels (VGSCs). Here we report the isolation of mu-conotoxins SIIIA and SIIIB by (125)I-TIIIA-guided fractionation of milked Conus striatus venom. SIIIA and SIIIB potently displaced (125)I-TIIIA from native rat brain Na(v)1.2 (IC(50) values 10 and 5 nm, respectively) and muscle Na(v)1.4 (IC(50) values 60 and 3 nm, respectively) VGSCs, and both inhibited current through Xenopus oocyte-expressed Na(v)1.2 and Na(v)1.4. An alanine scan of SIIIA-(2-20), a pyroglutamate-truncated analogue with enhanced neuronal activity, revealed residues important for affinity and selectivity. Alanine replacement of the solvent-exposed Trp-12, Arg-14, His-16, Arg-18 resulted in large reductions in SIIIA-(2-20) affinity, with His-16 replacement affecting structure. In contrast, [D15A]SIIIA-(2-20) had significantly enhanced neuronal affinity (IC(50) 0.65 nm), while the double mutant [D15A/H16R]SIIIA-(2-20) showed greatest Na(v)1.2 versus 1.4 selectivity (136-fold). (1)H NMR studies revealed that SIIIA adopted a single conformation in solution comprising a series of turns and an alpha-helical motif across residues 11-16 that is not found in larger mu-conotoxins. The structure of SIIIA provides a new structural template for the development of neuronally selective inhibitors of TTX-sensitive VGSCs based on the smaller mu-conotoxin pharmacophore.

    Ziconotide - UPDATE & REVIEW

    Williams JA, Day M, Heavner JE (2008) Ziconotide: an update and review. Expert Opin Pharmacother. 9:1575-1583.

    Texas Tech University Health Sciences Center, 3601 Fourth Street, MS 818, Lubbock, TX 79430, USA.

    Abstract: BACKGROUND: Ziconotide is the only N-type calcium channel blocker approved by the US FDA for the treatment of chronic pain. The approved indication is for the management of severe chronic pain in patients for whom intrathecal therapy is warranted and who are intolerant of or refractory to other treatments such as systemic analgesics, adjunctive therapies or intrathecal morphine. OBJECTIVE: The purpose of this article was to review the available safety, efficacy and dosing information for ziconotide. METHODS: The sources searched for literature from 1980 to January 2008 included Pub Med, MEDLINE and PREMEDLINE using the words ziconotide, conotoxins and pain. RESULTS/CONCLUSION: Ziconotide is administered intrathecally by infusion pump to block nociceptive signal transmission in the spinal cord. It is a synthetic neuroactive peptide equivalent to the omega conotoxin MVIIA, a constituent of the venom of the fish-hunting marine snail Conus magus. It is highly potent, has a steep dose-response curve, a slow onset of action and a narrow margin of safety and responses to dose adjustments are slow. Patients receiving ziconotide should be under the care of physicians experienced in the management of intrathecal infusion therapy for pain control and should have convenient access to medical facilities.

    Conopeptide Y family of Conus planorbis and Conus ferrugineus

    Imperial JS, Chen P, Sporning A, Terlau H, Daly NL, Craik DJ, Alewood PF, Olivera BM (2008) Tyrosine-rich conopeptides affect voltage-gated K+ channels. J Biol Chem. 283: 23026-23032.

    Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA. imperial@biology.utah.edu

    Abstract:Two venom peptides, CPY-Pl1 (EU000528) and CPY-Fe1 (EU000529), characterized from the vermivorous marine snails Conus planorbis and Conus ferrugineus, define a new class of conopeptides, the conopeptide Y (CPY) family. The peptides have no disulfide cross-links and are 30 amino acids long; the high content of tyrosine is unprecedented for any native gene product. The CPY peptides were chemically synthesized and shown to be biologically active upon injection into both mice and Caenorhabditis elegans; activity on mammalian Kv1 channel isoforms was demonstrated using an oocyte heterologous expression system, and selectivity for Kv1.6 was found. NMR spectroscopy revealed that the peptides were unstructured in aqueous solution; however, a helical region including residues 12-18 for one peptide, CPY-Pl1, formed in trifluoroethanol buffer. Clones obtained from cDNA of both species encoded prepropeptide precursors that shared a unique signal sequence, indicating that these peptides are encoded by a novel gene family. This is the first report of tyrosine-rich bioactive peptides in Conus venom.

    Conotoxin gene family evolution

    Duda TF Jr, Remigio EA (2008) Variation and evolution of toxin gene expression patterns of six closely related venomous marine snails. Mol Ecol. 17:3018-3032.

    Department of Ecology and Evolutionary Biology & Museum of Zoology, University of Michigan, 1109 Geddes Avenue, Ann Arbor, MI 48109, USA. tfduda@umich.edu

    Abstract: Venoms of predatory marine gastropods of the genus Conus show amazing levels of interspecific diversity and are comprised of a cocktail of peptide neurotoxins, termed conotoxins, that are encoded by large gene families. Conotoxin gene family evolution is characterized by gene duplications and high rates of nonsynonymous substitution among paralogues; yet, what controls the differentiation of venoms among species is not clear. We compared four-loop conotoxin transcripts of six closely related Conus species to examine conotoxin expression patterns among species. The species examined appear to express different numbers of four-loop conotoxin loci and similarity in expression patterns does not seem to correspond with phylogenetic affinity. Moreover, several loci appear to have been independently silenced while others appear to have been revived from previously silenced states. Some loci also appear to exhibit coordinated expression patterns. These results suggest that the evolution of conotoxin expression patterns is incredibly dynamic and the differentiation of venoms of Conus is controlled in part by the evolution of unique conotoxin expression patterns.

    iota-Conotoxin RXIA

    Fiedler B, Zhang MM, Buczek O, Azam L, Bulaj G, Norton RS, Olivera BM, Yoshikami D (2008) Specificity, affinity and efficacy of iota-conotoxin RXIA, an agonist of voltage-gated sodium channels Na(V)1.2, 1.6 and 1.7. Biochem Pharmacol. 75: 2334-2344.

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

    Abstract: The excitotoxic conopeptide iota-RXIA induces repetitive action potentials in frog motor axons and seizures upon intracranial injection into mice. We recently discovered that iota-RXIA shifts the voltage-dependence of activation of voltage-gated sodium channel Na(V)1.6 to a more hyperpolarized level. Here, we performed voltage-clamp experiments to examine its activity against rodent Na(V)1.1 through Na(V)1.7 co-expressed with the beta1 subunit in Xenopus oocytes and Na(V)1.8 in dissociated mouse DRG neurons. The order of sensitivity to iota-RXIA was Na(V)1.6 > 1.2 > 1.7, and the remaining subtypes were insensitive. The time course of iota-RXIA-activity on Na(V)1.6 during exposure to different peptide concentrations were well fit by single-exponential curves that provided k(obs). The plot of k(obs)versus [iota-RXIA] was linear, consistent with a bimolecular reaction with a K(d) of approximately 3 microM, close to the steady-state EC(50) of approximately 2 microM. iota-RXIA has an unusual residue, D-Phe, and the analog with an L-Phe instead, iota-RXIA[L-Phe44], had a two-fold lower affinity and two-fold faster off-rate than iota-RXIA on Na(V)1.6 and furthermore was inactive on Na(V)1.2. iota-RXIA induced repetitive action potentials in mouse sciatic nerve with conduction velocities of both A- and C-fibers, consistent with the presence of Na(V)1.6 at nodes of Ranvier as well as in unmyelinated axons. Sixteen peptides homologous to iota-RXIA have been identified from a single species of Conus, so these peptides represent a rich family of novel sodium channel-targeting ligands.

    Receptor subtype expression changes with age

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

    Department of Pharmacology and Physiology, George Washington University Medical Center, Washington, DC 20037, USA.

    Abstract: Adolescence is a time of significant brain development, and exposure to nicotine during this period is associated with higher subsequent rates of dependence. Chronic nicotine exposure alters expression of nicotinic acetylcholine receptors (nAChRs), changing the pattern of nicotine responsiveness. We used quantitative autoradiography to measure three major subtypes of nAChRs after chronic nicotine exposure by osmotic minipump in adult and periadolescent rats. Comparison of control animals at the two different ages revealed that periadolescents express consistently greater numbers of alpha4beta2* nAChRs compared to the same brain regions of adults. Similar but less pronounced increases in alpha7 nAChRs were found in control periadolescent rats compared to adults. Binding of [(125)I]alpha-conotoxin MII (largely to alpha6* nAChRs) did not systematically differ between adults and periadolescents. The response to chronic nicotine exposure also differed by age. Up-regulation of alpha4beta2* nAChRs was prominent and widespread in adult animals; in periadolescents, alpha4beta2* up-regulation also occurred, but in fewer regions and to a lesser extent. A similar pattern of response was seen with alpha7 receptors: adults were more responsive than periadolescents to nicotine-induced up-regulation. In adult animals, chronic nicotine exposure did not cause up-regulation of alpha6* nAChRs; binding was down-regulated in three regions. Unlike the other subtypes, the response of alpha6* nAChRs to chronic nicotine was greater in periadolescents, with more regions showing greater down-regulation compared to adults. These differences in receptor expression and regulation between age groups are likely to be important given the unique vulnerability of adolescents to nicotine-induced behavioral changes and susceptibility to drug abuse.

    conotoxin mr1e from Conus marmoreus

    Wang Y, Shao X, Li M, Wang S, Chi C, Wang C (2008) mr1e, a conotoxin from Conus marmoreus with a novel disulfide pattern. Acta Biochim Biophys Sin (Shanghai). 40: 391-396.

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

    Abstract: Conotoxins are well known for their highly variable structures and functions. Here we report the identification of a novel conotoxin named mr1e from Conus marmoreus. mr1e is composed of 11 amino acid residues cross-linked by two disulfide bonds (CCHSSWCKHLC). The spacing of intercysteine loops in mr1e is exactly the same as that in alpha4/3 conotoxins. However, the native mr1e peptide co-eluted on reverse-phase HPLC with the regioselectively synthesized ribbon disulfide linkage isomer (C1-C4, C2-C3) but not the globular linkage isomer (C1-C3, C2-C4). Although this peptide has the same disulfide connectivity as the chi-conotoxins, their sequences do not share significant homology. Thus, mr1e could be defined as a novel conotoxin family. By intracranial injection into mice, mr1e showed an excitatory effect. The characterization of mr1e certainly enriches our understanding of conotoxins, and also opens an avenue for further structural and functional investigation.

    alpha-conotoxin MII receptor subtypes

    Dwoskin LP, Wooters TE, Sumithran SP, Siripurapu KB, Joyce BM, Lockman PR, Manda VK, Ayers JT, Zhang Z, Deaciuc AG, McIntosh JM, Crooks PA, Bardo MT (2008) N,N'-Alkane-diyl-bis-3-picoliniums as Nicotinic Receptor Antagonists: Inhibition of Nicotine-induced Dopamine Release and Hyperactivity. J Pharmacol Exp Ther. 326: 563-576

    Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA. ldwoskin@email.uky.edu

    Abstract: The current study evaluated a new series of N,N'-alkane-diyl-bis-3-picolinium (bAPi) analogs with C6-C12 methylene linkers as nicotinic acetylcholine receptor (nAChR) antagonists, for nicotine-evoked [3H]dopamine (DA) overflow, for blood-brain barrier choline transporter affinity, and for attenuation of discriminative stimulus and locomotor stimulant effects of nicotine. bAPi analogs exhibited little affinity for alpha4beta2* (* indicates putative nAChR subtype assignment) and alpha7* high-affinity ligand binding sites and exhibited no inhibition of DA transporter function. With the exception of C6, all analogs inhibited nicotine-evoked [3H]DA overflow (IC50 = 2 nM-6 microM; Imax = 54-64%), with N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB; C12) being most potent. bPiDDB did not inhibit electrically evoked [3H]DA overflow, suggesting specific nAChR inhibitory effects and a lack of toxicity to DA neurons. Schild analysis suggested that bPiDDB interacts in an orthosteric manner at nAChRs mediating nicotine-evoked [3H]DA overflow. To determine whether bPiDDB interacts with alpha-conotoxin MII-sensitive alpha6beta2-containing nAChRs, slices were exposed concomitantly to maximally effective concentrations of bPiDDB (10 nM) and alpha-conotoxin MII (1 nM). Inhibition of nicotine-evoked [3H]DA overflow was not different with the combination compared with either antagonist alone, suggesting that bPiDDB interacts with alpha6beta2-containing nAChRs. C7, C8, C10, and C12 analogs exhibited high affinity for the blood-brain barrier choline transporter in vivo, suggesting brain bioavailability. Although none of the analogs altered the discriminative stimulus effect of nicotine, C8, C9, C10, and C12 analogs decreased nicotine-induced hyperactivity in nicotine-sensitized rats, without reducing spontaneous activity. Further development of nAChR antagonists that inhibit nicotine-evoked DA release and penetrate brain to antagonize DA-mediated locomotor stimulant effects of nicotine as novel treatments for nicotine addiction is warranted.

    Protein kinases effect a switch in ACh subunit composition

    Arredondo J, Chernyavsky AI, Jolkovsky DL, Pinkerton KE, Grando SA (2008) Receptor-mediated tobacco toxicity: acceleration of sequential expression of alpha5 and alpha7 nicotinic receptor subunits in oral keratinocytes exposed to cigarette smoke. FASEB J. 22: 1356-1368.

    Department of Dermatology, University of California, Irvine, C340 Medical Sciences I, Irvine, CA 92697, USA.

    Abstract: Tobacco products and nicotine alter the cell cycle and lead to squamatization of oral keratinocytes (KCs) and squamous cell carcinoma. Activation of nicotinic acetylcholine receptors (nAChRs) elicits Ca(2+) influx that varies in magnitude between different nAChR subtypes. Normal differentiation of KCs is associated with sequential expression of the nAChR subtypes with increasing Ca(2+) permeability, such as alpha5-containing alpha3 nAChR and alpha7 nAChR. Exposure to environmental tobacco smoke (ETS) or an equivalent concentration of nicotine accelerated by severalfold the alpha5 and alpha7 expression in KCs, which could be abolished by mecamylamine and alpha-bungarotoxin with different efficacies, suggesting the following sequence of autoregulation of the expression of nAChR subtypes: alpha3(beta2/beta4) > alpha3(beta2/beta4)alpha5 > alpha7 > alpha7. This conjecture was corroborated by results of quantitative assays of subunit mRNA and protein levels, using nAChR-specific pharmacologic antagonists and small interfering RNAs. The genomic effects of ETS and nicotine involved the transcription factor GATA-2 that showed a multifold increase in quantity and activity in exposed KCs. Using protein kinase inhibitors and dominant negative and constitutively active constructs, we characterized the principal signaling cascades mediating a switch in the nAChR subtype. Cumulative results indicated that the alpha3(beta2/beta4) to alpha3(beta2/beta4)alpha5 nAChR transition predominantly involved protein kinase C, alpha3(beta2/beta4)alpha5 to alpha7 nAChR transition-Ca(2+)/calmodulin-dependent protein kinase II and p38 MAPK, and alpha7 self-up-regulation-the p38 MAPK/Akt pathway, and JAK-2. These results provide a mechanistic insight into the genomic effects of ETS and nicotine on KCs and characterize signaling pathways mediating autoregulation of stepwise overexpression of nAChR subtypes with increasing Ca(2+) permeability in exposed cells. These observations have salient clinical implications, because a switch in the nAChR subunit composition can bring about a corresponding switch in receptor function, leading to profound pathobiologic effects observed in KCs exposed to tobacco products.

    alpha conotoxin PIA targets alpha6/4 subunit receptors

    Papke RL, Dwoskin LP, Crooks PA, Zheng G, Zhang Z, McIntosh JM, Stokes C (2008) Extending the analysis of nicotinic receptor antagonists with the study of alpha6 nicotinic receptor subunit chimeras. Neuropharmacology. 54: 1189-1200.

    Department of Pharmacology and Therapeutics, University of Florida, College of Medicine, P.O. Box 100267, JHMHSC, 1600 SW Archer Road, Gainesville, FL 32610-0267, USA. rlpapke@ufl.edu

    Abstract: Heterologous expression systems have increased the feasibility of developing selective ligands to target nicotinic acetylcholine receptor (nAChR) subtypes. However, the alpha6 subunit, a component in nAChRs that mediates some of the reinforcing effects of nicotine, is not easily expressed in systems such as the Xenopus oocyte. Certain aspects of alpha6-containing receptor pharmacology have been studied by using chimeric subunits containing the alpha6 ligand-binding domain. However, these chimeras would not be sensitive to an alpha6-selective channel blocker; therefore we developed an alpha6 chimera (alpha4/6) that has the transmembrane and intracellular domains of alpha6 and the extracellular domain of alpha4. We examined the pharmacological properties of alpha4/6-containing receptors and other important nAChR subtypes, including alpha7, alpha4beta2, alpha4beta4, alpha3beta4, alpha3beta2, and alpha3beta2beta3, as well as receptors containing alpha6/3 and alpha6/4 chimeras. Our data show that the absence or presence of the beta4 subunit is an important factor for sensitivity to the ganglionic blocker mecamylamine, and that dihydro-beta-erythroidine is most effective on subtypes containing the alpha4 subunit extracellular domain. Receptors containing the alpha6/4 subunit are sensitive to alpha-conotoxin PIA, while receptors containing the reciprocal alpha4/6 chimera are insensitive. In experiments with novel antagonists of nicotine-evoked dopamine release, the alpha4/6 chimera indicated that structural rigidity was a key element of compounds that could result in selectivity for noncompetitive inhibition of alpha6-containing receptors. Our data extend the information available on prototypical nAChR antagonists, and establish the alpha4/6 chimera as a useful new tool for screening drugs as selective nAChR antagonists.

    6 April 2008

      Conotoxins and receptors

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

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

    25 March 2008

      alpha conotoxin Vc1.1 receives US patent

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

    27 February, 2008

      US patent win for unlisted Xenome

      Nick Evans BioTechnologyNews.Net Tuesday, 25 March 2008

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

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

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

        Background notes

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

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

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

    18 February, 2008

      Disulfide-rich peptide toxins from Gemmula species

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

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

    15 February, 2008

      Venom peptides from Conus ventricosus

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

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

    7 February, 2008

      Biogeographical patterns in Cape Verde Conus

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

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

    6 February, 2008

      Structure and function of α-conotoxin RgIA and analogues

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

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

      Conotoxin presentations at Society for Neuroscience meeting November 2007

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

    4 February, 2008

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

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

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

    31 January, 2008

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

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

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

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

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

    29 January, 2008

      Conotoxin transcripts in Conus leopardus

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

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

    25 January, 2008

      Conotoxins from Conus austini

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

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

      3-D structure of conotoxin tx3a from Conus textile

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

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

    20 January, 2008

      Conorfamide-Sr2 from Conus spurius

      Aguilar MB, Luna-Ramírez KS, Echeverría D, Falcón A, Olivera BM, Heimer de la Cotera EP, Maillo M. (2008) Conorfamide-Sr2, a gamma-carboxyglutamate-containing FMRFamide-related peptide from the venom of Conus spurius with activity in mice and mollusks. Peptides. 2007 Dec 5; [Epub ahead of print]
      Laboratorio de Neurofarmacología Marina, Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro 76230, Mexico.

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

    17 January, 2008

      Do hydroxyprolines determine the folding and activity of conotoxins ?

      Lopez-Vera E, Walewska A, Skalicky JJ, Olivera BM, Bulaj G. (2008) Role of Hydroxyprolines in the in Vitro Oxidative Folding and Biological Activity of Conotoxins. Biochemistry. 47: 1741-1751.
      Department of Biology, Department of Medicinal Chemistry, and Department of Biochemistry, University of Utah, Salt Lake City, Utah 84108, and Faculty of Chemistry, University of Gdansk, 80-952 Gdansk, Poland.

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

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

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

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

      New Species of Cone Shells

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

      The Cone Collector Volume 5 January 2008

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

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

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

    5 January, 2008

      Folding of conotoxins

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

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

    1 January, 2008

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