Abstract: We report the preparation of gold nanoparticles (AuNPs) functionalized with the peptide-toxin conantokin-G and their selective binding to N-methyl-d-aspartate (NMDA) receptors recombinantly expressed by transfected HEK 293 cells. The AuNPs are passivated with a mixed self-assembled monolayer of omega-carboxy- and omega-amino-polyethylene glycol (PEG) thiols. We compare two different passivation systems: the alkyl-PEG600 system is characterized by a C(11)-alkyl chain between the thiol group and the PEG segment, whereas the PEG3000 system lacks this alkyl-chain. We show that only the alkyl-PEG600 passivation system allows selective conjugation of cysteine-terminated peptides to the periphery of the passivation layer via a heterobifunctional linker strategy. In contrast, using the PEG3000 passivation system, peptides are immobilized both on the passivation layer and directly on the gold surface via concurrent place-exchange reaction. We therefore recommend the use of the alkyl-PEG600 system to precisely control the number of immobilized peptides on AuNPs. In fact, we show that the number of conjugated peptides per particle can be varied with good control simply by varying the composition of the self-assembled monolayer. Finally, we demonstrate that conjugation of the conantokin-G peptide to the solvent-exposed interface of the passivation layer results in maximal binding interaction between the peptide-functionalized AuNPs and the targeted NMDA receptors on the cell surface. Conantokin G-coupled AuNP may be used to spatially restrict NMDA-receptor-blockade on neuronal surfaces.
PMID: 20939520 [PubMed - as supplied by publisher]

12 October 2010

South African cone shell toxins

Kauferstein S, Porth C, Kendel Y, Wunder C, Nicke A, Kordis D, Favreau P, Koua D, Stöcklin R, Mebs D (2010). Venomic study on cone snails (Conus spp.) from South-Africa. Toxicon. 2010 Oct 6. [Epub ahead of print] PubMed PMID: 20933537.

Institute of Legal Medicine, University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt, Germany.

Abstract: From six Conus species (C. coronatus, C. lividus, C. mozambicus f. lautus, C. pictus, C. sazanka, C. tinianus) collected off the eastern coast of South-Africa the venoms were analyzed using MALDI-TOF mass spectrometry. Between 56 to 151 molecular masses most in a range of 1,000 to 2,500 Da, were identified. Among the six venoms, between 0 to 27% (C. coronatus versus C. sazanka) of the peptide masses were found to be similar. In a study on venoms from 6 Conus species collected in the Philippines, the percentage of identical masses was between none to 9% only. The venoms from the South-African Conus species antagonized the rat neuronal nicotinic acetylcholine receptors (nAChRs) alpha3beta2, alpha4beta2, and alpha7, except for C. coronatus venom which blocked the alpha4beta2 and alpba7 nAChRs only. HPLC-fractionation of C. tinianus venom led to the isolation of a peptide that is active on all three receptor subtypes. It consists of 16 amino acid residues cross-linked by two disulfide bridges as revealed by de novo sequencing using tandem mass spectrometry: GGCCSHPACQNNPDYC. Posttranslational modifications include C-terminal amidation and tyrosine sulfation. The new peptide is a member of the alpha-conotoxin family which are competitive antagonists of nAChRs. Phylogenetic analysis of the 16S RNA from numerous Conus species has clarified the evolutionary position of endemic South African Conus species and provided the first evidence for their close genetic relationships. PMID: 20933537 [PubMed - as supplied by publisher]

Calcium channel toxin from Conus californicus

Bernaldez J, López O, Licea A, Salceda E, Arellano RO, Vega R, Soto E (2010). Electrophysiological characterization of a novel small peptide from the venom of Conus californicus that targets voltage-gated neuronal Ca(2+) channels. Toxicon 2010 Oct 5. [Epub ahead of print] PubMed PMID: 20920515.

Laboratorio de Inmunología Molecular y Biotoxinas, Departamento de Biotecnología Marina (C.I.C.E.S.E.), Ensenada, B.C., Mexico.

Abstract: Conus californicus belongs to a genus of marine gastropods with more than 700 extant species. C. californicus has been shown to be distantly related to all Conus species, but showing unusual biological features. We report a novel peptide isolated from C. californicus with a significant inhibitory action over neuronal voltage-gated calcium channels. The new toxin is formed by 13-amino acid residues with two disulfide bonds, whose sequence (NCPAGCRSQGCCM) is strikingly different from regular omega-conotoxins. In the HPLC purification procedure, the venom fraction eluted in the first 10-15 min produced a significant decrease (54% ± 3%) of the Ca(2+) current in Xenopus laevis oocytes transfected with purified rat-brain mRNA. A specific peptide obtained from the elution at 13 min decreased the Ca(2+) current in the adult rat dorsal-root ganglion neurons in a primary culture by 34% ± 2%. The cysteine pattern of this peptide corresponds to the framework XVI described for the M-superfamily of conopeptides and is unprecedented among Conus peptides acting on Ca(2+) channels.

5 October 2010

How do alpha-conotoxins relieve allodynia?

Klimis H, Adams DJ, Callaghan B, Nevin S, Alewood PF, Vaughan CW, Mozar CA, Christie MJ (2010). A novel mechanism of inhibition of high-voltage activated calcium channels by alpha-conotoxins contributes to relief of nerve injury-induced neuropathic pain. Pain. 2010 Oct 1. [Epub ahead of print] PubMed PMID: 20889259. doi:10.1016/j.pain/2010.09.007

Pain Management Research Institute, University of Sydney, Royal North Shore Hospital, St. Leonards NSW 2065, Australia; Brain and Mind Research Institute, University of Sydney, NSW 2006, Australia.

Abstract: alpha-Conotoxins that are thought to act as antagonists of nicotinic acetylcholine receptors (nAChRs) containing alpha3-subunits are efficacious in several preclinical models of chronic pain. Potent interactions of Vc1.1 with other targets have suggested that the pain-relieving actions of alpha-conotoxins might be mediated by either alpha9alpha10 nAChRs or a novel GABA(B) receptor-mediated inhibition of N-type calcium channels. Here we establish that three alpha-conotoxins, Vc1.1, AuIB and MII have distinct selectivity profiles for these three potential targets. Their potencies after intramuscular administration were then determined for reversal of allodynia produced by partial nerve ligation in rats. Vc1.1, which potently inhibits alpha9alpha10 nAChRs and GABA(B)/Ca(2+) channels but weakly blocks alpha3beta2 and alpha3beta4 nAChRs, produced potent, long-lasting reversal of allodynia that were prevented by pre-treatment with the GABA(B) receptor antagonist, SCH50911. alpha-Conotoxin AuIB, a weak alpha3beta4 nAChR antagonist, inhibited GABA(B)/Ca(2+) channels but did not act on alpha9alpha10 nAChRs. AuIB also produced reversal of allodynia. These findings suggest that GABA(B) receptor-dependent inhibition of N-type Ca(2+) channels can mediate the sustained anti-allodynic actions of some alpha-conotoxins. However, MII, a potent alpha3beta2 nAChR antagonist but inactive on alpha9alpha10 and alpha3beta4 nAChRs and GABA(B)/Ca(2+) channels, was demonstrated to have short-acting anti-allodynic action. This suggests that alpha3beta2 nAChRs may also contribute to reversal of allodynia. Together, these findings suggest that inhibition of alpha9alpha10 nAChR is neither necessary nor sufficient for relief of allodynia and establish that alpha-conotoxins selective for GABA(B) receptor-dependent inhibition of N-type Ca(2+) channels relieve allodynia, and could therefore be developed to manage chronic pain.

3 October 2010

Proposed mechanism(s) for the antiallodynic conopeptide Vc1.1

Zamponi GW (2010). Antiallodynic effects of a confused alpha-conotoxin: Vc1.1 relieves neuropathic pain via off target actions on GABA(B) receptors and N-type channels. COMMENTARY Pain Sep 27. [Epub ahead of print] PubMed PMID: 20880631. doi:10.1016/j.pain.2010.09.019

Department of Physiology and Pharmacology, Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Dr. NW, Calgary, Canada T2N 4N1.

Abstract: No Abstract. EXTRACT follows: "Venoms from predatory marine organisms such as fish-hunting cone snails are a rich source of bioactive compounds that bind to, and regulate a wide range of voltage- and ligand-gated ion channels. These snails use a cocktail of peptide toxins to rapidly stun and ultimately kill their prey. The individual toxins contained within the venom selectively target specific ion channel subfamilies [3]. For example, mu-conotoxins and omega-conotoxins, respectively, block voltage gated sodium and calcium channels, whereas the alpha-conotoxins are typically thought of as antagonists of nicotinic acetylcholine receptors [5]. Of course, the primary purpose of cone snail venom is to kill fish. However, because there is sufficient conservation in ion channel sequences among fish and mammals, many types of cone snail toxins also potently interact with human ion channel proteins [3]. On one hand, this poses a danger to humans coming into contact with various Conus species, but on the other hand, the selectivity and potency of individual peptide toxins can potentially be harnessed for therapeutic purposes. A prime example is the clinical use of omega-conotoxin MVIIA, a potent inhibitor of N-type calcium channels that can be delivered intrathecally to mediate analgesia in patients with severe pain [1]. A number of biotechnology companies and academic research laboratories are actively seeking other conotoxin molecules for use as therapeutics in humans [3]. This includes Vc1.1, an alpha-conotoxin from Conus victoriae that is currently in phase II clinical trials for neuropathic pain." [Conotoxin Vc1.1 also known as ACV1, completed Phase IIa clinical trials with no deleterious effects (BGL)]

"Callaghan and colleagues recently reported a Vc1.1 mediated inhibition of N-type calcium channels in rat sensory neurons [2] These authors showed that this inhibition was dependent on a Vc1.1 action on GABAB receptors, rather than on its canonical target (i.e., the nicotinic receptor). While this is in itself unusual, what is even more perplexing is that when activated by Vc1.1, GABAB receptors appear to signal by a novel and still undefined pathway to the N-type channel protein. But irrespective of the precise mode of action, the fact that N-type channels are inhibited by Vc1.1 suggested the possibility that its therapeutic actions are also mediated via N-types, rather than an effect on nicotinic receptors."

"In this issue of Pain, Kimlis and colleagues [4] examine the mechanism of Vc1.1 mediated analgesia in rats, along with that of two related alpha-conotoxins AuIB (Conus aulicus) and MII (Conus magus). The authors first characterized the effect of the three toxins on recombinant acetylcholine receptors expressed in Xenopus oocytes, and found that only Vc1.1 significantly inhibited a9a10 receptors The authors then demonstrated that Vc1.1 and AuIB, but not MII, triggered a GABAB receptor-mediated inhibition of native N-type currents in dorsal root ganglion neurons. The distinct target selectivities of the three toxins thus provided the authors with a unique tool kit to probe for the involvement of nicotinic and GABAB receptors in the afferent pain pathway. For this purpose, Kimlis and colleagues examined the effects of the three toxins on mechanical allodynia in a peripheral nerve ligation model. When injected intramuscularly, all three toxins produced an increase in mechanical paw withdrawal threshold with Vc1.1 being the most potent inhibitor, followed by AuIB and MII, and the effects of Vc1.1 were blocked by a GABAB receptor antagonist. Collectively, these data show that the antiallodynic actions of all three alpha-conotoxins are not mediated by a9a10 receptors and that a9a10 receptors are not significantly involved in the transmission of peripheral pain signals. Furthermore, the data provide compelling evidence that molecules such as Vc1.1 mediate their therapeutic actions via GABAB receptor induced inhibition of N-type calcium channels."

"It is well established that inhibiting N-type calcium channels is a suitable objective for the development of analgesics, either directly, or indirectly via the activation of G protein coupled receptors [1]. The new findings with Vc1.1 fit with the latter notion, despite the fact that the Vc1.1 induced effect appears to involve a novel, and as yet not fully characterized mechanism of coupling between the receptor and the channel. The observation that MII also produced antiallodynic effects even though it did not act on either N-type channels or a9a10 receptors suggests that inhibition of other types of nicotinic receptors such as alpha3beta2 [5] may help to alleviate mechanical allodynia. If so, then it may well be possible that inhibition of both GABAB and nicotinic receptors could synergistically mediate analgesia. Future studies involving co-administration of compounds such as Vc1.1 and MII, or the identification of novel alpha-conotoxins with a combined action on both receptor types may be useful in testing such a possibility."

REFERENCES

[1] Altier C, Zamponi GW (2004). Targeting Ca2+ channels to treat pain: T-type versus Ntype. Trends Pharmacol Sci 25:465–467

. [2] 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.

[3] Han TS, Teichert RW, Olivera BM, Bulaj G. (2008) Conus venoms – a rich source of peptide-based therapeutics. Curr Pharm Des 14: 2462–2479.

[4] Klimis H, Adams DJ, Callghan B, Nevin S, Alewood PF, Vaughan CW, Mozar CA, Christie MJ (2010). A novel mechanism of inhibition of high-voltage activated calcium channels by alpha-conotoxins contributes to relief of nerve injury – induced neuropathic pain. Pain 2010; doi:10.1016/j.pain/2010.09.007.

[5] Olivera BM, Quik M, Vincler M, McIntosh JM (2008). Subtype-selective conopeptides targeted to nicotinic receptors: concerted discovery and biomedical applications. Channels (Austin) 2:143–152.

BUT see critique in the review by McIntosh JM, Absalom N, Chebib M, Elgoyhen AB, Vincler M.(2009) Alpha9 nicotinic acetylcholine receptors and the treatment of pain. Biochem Pharmacol. 78: 693-702.

Abstract: ......Pharmacological analysis indicates that RgIA and Vc1.1 are selective antagonists of alpha9alpha10 nAChRs. A recent study also reported that these alpha9alpha10 antagonists are also potent GABA-B agonists. In the current study, we were unable to detect RgIA or Vc1.1 binding to or action on cloned GABA-B receptors expressed in HEK cells or Xenopus oocytes. We review the background, findings and implications of use of compounds that act on alpha9* nAChRs.(1).

Molluscan literature

KOHN, 1991. Paintings of paralectotypes?: Conus of the Museum Ludovicae Ulricae. 12pp., 1fig., 2tabs., 2pls. Euro 2.25
COOMANS & al., 1979. Alphabetical revision of the (sub) sp. in recent Conidae 1. Abbas to adansonii. 18pp., 25figs. Euro 2.70
COOMANS & al., 1979. Alphabetical revision of the (sub) sp. in recent Conidae 2. adansoni to albuquerquei. 25pp., 50figs. Euro 3.75
COOMANS & al., 1980. Alphabetical revision of the (sub) sp. in recent Conidae 3. albus to antillarum with the description of Conus algoensis agulhasi, n.ssp. 33pp., 43figs. Euro 4.95
COOMANS, 1966. Boekbespreking [Marsh, 1964. Cone shells of the world]. 2pp. Euro 0.50

Available from A.N. van der Bijl, Burgemeester van Bruggenstraat 41 1165 NV Halfweg, The Netherlands +31-20-4977772 (phone), anvdbijl@xs4all.nl (e-mail)

24 September 2010

Folding of cyclotides and conotoxins - REVIEW

Craik DJ (2010). The Folding of Disulfide-Rich Proteins. Antioxid Redox Signal. 2010 Sep 20. [Epub ahead of print] PubMed PMID: 20849378.

The Unversity of Queensland, Institute for Molecular Bioscience, Carmody Road, Brisbane, Queensland, Australia, 4069; d.craik@imb.uq.edu.au

Abstract: The articles in this forum issue describe various aspects of the folding of disulfide-rich proteins. They include review articles using proteins such as bovine pancreatic trypsin inhibitor as models to highlight the range of folding pathways seen in disulfide-rich proteins, along with a detailed analysis of the methods used to study them. Following two comprehensive reviews on the methods and applications of protein folding, three original articles in this issue focus on two specific classes of disulfide-rich proteins that have applications in drug design and development, namely cyclotides and conotoxins. Cyclotides are head-to-tail cyclic and disulfide-rich proteins from plants and function as a defence against insect attack. Conotoxins are the disulfide-rich components of the venom of marine cone snails that is used to capture prey. These research articles report on factors that modulate protein folding pathways in these molecules and determine the outcomes of protein folding- i.e. yield and heterogeneity of products. Finally the issue concludes with a comprehensive review on a different type of disulfide bond, namely those that have a functional, rather than structural role in proteins, with a particular focus on allosteric disulfide bonds that modify protein function.

The Conoidea superfamily: Teretoxins and conotoxins

Puillandre N, Holford M (2010). The Terebridae and teretoxins: combining phylogeny and anatomy for concerted discovery of bioactive compounds. BMC Chem Biol. 2010 Sep 17;10(1):7. [Epub ahead of print] PubMed PMID: 20849634.

Abstract: The Conoidea superfamily, comprised of cone snails, terebrids, and turrids, is an exceptionally promising group for the discovery of natural peptide toxins. The potential of conoidean toxins has been realized with the distribution of the first Conus (cone snail) drug, Prialt (ziconotide), an analgesic used to alleviate chronic pain in HIV and cancer patients. Cone snail toxins (conotoxins) are highly variable, a consequence of a high mutation rate associated to duplication events and positive selection. As Conus and terebrids diverged in the early Paleocene, the toxins from terebrids (teretoxins) may demonstrate highly divergent and unique functionalities. Recent analysis of the Terebridae, a largely distributed family with more than 300 described species, indicate they have evolutionary and pharmacological potential. Based on a three gene (COI, 12S and 16S) molecular phylogeny, including ~50 species from the West-Pacific, five main terebrid lineages were discriminated: Two of these lineages independently lost their venom apparatus, and one venomous lineage was previously unknown. Knowing the phylogenetic relationship of the Terebridae aids in effectively targeting divergent lineages with novel peptide toxins. Preliminary results indicate that teretoxins are similar in structure and composition to conotoxins, suggesting teretoxins are an attractive line of research to discover and develop new therapeutics that target ion channels and receptors. Using conotoxins as a guideline, and innovative natural products discovery strategies, such as the Concerted Discovery Strategy, the potential of the Terebridae and their toxins are explored as a pioneering pharmacological resource.

A novel linker resin system for synthesis of 'difficult' conotoxins

Alewood D, Hopping G, Brust A, Reid RC, Alewood PF (2010). Benzhydrylamine linker grafting: a strategy for the improved synthesis of C-terminal peptide amides. J Pept Sci. 2010 Oct;16(10):551-7. PubMed PMID: 20862722.

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

Abstract: The standard p-MBHA resin used during Boc-chemistry synthesis of peptides carrying C-terminal carboxamides is compromised by batch-to-batch variations in its performance. This can cause artificially 'difficult' couplings during peptide chain assembly, which may ultimately lead to failed syntheses given the inability to achieve acceptable coupling yields. To overcome these problems, we have developed a new approach by grafting a functionalized benzhydrylamine linker onto well-characterized and well-performing PAM resins. We combine optimized Boc-chemistry, high-performing PAM resins and new benzhydrylamine-based linkers to achieve improved syntheses of peptide amides. Here we present the synthesis of two new benzhydrylamine linkers and their attachment to selected PAM resins. This novel solid support was evaluated through the synthesis of selected 'difficult' conotoxins and monitoring the coupling efficiency using quantitative ninhydrin assay. The results show a superior performance of the novel linker solid support compared to the standard p-MBHA resins routinely used. In summary, we describe an alternative linker-resin system that allows improved access to C-terminal amide peptides employing Boc/Bzl chemistry. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.

21 September 2010

Determination of disulfide pairing in conotoxins by MS.

Gupta K, Kumar M, Balaram P (2010). Disulfide Bond Assignments by Mass Spectrometry of Native Natural Peptides: Cysteine Pairing in Disulfide Bonded Conotoxins. Anal Chem. 2010 Sep 15. [Epub ahead of print] PubMed PMID: 20843009

Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012, India, and National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore-560065, India.

Abstract: The critical, and often most difficult, step in structure elucidation of diverse classes of natural peptides is the determination of correct disulfide pairing between multiple cysteine residues. Here, we present a direct mass spectrometric analytical methodology for the determination of disulfide pairing. Protonated peptides, having multiple disulfide bonds, fragmented under collision induced dissociation (CID) conditions and preferentially cleave along the peptide backbone, with occasional disulfide fragmentation either by C(?)-S bond cleavage through H(?) abstraction to yield dehydroalanine and cysteinepersulfide, or by S-S bond cleavage through H(?) abstraction to yield the thioaldehyde and cysteine. Further fragmentation of the initial set of product ions (MS(n)) yields third and fourth generation fragment ions, permitting a distinction between the various possible disulfide bonded structures. This approach is illustrated by establishing cysteine pairing patterns in five conotoxins containing two disulfide bonds. The methodology is extended to the Conus araneosus peptides Ar1446 and Ar1430, two 14 residue sequences containing 3 disulfide bonds. A distinction between 15 possible disulfide pairing schemes becomes possible using direct mass spectral fragmentation of the native peptides together with fragmentation of enzymatically nicked peptides.

15 September 2010

alpha-Conotoxin ImI disrupts swimming in Hirudo spp.

Wagenaar DA, Gonzalez R, Ries DC, Jr WB, French KA (2010). Alpha-conotoxin ImI Disrupts Central Control of Swimming in the Medicinal Leech. Neurosci Lett. 2010 Sep 9. [Epub ahead of print] PubMed PMID: 20833225.

California Institute of Technology, Broad Fellows Program and Division of Biology, 1200 E California Blvd 216-76, Pasadena, CA91125, United States.

Abstract: Medicinal leeches (Hirudo spp.) swim using a metachronal, front-to-back undulation. The behavior is generated by central pattern generators (CPGs) distributed along the animal's midbody ganglia and is coordinated by both central and peripheral mechanisms. Here we report that a component of the venom of Conus imperialis, alpha-conotoxin ImI, known to block nicotinic acetyl-choline receptors in other species, disrupts swimming. Leeches injected with the toxin swam in circles with exaggerated dorsoventral bends and reduced forward velocity. Fictive swimming in isolated nerve cords was even more strongly disrupted, indicating that the toxin targets the CPGs and central coordination, while peripheral coordination partially rescues the behavior in intact animals.

Calcium channel modulation in the auditory system

Zorrilla de San Martín J, Pyott S, Ballestero J, Katz E (2010). Ca2+ and Ca2+-activated k+ channels that support and modulate transmitter release at the olivocochlear efferent-inner hair cell synapse. J Neurosci. 2010 Sep 8;30(36):12157-67. PubMed PMID: 20826678.

Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, 1428 Buenos Aires, Argentina, Department of Biology and Marine Biology, University of North Carolina Wilmington, Wilmington, North Carolina 28403, and Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EGA Buenos Aires, Argentina.

Abstract: In the mammalian auditory system, the synapse between efferent olivocochlear (OC) neurons and sensory cochlear hair cells is cholinergic, fast, and inhibitory. This efferent synapse is mediated by the nicotinic alpha9alpha10 receptor coupled to the activation of SK2 Ca(2+)-activated K(+) channels that hyperpolarize the cell. So far, the ion channels that support and/or modulate neurotransmitter release from the OC terminals remain unknown. To identify these channels, we used an isolated mouse cochlear preparation and monitored transmitter release from the efferent synaptic terminals in inner hair cells (IHCs) voltage clamped in the whole-cell recording configuration. Acetylcholine (ACh) release was evoked by electrically stimulating the efferent fibers that make axosomatic contacts with IHCs before the onset of hearing. Using the specific antagonists for P/Q- and N-type voltage-gated calcium channels (VGCCs), ?-agatoxin IVA and omega-conotoxin GVIA, respectively, we show that Ca(2+) entering through both types of VGCCs support the release process at this synapse. Interestingly, we found that Ca(2+) entering through the dihydropiridine-sensitive L-type VGCCs exerts a negative control on transmitter release. Moreover, using immunostaining techniques combined with electrophysiology and pharmacology, we show that BK Ca(2+)-activated K(+) channels are transiently expressed at the OC efferent terminals contacting IHCs and that their activity modulates the release process at this synapse. The effects of dihydropiridines combined with iberiotoxin, a specific BK channel antagonist, strongly suggest that L-type VGCCs negatively regulate the release of ACh by fueling BK channels that are known to curtail the duration of the terminal action potential in several types of neurons.

Conotoxin-like proteins revealed among short viral proteins

Naamati G, Askenazi M, Linial M (2010). A predictor for toxin-like proteins exposes cell modulator candidates within viral genomes. Bioinformatics. 2010 Sep 15;26(18):i482-i488. PubMed PMID: 20823311; PubMed Central PMCID: PMC2935411.

School of Computer Science and Engineering, Department of Biological Chemistry, Sudarsky Center for Computational Biology, Hebrew University of Jerusalem, Israel and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USA.

Abstract: MOTIVATION: Animal toxins operate by binding to receptors and ion channels. These proteins are short and vary in sequence, structure and function. Sporadic discoveries have also revealed endogenous toxin-like proteins in non-venomous organisms. Viral proteins are the largest group of quickly evolving proteomes. We tested the hypothesis that toxin-like proteins exist in viruses and that they act to modulate functions of their hosts. RESULTS: We updated and improved a classifier for compact proteins resembling short animal toxins that is based on a machine-learning method. We applied it in a large-scale setting to identify toxin-like proteins among short viral proteins. Among the approximately 26 000 representatives of such short proteins, 510 sequences were positively identified. We focused on the 19 highest scoring proteins. Among them, we identified conotoxin-like proteins, growth factors receptor-like proteins and anti-bacterial peptides. Our predictor was shown to enhance annotation inference for many 'uncharacterized' proteins. We conclude that our protocol can expose toxin-like proteins in unexplored niches including metagenomics data and enhance the systematic discovery of novel cell modulators for drug development. AVAILABILITY: ClanTox is available at http://www.clantox.cs.huji.ac.il CONTACT: michall@cc.huji.ac.il.

Optimization of oxidative folding of conotoxins

Steiner AM, Bulaj G (2010). Optimization of oxidative folding methods for cysteine-rich peptides: A study of conotoxins containing three disulfide bridges. J Pept Sci. 2010 Sep 2. [Epub ahead of print] PubMed PMID: 20814907.

Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84108, USA.

Abstract: The oxidative folding of small, cysteine-rich peptides to selectively achieve the native disulfide bond connectivities is critical for discovery and structure-function studies of many bioactive peptides. As the propensity to acquire the native conformation greatly depends on the peptide sequence, numerous empirical oxidation methods are employed. The context-dependent optimization of these methods has thus far precluded a generalized oxidative folding protocol, in particular for peptides containing more than two disulfides. Herein, we compare the efficacy of optimized solution-phase and polymer-supported oxidation methods using three disulfide-bridged conotoxins, namely micro-SIIIA, micro-KIIIA and omega-GVIA. The use of diselenide bridges as proxies for disulfide bridges is also evaluated. We propose the ClearOx-assisted oxidation of selenopeptides as a fairly generalized oxidative folding protocol. Copyright (c) 2010 European Peptide Society and John Wiley & Sons, Ltd. PMID: 20814907 [PubMed - as supplied by publisher]

Chemical synthesis of conotoxins Mr1.1 and Lp1.4

Peng C, Chen W, Sanders T, Chew G, Liu J, Hawrot E, Chi C (2010). Chemical synthesis and characterization of two {alpha}4/7-conotoxins. Acta Biochim Biophys Sin (Shanghai). 2010 Aug 27. [Epub ahead of print] PubMed PMID: 20801929.

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

Abstract: alpha-Conotoxins are small disulfide-constrained peptides that act as potent and selective antagonists on specific subtypes of nicotinic acetylcholine receptors (nAChRs). We previously cloned two alpha-conotoxins, Mr1.1 from the molluscivorous Conus marmoreus and Lp1.4 from the vermivorous Conus leopardus. Both of them have the typical 4/7-type framework of the subfamily of alpha-conotoxins that act on neuronal nAChRs. In this work, we chemically synthesized these two toxins and characterized their functional properties. The synthetic Mr1.1 could primarily inhibit acetylcholine (ACh)-evoked currents reversibly in the oocyte-expressed rat alpha7 nAChR, whereas Lp1.4 was an unexpected specific blocker of the mouse fetal muscle alpha1beta1gammadelta receptor. Although their inhibition affinities were relatively low, their unique receptor recognition profiles make them valuable tools for toxin-receptor interaction studies. Mr1.1 could also suppress the inflammatory response to pain in vivo, suggesting that it should be further investigated with respect to its molecular role in analgesia and its mechanism or therapeutic target for the treatment of pain.

9 September 2010

Venom Peptides: Bachem and Atheris to collaborate on Melusine

Press Release: Bachem (SIX: BANB) today announced that the company concluded an agreement with Atheris Laboratories to market their Melusine® libraries, consisting of natural products isolated from animal venoms. The agreement includes services to assist customers with the identification of the active ingredients and to synthesize individual compounds.

These libraries, isolated from venomous animals, have been fractionated to provide well-defined mixtures. The main components are peptide toxins, a class of interest for the development of peptide based therapeutics and cosmetics. Atheris and Bachem intend to encourage the screening of these compounds to expand and support their applications as drugs.

Melusine libraries are offered on microplates (96 or 384-wells plates) in multiple formats, from raw extracts to highly purified molecules.

From the Melusine Shop you can obtain from their Royal Menu fractionated venoms for testing from the following Conus species: Conus ammiralis, Conus coelinae, Conus eburneus, Conus imperialis, Conus leopardis, Conus miles, Conus textile, Conus vexillum and Conus virgo

For each species, the venom sample is submitted to a specific handling step, followed by a high resolution chromatographic separation into 80 fractions that are dispatched in ready-to-use microplates for HTS. For more information go to the Melusine web site at www.melusine.com

31 August 2010

Selectivity of {alpha}-Conotoxin BuIA[T5A;P6O]

Azam L, Maskos U, Changeux JP, Dowell CD, Christensen S, De Biasi M, McIntosh JM (2010). {alpha}-Conotoxin BuIA[T5A;P6O]: a novel ligand that discriminates between {alpha}6{beta}4 and {alpha}6{beta}2 nicotinic acetylcholine receptors and blocks nicotine-stimulated norepinephrine release. FASEB J. 2010 Aug 25. [Epub ahead of print] PubMed PMID: 20739611.

*Department of Biology andDepartment of Psychiatry, University of Utah, Salt Lake City, Utah, USA;Unité Neurobiologie Intégrative des Systèmes Cholinergiques, Institut Pasteur, Paris, France; and Department of Neuroscience and Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas, USA.

Abstract: alpha6* (asterisk indicates the presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) are broadly implicated in catecholamine-dependent disorders that involve attention, motor movement, and nicotine self-administration. Different molecular forms of alpha6 nAChRs mediate catecholamine release, but receptor differentiation is greatly hampered by a paucity of subtype selective ligands. alpha-Conotoxins are nAChR-targeted peptides used by Conus species to incapacitate prey. We hypothesized that distinct conotoxin-binding kinetics could be exploited to develop a series of selective probes to enable study of native receptor subtypes. Proline6 of alpha-conotoxin BuIA was found to be critical for nAChR selectivity; substitution of proline6 with 4-hydroyxproline increased the IC50 by 2800-fold at alpha6/alpha3beta2beta3 but only by 6-fold at alpha6/alpha3beta4 nAChRs (to 1300 and 12 nM, respectively). We used conotoxin probes together with subunit-null mice to interrogate nAChR subtypes that modulate hippocampal norepinephrine release. Release was abolished in alpha6-null mutant mice. alpha-Conotoxin BuIA[T5A;P6O] partially blocked norepinephrine release in wild-type controls but failed to block release in beta4(-/-) mice. In contrast, BuIA[T5A;P6O] failed to block dopamine release in the wild-type striatum known to contain alpha6beta2* nAChRs. BuIA[T5A;P6O] is a novel ligand for distinguishing between closely related alpha6* nAChRs; alpha6beta4* nAChRs modulate norepinephrine release in hippocampus but not dopamine release in striatum.

In vivo screening of toxins

Mejia M, Heghinian MD, Busch A, Armishaw CJ, Marí F, Godenschwege TA (2010). A novel approach for in vivo screening of toxins using the Drosophila Giant Fiber circuit. Toxicon. 2010 Aug 17. [Epub ahead of print] PubMed PMID: 20723555.

Department of Biological Sciences, Florida Atlantic University, 777 Glades Road, Boca Raton Fl 33431, USA.

Abstract: Finding compounds that affect neuronal or muscular function is of great interest as potential therapeutic agents for a variety of neurological disorders. Alternative applications for these compounds include their use as molecular probes as well as insecticides. We have developed a bioassay that requires small amounts of compounds and allows for unbiased screening of biological activity in vivo. For this, we paired administering compounds in a non-invasive manner with simultaneous electrophysiological recordings from a well-characterized neuronal circuit, the Giant Fiber System of Drosophila melanogaster, which mediates the escape response of the fly. The circuit encompasses a variety of neurons with cholinergic, glutamatergic, and electrical synapses as well as neuromuscular junctions. Electrophysiological recordings from this system allow for the detection of compound-related effects against any molecular target on these components. Here, we provide evidence that this novel bioassay works with small molecules such as the cholinergic receptor blocker mecamylamine hydrochloride and the potassium channel blocker tetraethylammonium hydroxide, as well as with venom from Conus brunneus and isolated conopeptides. Conopeptides have been developed into powerful drugs, such as the painkillers Prialt and Xen2174. However, most conopeptides have yet to be characterized, revealing the need for a rapid and straightforward screening method. Our findings show that mecamylamine hydrochloride, as well as the alpha-conotoxin ImI, which is known to be an antagonist of the human alpha7 nicotinic acetylcholine receptor, efficiently disrupted the synaptic transmission of a Drosophila alpha7 nicotinic acetylcholine receptor-dependent pathway in our circuit but did not affect the function of neurons with other types of synapses. This demonstrates that our bioassay is a valid tool for screening for compounds relevant to human health.

26 August 2010

A new superfamily of E-conotoxins

Liu Z, Yu Z, Liu N, Zhao C, Hu J, Dai Q (2010). cDNA cloning of conotoxins with framework XII from several Conus species. Acta Biochim Biophys Sin (Shanghai). 42: 656-661.

Beijing Institute of Biotechnology, Beijing 100071, China.

Abstract: In our efforts for cloning novel I(2)-superfamily conotoxins using the signal peptide sequence, we identified a novel conotoxin Lt12.4 from Conus litteratus. This gene has a framework XII (-C-C-C-C-CC-C-C-), which is distinct from the cysteine pattern I(2)-superfamily conotoxin (-C-C-CC-CC-C-C-). Subsequently, we found the signal peptide sequence of Lt12.4 by 5'-RACE. Using this new sequence, we identified another five novel conotoxins with this cysteine pattern from four Conus species (Conus eburneus, Conus imperialis, Conus marmoreus, and C. litteratus). These novel conotoxins have the same cysteine pattern as the reported Gla-TxX and Gla-MII, and may contain Gla residues. Furthermore, they have the highly conserved signal peptide and hypervariable mature peptide sequences, and widely exist in Conus species. Therefore, it could be defined as a new superfamily of E-conotoxins.

Binding of mu-conotoxin KIIIA and TTX to sodium channels

French RJ, Yoshikami D, Sheets MF, Olivera BM. (2010) The tetrodotoxin receptor of voltage-gated sodium channels--perspectives from interactions with mu-conotoxins. Mar Drugs. 8: 2153-2161

Department of Physiology and Pharmacology, University of Calgary, and Hotchkiss Brain Institute, 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4N1, Canada. french@ucalgary.ca

Abstract: Neurotoxin receptor site 1, in the outer vestibule of the conducting pore of voltage-gated sodium channels (VGSCs), was first functionally defined by its ability to bind the guanidinium-containing agents, tetrodotoxin (TTX) and saxitoxin (STX). Subsequent studies showed that peptide mu-conotoxins competed for binding at site 1. All of these natural inhibitors block single sodium channels in an all-or-none manner on binding. With the discovery of an increasing variety of mu-conotoxins, and the synthesis of numerous derivatives, observed interactions between the channel and these different ligands have become more complex. Certain mu-conotoxin derivatives block single-channel currents partially, rather than completely, thus enabling the demonstration of interactions between the bound toxin and the channel's voltage sensor. Most recently, the relatively small mu-conotoxin KIIIA (16 amino acids) and its variants have been shown to bind simultaneously with TTX and exhibit both synergistic and antagonistic interactions with TTX. These interactions raise new pharmacological possibilities and place new constraints on the possible structures of the bound complexes of VGSCs with these toxins.

The alpha7 nAChR agonist, PNU-282987, protects against neuropathic pain

Pacini A, Di Cesare Mannelli L, Bonaccini L, Ronzoni S, Bartolini A, Ghelardini C (2010). Protective effect of alpha7 nAChR: behavioural and morphological features on neuropathy. Pain. 150: 542-549.

Dept of Anatomy Histology and Forensic Medicine, University of Florence, Viale Morgagni 85, 50139 Florence, Italy.

Abstract: Traumatic, toxic or metabolic damage to the nervous system is the etiological foundation of neuropathic pain. Neuropathies are extremely difficult to treat and available drugs rarely joint an anti-hyperalgesic with a neurorestorative effect. From the literature, evidences support the alpha7 nicotinic receptor (nAChR) subtype as having a role in neuropathic pain as well as possessing neuroprotective properties. Aimed to inquire the anti-neuropathic effect of the alpha7 nAChR stimulation, we evaluated the pharmacological profile of the alpha7 nAChR agonist PNU-282987 on pain and on morphological alterations induced in the rat sciatic nerve by loose ligation (CCI). Acute administration of PNU-282987, 10 and 30 mg kg(-1) p.o. (15 days after ligation), was able to reduce hyperalgesia in a methyllicaconitine-reversed manner. This alpha7 nAChR agonist exerted no analgesic effects. Chronic PNU-282987 treatments, 30 mg kg(-1) once a day for 7 days and 10 mg kg(-1) for 28 days, were able to decrease pain perception. The histological studies highlighted that the ligation induces oedema and macrophagic infiltrate. Moreover, osmicated preparations revealed a decrease in axons' compactness and diameter, together with a significant loss of myelin sheath. Repeated treatment with PNU-282987 reduced the presence of oedema and macrophagic infiltrate and, on the coronal sections of the nerve, a significant higher myelin sheath, axonal diameter and number of fibers were observable. These results strongly suggest the pivotal role of alpha7 nAChR in the neuroprotection during neuropathy.

18 August 2010

Exploring Biodiversity: The Search for New Medicines

The Howard Hughes Medical Institute has produced a DVD in which two leading biologists, Bonnie L. Bassler PhD and Baldomero Olivera PhD chart paths of medical discovery through a deeper understanding of biodiversity. These lectures are the outcome of the Howard Hughes Medical Institute 'Holiday Lectures on Science', for December 2009.

Dr. Baldomero Olivera's presentation includes 4 animations:

Abstract: The alpha7* (*denotes the possible presence of additional subunits) nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the vertebrate nervous system and implicated in neuropsychiatric disorders that compromise thought and cognition. In this report, we demonstrate that the recently developed fluorescent ligand Cy3-ArIB[V11L;V16A] labels alpha7 nAChRs in cultured hippocampal neurons. However, photobleaching of this ligand during long image acquisition times prompted us to develop a new derivative. In photostability studies, this new ligand, Alexa Fluor 546-ArIB[V11L;V16A], was significantly more resistant to bleaching than the Cy3 derivative. The classic alpha7 ligand alpha-bungarotoxin binds to alpha1* and alpha9* nAChRs. In contrast, Alexa Fluor 546-ArIB[V11L;V16A] potently (IC(50) 1.8 nM) and selectively blocked alpha7 nAChRs but not alpha1* or alpha9* nAChRs expressed in Xenopus oocytes. Selectivity was further confirmed by competition binding studies of native nAChRs in rat brain membranes. The fluorescence properties of Alexa Fluor 546-ArIB[V11L;V16A] were assessed using human embryonic kidney-293 cells stably transfected with nAChRs; labeling was observed on cells expressing alpha7 but not cells expressing alpha3beta2, alpha3beta4, or alpha4beta2 nAChRs. Further imaging studies demonstrate that Alexa Fluor 546-ArIB[V11L;V16A] labels hippocampal neurons from wild-type mice but not from nAChR alpha7 subunit-null mice. Thus, Alexa Fluor 546-ArIB[V11L;V16A] represents a potent and selective ligand for imaging alpha7 nAChRs.

4 August 2010

Efficient regioselective folding of conopeptides

Han TS, Zhang MM, Gowd KH, Walewska A, Yoshikami D, Olivera BM, Bulaj G (2010). Disulfide-Depleted Selenoconopeptides: a Minimalist Strategy to Oxidative Folding of Cysteine-Rich Peptides. ACS Med Chem Lett. 1: 140-144.

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

Abstract: Despite the therapeutic promise of disulfide-rich, peptidic natural products, their discovery and structure/function studies have been hampered by inefficient oxidative folding methods for their synthesis. Here we report that converting the three disulfide-bridged mu-conopeptide KIIIA into a disulfide-depleted selenoconopeptide (by removal of a noncritical disulfide bridge and substitution of a disulfide- with a diselenide-bridge) dramatically simplified its oxidative folding while preserving the peptide's ability to block voltage-gated sodium channels. The simplicity of synthesizing disulfide-depleted selenopeptide analogs containing a single disulfide bridge allowed rapid positional scanning at Lys7 of mu-KIIIA, resulting in the identification of K7L as a mutation that improved the peptide's selectivity in blocking a neuronal (Na(v)1.2) over a muscle (Na(v)1.4) subtype of sodium channel. The disulfide-depleted selenopeptide strategy offers regioselective folding compatible with high throughput chemical synthesis and on-resin oxidation methods, and thus shows great promise to accelerate the use of disulfide-rich peptides as research tools and drugs.

Ziconotide combination therapy: Review

Wallace MS, Rauck RL, Deer T (2010). Ziconotide Combination Intrathecal Therapy: Rationale and Evidence. Clin J Pain. 2010 Jul 15. [Epub ahead of print]

University of California, San Diego, School of Medicine, La Jolla, CA Wake Forest University Health Sciences, Winston-Salem, NC; Center for Pain Relief, Charleston, WV.

Abstract: BACKGROUND: Ziconotide is a nonopioid intrathecal analgesic used to manage moderate to severe chronic pain. Although ziconotide is approved in the United States for intrathecal monotherapy only, it is often used in combination with other intrathecal drugs in clinical practice. OBJECTIVES: The need exists for a critical assessment of the currently available published literature on ziconotide combination therapy. This review summarizes and evaluates the publications from preclinical and clinical peer-reviewed experiments that have investigated the safety and effectiveness of ziconotide in combination with a variety of other drugs. METHODS/RESULTS: Eleven relevant publications were identified through a systematic search of multiple databases. DISCUSSION: In preclinical studies, additive or synergistic antinociceptive effects were discovered when ziconotide was used in combination with morphine, clonidine, or baclofen; however, no additional antinociceptive effects were observed when bupivacaine was added to ziconotide therapy. Safety data from animal studies revealed that ziconotide did not exacerbate morphine-induced respiratory depression, or clonidine-induced hypotension or bradycardia; however, ziconotide did potentiate morphine-induced hypotension and inhibition of gastrointestinal tract motility. Results from 2 open-label trials indicated that combination ziconotide and morphine therapy produced greater analgesia than was produced by the use of either drug alone. Preliminary support for the use of ziconotide in combination with morphine, baclofen, or hydromorphone was provided by case studies. CONCLUSIONS: Although clinical and preclinical studies provide some support for the use of ziconotide in combination with morphine, hydromorphone, clonidine, or baclofen, strong evidence-based data are limited. Controlled, long-term clinical trials are warranted.

26 July 2010

Scientists engineer an orally active conotoxin from Conus victoriae

Conus victoriae Conotoxin cVc1.1

Article by Bethany Halford, "Pain Relief From Snail Spit" reported in Chemical & Engineering News 88 (30) 39-40, July 26, 2010

Researchers led by David J. Craik of the Insittute for Molecular Bioscience at the University of Queensland discovered that by linking the N-terminus of alpha-conotoxin Vc1.1 - a compound derived from Conus victoriae to its C-terminus, they could make the 16-residue peptide orally active (Angew Chem Int Ed Engl. 122: 1-5 DOI:10.1002/anie.201000620). Craik's cyclized conotoxin "is a prime example of how peptide bioengineering can enhance bioavailability", comments Jon-Paul Bingham, a professor of molecular biosciences and bioengineering at the University of Hawaii, Manoa, noting that an orally active drug with the painkilling power of Prialt "would absolutely revolutionize how we manage chronic and terminal pain".

For further information scroll down to 15 June entry.

22 July 2010

Western Atlantic Cones

The Cone Collector: Special Issue, Volume 14A July 2010 contains a single important paper (166 pages) by John Tucker on Western Atlantic Cones.

Background: John Tucker writes in his Introduction "In 1984, Danker Vink published part I of 'The Conidae of the Western Atlantic' in La Conchiglia. A total of 15 parts appeared between 1984 and 1990. It, thus, has been 20 years or longer since these parts appeared. Understanding of what Vink actually said appears to be fading among collectors. Because I think that the Vink papers are an important contribution to the systematics of Western Atlantic cone shells, I decided to review these papers. The goal is to review the 62 taxa that Vink covered in his 15 parts (Table 1), and to present images that are as close as possible to those used by Vink in his papers. I do suggest revisions, but I have tried to keep those in the context of the Vink species".

Edited by António Monteiro, with layout by André Poremski.
(See interview with António Monteiro on the Seashell Collector.com)

Click here to download The Cone Collector #14A (PDF - 6 MB).
[Earlier issues, #0 - #14, are available for download at http://www.conchology.be/?t=42]

7 July 2010

NPY-like conopeptides in the venom of Conus betulinus

Wu X, Shao X, Guo ZY and Chi CW (2010) Identification of neuropeptide Y-like conopeptides from the venom of Conus betulinus Acta Biochim Biophys Sin. 42: 502-505

Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China; State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China

Correspondence E-mail: zhan-yun.guo@tongji.edu.cn (Z.-Y.G.). zwqi@sibs.ac.cn (C.-W.C.)

Abstract: Neuropeptide Y (NPY) is a ubiquitous endocrine neuropeptide found in vertebrate and invertebrate. In our present work, two NPY-like exocrine conopeptides (designated as cono-NPYs) were first identified in the venom of cone snails. Both cono-NPYs showed sequence characteristics of invertebrate NPYs, suggesting that some exocrine venom peptides are probably evolved from the preexisting endocrine peptides during the evolution of cone snails.

Development of mu-conotoxins as safe analgesics

Zhang MM, Han TS, Olivera BM, Bulaj G, Yoshikami D (2010). Mu-conotoxin KIIIA derivatives with divergent affinities versus efficacies in blocking voltage-gated sodium channels. Biochemistry 49: 4804-4812.

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

Abstract: The possibility of independently manipulating the affinity and efficacy of pore-blocking ligands of sodium channels is of interest for the development of new drugs for the treatment of pain. The analgesic mu-conotoxin KIIIA (KIIIA), a 16-residue peptide with three disulfide bridges, is a pore blocker of voltage-gated sodium channels, including neuronal subtype Na(V)1.2 (K(d) = 5 nM). At saturating concentrations, KIIIA incompletely blocks the sodium current of Na(V)1.2, leaving a 5% residual current (rI(Na)). Lys7 is an important residue: the K7A mutation decreases both the efficacy (i.e., increases rI(Na) to 23%) and the affinity of the peptide (K(d) = 115 nM). In this report, various replacements of residue 7 were examined to determine whether affinity and efficacy were inexorably linked. Because of their facile chemical synthesis, KIIIA analogues that had as a core structure the disulfide-depleted KIIIA[C1A,C2U,C9A,C15U] (where U is selenocysteine) or ddKIIIA were used. Analogues ddKIIIA and ddKIIIA[K7X], where X represents one of nine different amino acids, were tested on voltage-clamped Xenopus oocytes expressing rat Na(V)1.2 or Na(V)1.4. Their affinities ranged from 0.01 to 36 muM and rI(Na) values from 2 to 42%, and these two variables appeared to be uncorrelated. Instead, rI(Na) varied inversely with side chain size, and remarkably charge and hydrophobicity appeared to be inconsequential. The ability to manipulate a mu-conopeptide's affinity and efficacy, as well as its capacity to interfere with subsequent tetrodotoxin binding, greatly expands its scope as a reagent for probing sodium channel structure and function and may also lead to the development of mu-conotoxins as safe analgesics.

mu-conotoxins KIIIA and KIIIA[K7A] act synergistically with TTX

Zhang MM, Gruszczynski P, Walewska A, Bulaj G, Olivera BM, Yoshikami D (2010). Cooccupancy of the outer vestibule of voltage-gated sodium channels by mu-conotoxin KIIIA and saxitoxin or tetrodotoxin. J Neurophysiol. 104: 88-97.

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

Abstract: The guanidinium alkaloids tetrodotoxin (TTX) and saxitoxin (STX) are classic ligands of voltage-gated sodium channels (VGSCs). Like TTX and STX, mu-conotoxin peptides are pore blockers but with greater VGSC subtype selectivity. mu-conotoxin KIIIA blocks the neuronal subtype Na(V)1.2 with nanomolar affinity and we recently discovered that KIIIA and its mutant with one fewer positive charge, KIIIA[K7A], could act synergistically with TTX in a ternary peptide x TTX x Na(V) complex. In the complex, the peptide appeared to trap TTX in its normal binding site such that TTX could not readily dissociate from the channel until the peptide had done so; in turn, the presence of TTX accelerated the rate at which peptide dissociated from the channel. In the present study we examined the inhibition of Na(V)1.2, exogenously expressed in Xenopus oocytes, by STX (a divalent cation) and its sulfated congener GTX2/3 (with a net +1 charge). Each could form a ternary complex with KIIIA and Na(V)1.2, as previously found with TTX (a monovalent cation), but only when STX or GTX2/3 was added before KIIIA. The KIIIA x alkaloid x Na(V) complex was considerably less stable with STX than with either GTX2/3 or TTX. In contrast, ternary KIIIA[K7A] x alkaloid x Na(V) complexes could be formed with either order of ligand addition and were about equally stable with STX, GTX2/3, or TTX. The most parsimonious interpretation of the overall results is that the alkaloid and peptide are closely apposed in the ternary complex. The demonstration that two interacting ligands ("syntoxins") occupy adjacent sites raises the possibility of evolving a much more sophisticated neuropharmacology of VGSCs.

Conantokins enhance neuronal survival and plasticity

Huang L, Balsara RD, Sheng Z, Castellino FJ.(2010) Conantokins inhibit NMDAR-dependent calcium influx in developing rat hippocampal neurons in primary culture with resulting effects on CREB phosphorylation. Mol Cell Neurosci. June 21. [Epub ahead of print] PubMed PMID: 20600930.

W.M. Keck Center for Transgene Research and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556.

Abstract: The effects of conantokin (con)-G, con-R[1-17], and con-T on ion flow through N-methyl-D-aspartate receptor (NMDAR) ion channels, were determined in cultured primary rat hippocampal neurons. The potency of con-G diminished, whereas inhibition by con-R[1-17] and con-T did not change, as the neurons matured. Con-G, con-R[1-17], and con-T effectively diminished NMDA-induced Ca(2+) influx into the cells. A similar age-dependent decrease in con-G-mediated inhibition of the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) was observed, compared to con-R[1-17] and con-T. The effects of the conantokins on NMDA-induced cAMP response element-binding protein (CREB) phosphorylation in immature (DIV 9) and mature (DIV 16) neurons showed that, at DIV 9, con-G, con-R[1-17], and con-T inhibited NMDA-mediated P-CREB levels, whereas in DIV 16 neurons the conantokins did not inhibit overall levels of NMDA-induced P-CREB. In contrast, P-CREB levels were enhanced through inhibition of the protein phosphatases, PP1 and PP2B (calcineurin). This ability of conantokins to sustain CREB phosphorylation can thus enhance neuronal survival and plasticity.

4 July 2010

Synthesis of Cyclic Peptides : Review

Clark RJ, Craik DJ. (2010) Invited review : Native chemical ligation applied to the synthesis and bioengineering of circular peptides and proteins.Biopolymers. 94: 414-422

Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.

Abstract: Native chemical ligation methodology developed in the laboratory of Stephen Kent is a versatile approach to the linkage of peptide fragments using a native peptide bond. It is readily adaptable to the task of joining the N- and C-termini of peptides to produce cyclic molecules and we have used it for the cyclization of a range of disulfide-rich peptides. Specifically, it has been valuable for the synthesis of cyclotides, naturally occurring peptides characterized by a head-to-tail cyclized backbone and a knotted arrangement of three conserved disulfide bonds. Cyclotides have a diverse range of biological activities, including anti-HIV, antimicrobial, and insecticidal activities. They are ultrastable owing to their cyclic cystine knot motif, and native chemical ligation methodology has been invaluable in the synthesis of a range of native and modified cyclotides to explore their structure-activity relationships and applications in drug design. Similar studies have also been applied to a smaller cyclic peptide produced in sunflower seeds, sunflower trypsin inhibitor-1, which also shows promise as a template in drug design applications. We have also found native chemical ligation to be a valuable methodology for the cyclization of conotoxins, small disulfide-rich peptides from the venoms of marine cone snails. Conotoxins target a range of ions channels and receptors and are exciting leads in drug design applications. The synthetic cyclization of conotoxins with peptide linkers stabilizes them and improves their biopharmaceutical properties. In summary, this article illustrates the use of native chemical ligation technology in the cyclization of cyclotides, sunflower trypsin inhibitor-1, and conotoxins in our laboratory. (c) 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 414-422, 2010.

26 June 2010

Conantokin R1-A from Conus rolani

Gowd KH, Watkins M, Twede VD, Bulaj GW, Olivera BM. (2010) Characterization of conantokin Rl-A: molecular phylogeny as structure/function study. J Pept Sci. 16(8):375-82.

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

Abstract: A multidisciplinary strategy for discovery of new Conus venom peptides combines molecular genetics and phylogenetics with peptide chemistry and neuropharmacology. Here we describe application of this approach to the conantokin family of conopeptides targeting NMDA receptors. A new conantokin from Conus rolani, ConRl-A, was identified using molecular phylogeny and subsequently synthesized and functionally characterized. ConRl-A is a 24-residue peptide containing three gamma-carboxyglutamic acid residues with a number of unique sequence features compared to conantokins previously characterized. The HPLC elution of ConRl-A suggested that this peptide exists as two distinct, slowly exchanging conformers. ConRl-A is predominantly helical (estimated helicity of 50%), both in the presence and absence of Ca(++). The order of potency for blocking the four NMDA receptor subtypes by ConRl-A was NR2B > NR2D > NR2A > NR2C. This peptide has a greater discrimination between NR2B and NR2C than any other ligand reported so far. In summary, ConRl-A is a new member of the conantokin family that expands our understanding of structure/function of this group of peptidic ligands targeted to NMDA receptors. Thus, incorporating phylogeny in the discovery of novel ligands for the given family of ion channels or receptors is an efficient means of exploring the megadiverse group of peptides from the genus Conus. Copyright (c) 2010 European Peptide Society and John Wiley & Sons, Ltd.

Six novel conopeptides from Conus parius

Jimenez EC, Olivera BM. (2010) Divergent M- and O-superfamily peptides from venom of fish-hunting Conus parius. Peptides. 2010 Jun 4. [Epub ahead of print] PubMed PMID: 20570703.

Department of Physical Sciences, College of Science, University of the Philippines Baguio, Baguio City 2600, Philippines; Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.

Abstract: Six novel peptides from the piscivorous cone snail, Conus parius were purified by reverse-phase HPLC fractionation of crude venom. With the use of matrix-assisted laser desorption ionization mass spectrometry and standard Edman sequencing methods, the peptides were characterized. Two peptides were identified as members of the m-2 and m-4 branches of the M-superfamily and were designated as pr3a and pr3b, while four peptides were identified as members of the O-superfamily and were designated as pr6a, pr6b, pr6c and pr6d. Peptide pr3a differs from the majority of the M-superfamily peptides in the presence of two prolines, which are not modified to 4-trans-hydroxyproline. In peptide pr3b, five amino acids out of the 16 non-cysteine residues are identical with those of mu-GIIIA and mu-PIIIA, suggesting that pr3b may be a divergent mu-conotoxin. Peptide pr6a is notable because of its extreme hydrophobicity. Peptide pr6c has three prolines that are unhydroxylated. Peptides pr6b and pr6d differ from the previously characterized O-superfamily peptides in the presence of an extended N-terminus consisting of six amino acids. Peptides pr3a, pr3b, pr6a and pr6b were demonstrated to be biologically active when injected intraperitoneally in fish. The identification and characterization of these peptides in venom of a fish-hunting species establish the divergence of gene products and their patterns of post-translational modification within superfamilies in a single Conus species. Copyright © 2010 Elsevier Inc. All rights reserved.

Large conopeptides from the venom of Conus purpuascens

Möller C, Marí F. (2010) 9.3 kDa Components of the injected venom of Conus purpurascens define a New 5-disulfide conotoxin framework. Biopolymers. 2010 May 26. [Epub ahead of print] PubMed PMID: 20564010.

Department of Chemistry & Biochemistry, Florida Atlantic University, Boca Raton, Fl 33431, USA.

Abstract: The 83-residue conopeptide (p21a) and its corresponding non-hydroxylated analog were isolated from the injected venom of Conus purpurascens. The complete conopeptide sequences were derived from Edman degradation sequencing of fragments from tryptic, chymotryptic and cyanogen bromide digestions. p21a has a unique, ten-cystine/5-disulfide 7-loop framework with extended 10-residue N-terminus and a 5-residue C-terminus tails, respectively. p21a has a 48% sequence homology with a recently described 13-cystine conopeptide, con-ikot-ikot, isolated from the dissected venom of the fish-hunting snail Conus striatus. However, unlike con-ikot-ikot, p21a does not form a dimer of dimers. MALDI-TOF mass spectrometry suggests that p21a may form a non-covalent dimer. p21a is an unusually large conotoxin and insofar is the largest isolated from injected venom. p21a provides evidence that the Conus venom arsenal includes larger molecules that are directly injected into the prey. Therefore, cone snails can utilized toxins that are comparable in size to the ones commonly found in other venomous animals. (c) 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2010.

16 June 2010

Evolution of Conus californicus

Biggs JS, Watkins M, Puillandre N, Ownby JP, Lopez-Vera E, Christensen S, Moreno KJ, Bernaldez J, Licea-Navarro A, Corneli PS, Olivera BM (2010) Evolution of Conus peptide toxins: analysis of Conus californicus Reeve, 1844. Mol Phylogenet Evol. 56: 1-12.

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

Abstract: Conus species are characterized by their hyperdiverse toxins, encoded by a few gene superfamilies. Our phylogenies of the genus, based on mitochondrial genes, confirm previous results that C. californicus is highly divergent from all other species. Genetic and biochemical analysis of their venom peptides comprise the fifteen most abundant conopeptides and over 50 mature cDNA transcripts from the venom duct. Although C. californicus venom retains many of the general properties of other Conus species, they share only half of the toxin gene superfamilies found in other Conus species. Thus, in these two lineages, approximately half of the rapidly diversifying gene superfamilies originated after an early Tertiary split. Such results demonstrate that, unlike endogenously acting gene families, these genes are likely to be significantly more restricted in their phylogenetic distribution. In concordance with the evolutionary distance of C. californicus from other species, there are aspects of prey-capture behavior and prey preferences of this species that diverges significantly from all other Conus. Copyright 2010 Elsevier Inc. All rights reserved.

29 June 2010

US Patent on dicarba ImI

Robinson A and Jamana E (2010) Conotoxin analogues and methods for synthesis of intramolecular dicarba bridge-containing peptides. US Patent #7,745,573

Robinson, Andrea (St. Kilda, AU); Elaridi, Jomana (Endeavour Hills, AU)

Abstract: According to the present invention, there is provided a range of new conotoxin derivatives and methods for synthesizing these analogues and other intramolecular dicarba bridge-containing peptides, including dicarba-disulfide bridge-containing peptides.

Assignee: Polychip Pharmaceuticals Pty Ltd. (Toorak, AU) Monash University (Clayton, AU)

RELATED APPLICATIONS: This application claims the benefit of the filing date of Australian Patent Application No. 2006900798, which was filed Feb. 17, 2006. The contents of that application are hereby incorporated by reference in their entirety.

15 June 2010

Orally active cyclic Conotoxin Vc1.1

Clark RJ, Jensen J, Nevin ST, Callaghan BP, Adams DJ, Craik DJ. (2010) The Engineering of an Orally Active Conotoxin for the Treatment of Neuropathic Pain. Angew Chem Int Ed Engl. 122: 1-5 DOI:10.1002/anie.201000620

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072 (Australia), Fax: (+61) 7-3346-2101.

Abstract: From killers to curers: Peptides from cone snail venoms are potential therapeutic agents for the treatment of neuropathic pain. Unfortunately, these peptides suffer from the disadvantage of short biological half-lives and poor activity when taken orally. A new orally active conotoxin was developed to solve these problems.

Extract: "In summary, we have developed an orally active peptide based on a natural conotoxin, with exciting potential for the treatment of neuropathic pain. Our orally active peptide exhibits higher selectivity and potency for GABA(B) receptor-mediated N-type Ca2+ channel currents, the proposed target for analgesia, than the parent linear peptide...... By developing an orally active analogue of Vc1.1, we have substantially enhanced its potential as a new therapy for neuropathic pain."

See also: Article by Jessica Hamzelou, Sea snail venom provides potent pain relief reported in NewScientist 10 June 2010 No. 2765, page 12.

EXTRACT: "Sea snail venom could become the gold standard for the relief of nerve-related pain following the development of a pill that is 100 times as potent as leading treatments.
Current treatements for neuropathic pain include morphine, which is highly addictive, and gabapentin, which both act on nerve receptors. Sea snail venom had been suggested as a good alternative because it consistes of a cocktail of peptides, known as conotoxins. These act to immobilize the snail's prey by blocking nerve-cell conduction, but in mammals the peptides are an effective analgesic.
The only conotoxin-derived drug approved for human use is ziconotide. Unfortunately, it is susceptible to breakdown by enzymes in the saliva and gut, so it is administered by a pump surgically inserted into the abdominal wall, making it an invasive and expensive treatment.
To solve this problem, David Craik and his team at the University of Queensland in Brisbane, Australia, have developed the first "orally active" conotoxin drug. They started with a synthetic version of conotoxin. Since the enzymes that break down the drug usually act at the ends of the conotoxin molecule, the team used a chain of amino-acids to join up these ends, to form a circular structure. They found this version to be resistant to enzymes in the body.
Craik's team tested the conotoxin in rats with neruopathic pain. They found that a single oral dose significantly reduced pain using a standard test - how much pressure the rat could withstand before withdrawing its paw. Compared with gabapentin, conotoxin was judged to be 100 times as potent (Angewandte Chemie, DOI: 10.1002/anie.2010006.20).

Because conotoxin is so potent only very small doses are needed to provide pain relief, reducing the risk of side effects, says Craik. His team has applied for approval from the US Food and Drug Administration for a trial in humans."

See also: Article by Bethany Halford, Pain Relief From Snail Spit reported in Chemical & Engineering News 88 (30) 39-40, July 26, 2010

31 May 2010

Marine pharmaceuticals - Review

Mayer AM, Glaser KB, Cuevas C, Jacobs RS, Kem W, Little RD, McIntosh JM, Newman DJ, Potts BC, Shuster DE (2010). The odyssey of marine pharmaceuticals: a current pipeline perspective. Trends Pharmacol Sci. 31: 255-265.

Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA.

Abstract:The global marine pharmaceutical pipeline consists of three Food and Drug Administration (FDA) approved drugs, one EU registered drug, 13 natural products (or derivatives thereof) in different phases of the clinical pipeline and a large number of marine chemicals in the preclinical pipeline. In the United States there are three FDA approved marine-derived drugs, namely cytarabine (Cytosar-U((R)), Depocyt((R))), vidarabine (Vira-A((R))) and ziconotide (Prialt((R))). The current clinical pipeline includes 13 marine-derived compounds that are either in Phase I, Phase II or Phase III clinical trials. Several key Phase III studies are ongoing and there are seven marine-derived compounds now in Phase II trials. The preclinical pipeline continues to supply several hundred novel marine compounds every year and those continue to feed the clinical pipeline with potentially valuable compounds. From a global perspective the marine pharmaceutical pipeline remains very active, and now has sufficient momentum to deliver several additional compounds to the marketplace in the near future; this review provides a current view of the pipeline. Copyright © 2010 Elsevier Ltd. All rights reserved.

Extract: "Ziconotide (Prialt1) is the synthetic equivalent of a naturally occurring 25-amino acid peptide, w-conotoxin MVIIA (Figure 1), originally isolated from the venom of the fish-hunting marine snail Conus magus [7]. Ziconotide is a potent analgesic with a completely novel mechanism of action [8,9]. Various subtypes of voltage-gated calcium channels have been identified in the nervous system. Ziconotide reversibly blocks N-type calcium channels located on primary nociceptive afferent nerves in the superficial layers of the dorsal horn of the spinal cord. Binding of ziconotide to presynaptic N-type calcium channels reduces the release of excitatory neurotransmitter release from the primary afferent nerve terminals [10,11]. Tolerance to drug effects is a major limiting factor in opiate-based therapies; unlike opiates, ziconotide does not produce tolerance [12]. A recent search in PubMed (December 2009) using the search terms v-conotoxin MVIIA, SNX-111, ziconotide or Prialt1 retrieved 261 publications in the peer-reviewed literature. Ziconotide does not readily cross the blood–brain barrier and is therefore delivered intrathecally via an implantable pump or temporarily by an external microinfusion device [11,13,14]. Ziconotide received FDA approval in December 2004 and is currently labeled for the management of severe chronic pain in patients with cancer or AIDS [14,15] for whom intrathecal (IT) therapy is warranted, and who are intolerant of or refractory to other treatments, such as systemic analgesics, adjunctive therapies or IT morphine. Prialt1 is marketed by Elan Corporation, PLC (http://www.elan.com/therapies/products/prialt.asp). Ziconotide has also been approved by the EMEA [16]." Copyright © 2010 Elsevier Ltd. All rights reserved.

For data on Ziconotide, see also: http://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?id=6893
or Downlad the FDA offical download about Ziconotide

29 May 2010

Conantokin dimerization

Cnudde SE, Prorok M, Castellino FJ, Geiger JH. (2010) Metal ion determinants of conantokin dimerization as revealed in the X-ray crystallographic structure of the Cd(2+)/Mg (2+)-con-T[K7gamma] complex. J Biol Inorg Chem.15: 667-675.

Department of Chemistry, Michigan State University, East Lansing, MI, 48824, USA.

Abstract: Predatory sea snails from the Conus family produce a variety of venomous small helical peptides called conantokins that are rich in gamma-carboxyglutamic acid (Gla) residues. As potent and selective antagonists of the N-methyl-D: -aspartate receptor, these peptides are potential therapeutic agents for a variety of neurological conditions. The two most studied members of this family of peptides are con-G and con-T. Con-G has Gla residues at sequence positions 3, 4, 7, 10, and 14, and requires divalent cation binding to adopt a helical conformation. Although both Ca(2+) and Mg(2+) can fulfill this role, Ca(2+) induces dimerization of con-G, whereas the Mg(2+)-complexed peptide remains monomeric. A variant of con-T, con-T[K7gamma] (gamma is Gla), contains Gla residues at the same five positions as in con-G and behaves very similarly with respect to metal ion binding and dimerization; each peptide binds two Ca(2+) ions and two Mg(2+) ions per helix. To understand the difference in metal ion selectivity, affinity, and the dependence on Ca(2+) for dimer formation, we report here the structure of the monomeric Cd(2+)/Mg(2+)-con-T[K7gamma] complex, and, by comparison with the previously published con-T[K7gamma]/Ca(2+) dimer structure, we suggest explanations for both metal ion binding site specificity and metal-ion-dependent dimerization.

New way to screen alpha-conotoxins.

Check out the report by Stu Borman in Chemical and Engineering News, March 2010 here

A new way to synthesize snail-based peptides called alpha-conotoxins, fold them into their native structures, and screen them for biological activity has been developed by an Australian research group. The approach could lead to new medications for neurological disorders and other conditions.
alpha-Conotoxins are one of many families of conotoxins—neurotoxic peptides made by predatory cone snails, which use them to immobilize their prey. Conotoxins have attracted considerable attention for their bioactivity, especially pain relief. One conotoxin, Prialt (ziconotide), is an approved drug for severe chronic pain, and a conotoxin analog called Xen2174 is in Phase II human clinical trials, also as a pain reliever.
Although those two agents belong to other conotoxin families, alpha-conotoxins also relieve pain. But it has been difficult to synthesize alpha-conotoxins in their natively folded forms to assess their prospects as drug candidates. The peptides each have two disulfide links, and when they are synthesized as linear peptides, their disulfide-forming cysteines often combine with the “wrong” cysteine partners, yielding nonnative, misfolded alpha-conotoxins with impaired biological activity.

Bioactive-peptide specialist Paul F. Alewood of the University of Queensland and coworkers have now found a workaround (J. Am. Chem. Soc., DOI: 10.1021/ja910602h). They synthesize alpha-conotoxins in which one pair of cysteine residues has been replaced by a pair of selenocysteines. The selenocysteines combine to form diselenides more readily than cysteines react to form disulfides. So when a linear alpha-conotoxin is oxidized, the diselenide forms first, preventing mismatches, and the disulfide forms later, yielding a correct structure.
The diselenide analogs generally have conformations and bioactivities similar to those of the corresponding native alpha-conotoxins, and in some cases, the analogs’ bioactivities are better.
Alewood and coworkers also report the first method for synthesizing selenocysteine analogs of alpha-conotoxins on solid-support beads and then folding them while they are still on the resin. Having the folded analogs on solid-support particles makes it possible to screen the compounds for biological activity more quickly and conveniently than has been previously possible.
The use of diselenides as disulfide replacements in synthetic peptides was pioneered in the late 1990s by bioorganic chemist Luis Moroder of the Max Planck Institute of Biochemistry, in Martinsried, Germany. But the technique has only recently been applied to conotoxins — in one earlier study by the Alewood group and in another by conotoxin specialist Grzegorz Bulaj of the University of Utah and coworkers.
Of the new work, Bulaj says: “Having selenoconotoxins on-resin is a very nice advance. The solid-support-based approach can probably be extended to other di­sulfide-bridged peptides as well. It’s an important step toward the discovery and development of cysteine-rich peptides as future drugs.”

28 May 2010

Image collection of shells of Carl Linnaeus (1707-1778).

On May 24th 2010 the Linnean Society of London launched online the digitised images of the shell collection of the great Swedish naturalist Carl Linnaeus (1707-1778).The 3,054 new images represent the molluscan specimens held in the Linnean Society's strongroom collection. All major worldwide groups are represented, the coverage reflecting the stage of exploration that had been reached in the early 19th century; today of course, Linnaeus' species represent only a portion of the recent mollusc fauna now known but as the basis of all shell nomenclature the Linnaean specimens are invaluable to researchers across the globe. Molluscan taxonomy is in a constant state of revision and work continues on interpreting the Linnean collections.

The direct link to the collection of mollusks in the Linnaean collection is:
http://www.linnean-online.org/view/shells/shells.html

The direct link to the collection of cone shells in the Linnaean collection is:
http://www.linnean-online.org/view/shells/conus.html

If you have any comments to the Curator of Zoological Collections, The Linnaean Society, London (and collections manager of Higher Invertebrates, Natural History Museum), Ms. Kathie Way, (congratulations to her for this great achievement), her email is: k.way@nhm.ac.uk

An interesting query raised by Paul Monfils and answered on CONCH-L by Bill Fenzan relates to Conus aulicus:

Subject: Re: [CONCH-L] Photos of the Linnaean mollusk collection online.

A number of taxonomic questions came to mind in viewing the Linnaeus collection. Just a sample one. Perhaps an explanation on this one will help makes sense of some of my others. The shell labeled "Conus aulicus" is obviously not what we now know as Conus aulicus. If the pictured shell is the one Linnaeus assigned that name to, how did a different shell end up with that name?

The direct link to the collection of Conus aulicus in the Linnaean collection is: http://www.linnean-online.org/16950/

Bill Fenzan replied : There is information on your question in: Kohn, A. J. 1992. A Chronological Taxonomy of Conus, 1758-1840. Smithsonian Institution Press, Washington D. C., pp. 315.

The short version is that Linnaeus listed references to illustrations of many different cones in the original description. Each subsequent reviser (Hanley in the 1850s & Dodge in the 1950s) noted this but did not take action to clarify the situation. Alan Kohn finally designated a lectotype in 1963 when he studied the Linnaean cones and realized that the species needed to be better defined. Ironically, the shell desigated as the lectotype for Conus aulicus is not in the Linnaean collection on the website, but in the Royal (MLU) collection which is preserved at the university in Uppsala, Sweden.

Alan Kohn replied on 31 May 2010 in CONCH-L as follows:

Dear Bill, Paul, et al.,
Actually the lectotype of Conus aulicus is not in Sweden but in the Gualtieri Collection in the Museo di Storia Naturale, Università di Pisa, Italy, where Robert Moolenbeek discovered it, I believe around 1990. Here is the story: In his own copy of the Systema Naturae of 1758, Linnaeus indicated that he owned a specimen of C. aulicus in his collection. When I studied his collection 200 years later, I found no specimen of C. aulicus. Neither had Sylvanus Hanley a hundred years before (Ipsa Linnaei Conchylia, London, 1855). Thus in the absence of a type specimen, in my 1963 paper I selected as lectotype a specimen that Linnaeus had cited as representing his new species in his original description. This shell was described and illustrated by Gualtieri in 1742. Of course he could not have named it according to Linnaeus's system, which was not yet invented. However, Gualtieri described his specimen and illustrated it in a fine engraving that Linnaeus also cited. I reproduced Gualtieri's image as the representation of the lectotype of C. aulicus. At that time we did not know the whereabouts of Gualtieri's specimen either. However, as mentioned above Robert Moloenbeek located it in the Pisa Museum and kindly provided me with photographs of it. They appear, along with a reproduction of Gualtieri's original illustration, in my 1992 book that Bill cited.
Alan

27 May 2010

alpha-Conotoxin RgIA reveals a role for alpha-9 nAChRs in adrenal stress response in the rat

Colomer C, Olivos-Oré LA, Vincent A, McIntosh JM, Artalejo AR, Guérineau NC (2010). Functional characterization of alpha9-containing cholinergic nicotinic receptors in the rat adrenal medulla: implication in stress-induced functional plasticity. J Neurosci. 30: 6732-6742.

Institute of Functional Genomics, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5203, Institut National de la Santé et de la Recherche Médicale U661, University of Montpellier, 34094 Montpellier, France.

Abstract:An increase in circulating adrenal catecholamine levels constitutes one of the mechanisms whereby organisms cope with stress. Accordingly, stimulus-secretion coupling within the stressed adrenal medullary tissue undergoes persistent remodeling. In particular, cholinergic synaptic neurotransmission between splanchnic nerve terminals and chromaffin cells is upregulated in stressed rats. Since synaptic transmission is mainly supported by activation of postsynaptic neuronal acetylcholine nicotinic receptors (nAChRs), we focused our study on the role of alpha9-containing nAChRs, which have been recently described in chromaffin cells. Taking advantage of their specific blockade by the alpha-conotoxin RgIA (alpha-RgIA), we unveil novel functional roles for these receptors in the stimulus-secretion coupling of the medulla. First, we show that in rat acute adrenal slices, alpha9-containing nAChRs codistribute with synaptophysin and significantly contribute to EPSCs. Second, we show that these receptors are involved in the tonic inhibitory control exerted by cholinergic activity on gap junctional coupling between chromaffin cells, as evidenced by an increased Lucifer yellow diffusion within the medulla in alpha-RgIA-treated slices. Third, we unexpectedly found that alpha9-containing nAChRs dominantly (>70%) contribute to acetylcholine-induced current in cold-stressed rats, whereas alpha3 nAChRs are the main contributing channels in unstressed animals. Consistently, expression levels of alpha9 nAChR transcript and protein are overexpressed in cold-stressed rats. As a functional relevance, we propose that upregulation of alpha9-containing nAChR channels and ensuing dominant contribution in cholinergic signaling may be one of the mechanisms whereby adrenal medullary tissue appropriately adapts to increased splanchnic nerve electrical discharges occurring in stressful situations.

15 May 2010

alpha-Conotoxin AuIB isomers. Action on nicotinic receptors

Grishin AA, Wang C-IA, Muttenthaler M, Alewood PF, Lewis RJ and Adams DJ (2010) {alpha}-Conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity to stoichiometry of {alpha}3{beta}4 nAChRs. J. Biol. Chem. 285: 22254-22263

RMIT University, Australia; University of Queensland, Australia Corresponding author; email: david.adams@rmit.edu.au

Abstract: Non-native disulfide isomers of alpha-conotoxins are generally inactive although some unexpectedly demonstrate comparable or enhanced bioactivity. The actions of 'globular' and 'ribbon' isomers of alpha-conotoxin AuIB have been characterized on alpha3beta4 nAChRs heterologously expressed in Xenopus oocytes. Using two electrode voltage clamp recording, we showed that inhibitory efficacy of the ribbon isomer of AuIB is limited to ~50%. The maximal inhibition was stoichiometry-dependent since altering alpha3:beta4 RNA injection ratios either increased AuIB[ribbon] efficacy (10alpha:1beta) or completely abolished blockade (1alpha:10beta). In contrast, inhibition by AuIB[globular] was independent of injection ratios. ACh-evoked current amplitude was largest for 1:10 injected oocytes and smallest for 10:1 ratio. ACh concentration-response curves revealed high (HS, 1:10) and low (LS, 10:1) sensitivity alpha3beta4 nAChRs with corresponding EC50's of 22.6 uM and 176.9 uM, respectively. Increasing the agonist concentration antagonized the inhibition of LS alpha3beta4 nAChRs by AuIB[ribbon] whereas inhibition of HS and LS alpha3beta4 nAChRs by AuIB[globular] was unaffected. Inhibition of LS and HS alpha3beta4 nAChRs by AuIB[globular] was insurmountable and independent of membrane potential. Molecular docking simulation suggested that AuIB[globular] is likely to bind to both ?3?4 nAChR stoichiometries outside of the ACh-binding pocket whereas AuIB[ribbon] binds to the classical agonist-binding site of the LS alpha3beta4 nAChR only. In conclusion, the two isomers of AuIB differ in their inhibitory mechanisms such that AuIB[ribbon] inhibits only LS alpha3beta4 nAChRs competitively whereas AuIB[globular] inhibits alpha3beta4 nAChRs irrespective of receptor stoichiometry, primarily by a non-competitive mechanism.

12 May 2010

An omega conotoxin from Conus striatus is analgesic in rodents

Yan LD, Liu YL, Zhang L, Dong HJ, Zhou PL, Su RB, Gong ZH, Huang PT. (2010) Spinal antinociception of synthetic omega-conotoxin SO-3, a selective N-type neuronal voltage-sensitive calcium channel blocker, and its effects on morphine analgesia in chemical stimulus tests in rodent. Eur J Pharmacol. 636): 73-81.

Beijing Institute of Pharmacology and Toxicology, 27 Tai-Ping Road, Haidian District, Beijing 100850, China.

Abstract: SO-3, a novel Omega-superfamily conotoxin derived from Conus striatus, selectively inhibits N-type neuronal voltage-sensitive calcium channels. In current study, antinociception of SO-3 compared with MVIIA or morphine and its effects on morphine analgesia were investigated in rodent chemical stimulus tests after acute or repeated intrathecal administration. In mice acetic acid writhing test, similar to MVIIA, SO-3 caused dose- and time-dependent spinal antinociception with ED(50) of 0.25 microg/kg and t(1/2) of 4h, which was more potent and longer-acting than morphine. In rat formalin test after intrathecal bolus injection, SO-3 produced dose- and time-dependent antinociception by suppressing acute (ED(50), 1.79 microg/kg) and tonic phases (ED(50), 0.41 microg/kg), which was similar to MVIIA and approximately 10-fold potency and twice longer-acting of morphine in blocking tonic phase responses. After repeated intrathecal injections twice daily for 5 consecutive days, SO-3 produced analgesia without loss of potency whereas morphine produced analgesia tolerance in rat formalin test; further, SO-3 still produced potent analgesia in morphine-tolerant rats. SO-3 co-administered with morphine left-shift the dose-response curve of morphine in mice acetic acid writhing test and significantly potentiated morphine analgesia in rat formalin test. No changes in motor function were seen in mice or rats receiving antinociceptive doses of SO-3 whereas MVIIA caused motor dysfunction at doses of 1.0-2.0 microg/kg in rats. This study showed that (1) novel SO-3 produced potent and long-acting spinal antinociception without observable motor dysfunction, (2) SO-3 significantly potentiated morphine analgesia, (3) After repeated intrathecal administration, SO-3 produced neither tolerance nor cross-tolerance to morphine analgesia.

11 May 2010

Differential expression of conotoxins along venom duct

Tayo LL, Lu B, Cruz LJ, Yates JR 3rd. (2010) Proteomic analysis provides insights on venom processing in Conus textile. J Proteome Res. 9: 2292-2301

School of Chemical Engineering and Chemistry, Mapua Institute of Technology, Muralla Street Intramuros, Manila 1002, Philippines.

Abstract: Conus species of marine snails deliver a potent collection of toxins from the venom duct via a long proboscis attached to a harpoon tooth. Conotoxins are known to possess powerful neurological effects and some have been developed for therapeutic uses. Using mass-spectrometry based proteomics, qualitative and quantitative differences in conotoxin components were found in the proximal, central and distal sections of the Conus textile venom duct suggesting specialization of duct sections for biosynthesis of particular conotoxins. Reversed phase HPLC followed by Orbitrap mass spectrometry and data analysis using SEQUEST and ProLuCID identified 31 conotoxin sequences and 25 post-translational modification (PTM) variants with King-Kong 2 peptide being the most abundant. Several previously unreported variants of known conopeptides were found and this is the first time that HyVal is reported for a disulfide rich Conus peptide. Differential expression along the venom duct, production of PTM variants, alternative proteolytic cleavage sites, and venom processing enroute to the proboscis all appear to contribute to enriching the combinatorial pool of conopeptides and producing the appropriate formulation for a particular hunting situation. The complementary tools of mass spectrometry-based proteomics and molecular biology can greatly accelerate the discovery of Conus peptides and provide insights on envenomation and other biological strategies of cone snails.

9 May 2010

Book Review in The Cone Collector, April 2010

John Tucker and Manuel Tenorio.(2009) Systematic Classification of Recent and Fossil Conoidean Gastropods.

Conchbooks 2009

Abstract: This book review by Paul Kersten.in The Cone Collector Vol. 14 (2010) provides an account of the the joint efforts of two well-known scientists and cone experts, John Tucker and Manuel Tenorio, that has resulted in a new classification of what we are used to call Conus species. The new scheme is based primarily on the radula, with morphology and other factors used (eg. fossils) when the radula is not known.

Individual issues of "The Cone Collector" - a Newsletter devoted to Cones, can be downloaded from this site

mu-conotoxins as analgesics

Norton RS (2010) mu-conotoxins as leads in the development of new analgesics. Molecules 15:2825-2844

Walter and Eliza Hall Institute of Medical Research, Victoria, Australia. ray.norton@wehi.edu.au

Abstract: Voltage-gated sodium channels (VGSCs) contain a specific binding site for a family of cone shell toxins known as mu-conotoxins. As some VGSCs are involved in pain perception and mu-conotoxins are able to block these channels, mu-conotoxins show considerable potential as analgesics. Recent studies have advanced our understanding of the three-dimensional structures and structure-function relationships of the mu-conotoxins, including their interaction with VGSCs. Truncated peptide analogues of the native toxins have been created in which secondary structure elements are stabilized by non-native linkers such as lactam bridges. Ultimately, it would be desirable to capture the favourable analgesic properties of the native toxins, in particular their potency and channel sub-type selectivity, in non-peptide mimetics. Such mimetics would constitute lead compounds in the development of new therapeutics for the treatment of pain.

7 May 2010

Ziconotide for pain treatment - Review

Schmidtko A, Lötsch J, Freynhagen R, Geisslinger G. (2009) Ziconotide for treatment of severe chronic pain. Lancet 375:1569-1577

Pharmazentrum Frankfurt/ZAFES, Institut für Klinische Pharmakologie, Goethe-Universität, Frankfurt am Main, Germany.

Abstract:Pharmacological management of severe chronic pain is difficult to achieve with currently available analgesic drugs, and remains a large unmet therapeutic need. The synthetic peptide ziconotide has been approved by the US Food and Drug Administration and the European Medicines Agency for intrathecal treatment of patients with severe chronic pain that is refractory to other treatment modalities. Ziconotide is the first member in the new drug class of selective N-type voltage-sensitive calcium-channel blockers. The ziconotide-induced blockade of N-type calcium channels in the spinal cord inhibits release of pain-relevant neurotransmitters from central terminals of primary afferent neurons. By this mechanism, ziconotide can effectively reduce pain. However, ziconotide has a narrow therapeutic window because of substantial CNS side-effects, and thus treatment with ziconotide is appropriate for only a small subset of patients with severe chronic pain. We provide an overview of the benefits and limitations of intrathecal ziconotide treatment and review potential future developments in this new drug class.

Marine Pharmaceuticals - Review

Mayer AM, Glaser KB, Cuevas C, Jacobs RS, Kem W, Little RD, McIntosh JM, Newman DJ, Potts BC, Shuster DE. (2010) The odyssey of marine pharmaceuticals: a current pipeline perspective. Trends Pharmacol Sci. 31: 255-265. .

Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA.

Abstract:The global marine pharmaceutical pipeline consists of three Food and Drug Administration (FDA) approved drugs, one EU registered drug, 13 natural products (or derivatives thereof) in different phases of the clinical pipeline and a large number of marine chemicals in the preclinical pipeline. In the United States there are three FDA approved marine-derived drugs, namely cytarabine (Cytosar-U((R)), Depocyt((R))), vidarabine (Vira-A((R))) and ziconotide (Prialt((R))). The current clinical pipeline includes 13 marine-derived compounds that are either in Phase I, Phase II or Phase III clinical trials. Several key Phase III studies are ongoing and there are seven marine-derived compounds now in Phase II trials. The preclinical pipeline continues to supply several hundred novel marine compounds every year and those continue to feed the clinical pipeline with potentially valuable compounds. From a global perspective the marine pharmaceutical pipeline remains very active, and now has sufficient momentum to deliver several additional compounds to the marketplace in the near future; this review provides a current view of the pipeline.

Mechanisms of action of analgesics

Tsukamoto M, Kiso T, Shimoshige Y, Aoki T, Matsuoka N. (2010) Spinal mechanism of standard analgesics: evaluation using mouse models of allodynia. Eur J Pharmacol. 634: 40-45.

Pharmacology Research Laboratories, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan. mina.tsukamoto@jp.astellas.com

Abstract: Spinal neurotransmission plays an important role in the perception of pain signaling. In the present study, we investigated the spinal anti-nociceptive mechanism of current standard analgesics in mouse models of tactile allodynia induced by intrathecal administration of N-methyl-D-aspartic acid (NMDA), prostaglandin E2 (PGE2), and bicuculline. NMDA-induced allodynia is induced by postsynaptic NMDA receptor activation, while PGE2-induced allodynia is triggered by the enhancement of presynaptic glutamate release via EP1 receptor activation. In contrast, bicuculline induces allodynia by the blockade of gamma-aminobutyric acid (GABA)A receptor-mediated inhibitory system. As the clinically available analgesics, pregabalin (alpha2delta-subunit calcium channel ligand), ziconotide (N-type calcium channel blocker), mexiletine (sodium channel blocker), and duloxetine (serotonin and norepinephrine reuptake inhibitors) were evaluated in these neurochemically-induced allodynia models. Pregabalin almost completely alleviated NMDA-, PGE2-, and bicuculline-induced allodynia. Despite being classified as an agent with a similar molecular target mechanism, ziconotide could only alleviate PGE2-induced allodynia, but not NMDA- or bicuculline-induced allodynia, as did mexiletine and duloxetine. These results taken together suggest that ziconotide, mexiletine, and duloxetine suppress spinal hyperactivity via the presynaptic site mechanism. In contrast, pregabalin could suppress via the downstream step during spinal hyperactivation such as postsynaptic NMDA activation or dysfunction of GABAergic control in addition to presynaptic mechanism. In conclusion, present findings provide implication that the spinal anti-nociceptive mechanistic site of pregabalin is different from that of ziconotide, mexiletine, and duloxetine, and pregabalin could have a broader anti-nociceptive mechanism other than N-type calcium channel blockade.

2 May 2010

Conus glorioceanus Poppe & Tagaro, 2009

Guido T. Poppe announced via the newsgroup CONCH-L the discovery of a second specimen of Conus glorioceanus. As reported, "The shell measures 41.1 mm in length, is fresh dead and has a defect on the ventral side. Definitely fresh but dead taken with a hermit crab in a fish trap on the same place as the holotype: Zamboanga, between Recodo and Perlas Island in about 100 m depth. This C. glorioceanus is adult, but 8.5 mm smaller than the holotype.

The shell confirms the size and conchological characteristics of the holotype as to be stable. Indeed, the description, based on a single specimen raised rumors as this should be a freak from another Conus-species. Despite a triple expertise of Gabriella Raybaudi, Sheila Tagaro and myself who all agreed that the species was new.

Dozens of local fishermen got photos from the holotype and the sums offered locally are high enough so that we can positively expect more shells to reach Cebu in the future. Getting a shell out of 100 m deep may sound simple, in reality it is not. It is intensive in work and high in investment: one needs a boat, 160 m of rope, nets and a lot of muscles and time to get the gear to the surface daily.

The topotype is now in the collection of Conchology, Inc., the holotype indeed belongs to the National Museum of the Philippines, Manila."

You can view the shell in the Encyclopedia

17 April 2010

Bioactives from marine sponges

Ebada SS, Lin W, Proksch P. (2010) Bioactive sesterterpenes and triterpenes from marine sponges: occurrence and pharmacological significance. Mar Drugs. 8:313-346.

Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine University, Duesseldorf, Germany. sherif.Elsayed@uni-duesseldorf.de

Abstract:Marine ecosystems (>70% of the planet's surface) comprise a continuous resource of immeasurable biological activities and immense chemical entities. This diversity has provided a unique source of chemical compounds with potential bioactivities that could lead to potential new drug candidates. Many marine-living organisms are soft bodied and/or sessile. Consequently, they have developed toxic secondary metabolites or obtained them from microorganisms to defend themselves against predators [1]. For the last 30-40 years, marine invertebrates have been an attractive research topic for scientists all over the world. A relatively small number of marine plants, animals and microbes have yielded more than 15,000 natural products including numerous compounds with potential pharmaceutical potential. Some of these have already been launched on the pharmaceutical market such as Prialt (ziconotide; potent analgesic) and Yondelis (trabectedin or ET-743; antitumor) while others have entered clinical trials, e.g., alpidin and kahalalide F. Amongst the vast array of marine natural products, the terpenoids are one of the more commonly reported and discovered to date. Sesterterpenoids (C(25)) and triterpenoids (C(30)) are of frequent occurrence, particularly in marine sponges, and they show prominent bioactivities. In this review, we survey sesterterpenoids and triterpenoids obtained from marine sponges and highlight their bioactivities.

alpha-conotoxin from Conus litteratus

Luo S, Akondi KB, Zhangsun D, Wu Y, Zhu X, Hu Y, Christensen S, Dowell C, Daly NL, Craik DJ, Wang CI, Lewis RJ, Alewood PF, Michael McIntosh J. (2010) Atypical alpha-Conotoxin LtIA from Conus litteratus targets a novel microsite of the alpha3beta2 nicotinic receptor. J Biol Chem. 285:12355-12366.

Key Laboratory of Tropical Biological Resources, Ministry of Education, Ocean College, College of Materials and Chemical Engineering, Center for Experimental Biotechnology, Hainan University, Haikou Hainan 570228, China. luosulan2003@163.com

Abstract: Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. alpha-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of alpha-conotoxins may serve as a platform for development of new therapies. Previously characterized alpha-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel alpha-conotoxin LtIA, which lacks this highly conserved motif but potently blocked alpha3beta2 nAChRs with a 9.8 nm IC(50) value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing alpha-conotoxin MII. Nevertheless, pre-block of alpha3beta2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR beta subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for alpha3beta2 versus alpha3beta4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC(50) value on alpha3beta2(F119Q) versus wild type nAChRs, whereas T59K and V111I beta2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the alpha3beta2 nAChR that includes beta2 Lys-79. The K79A mutant disrupted LtIA binding but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.

Evolution of Conus peptide genes

Puillandre N, Watkins M, Olivera BM. (2010) Evolution of Conus Peptide Genes: Duplication and Positive Selection in the A-Superfamily. J Mol Evol. 2010 Feb 9. [Epub ahead of print]

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

Abstract: A remarkable diversity of venom peptides is expressed in the genus Conus (known as "conotoxins" or "conopeptides"). Between 50 and 200 different venom peptides can be found in a single Conus species, each having its own complement of peptides. Conopeptides are encoded by a few gene superfamilies; here we analyze the evolution of the A-superfamily in a fish-hunting species clade, Pionoconus. More than 90 conopeptide sequences from 11 different Conus species were used to build a phylogenetic tree. Comparison with a species tree based on standard genes reveals multiple gene duplication events, some of which took place before the Pionoconus radiation. By analysing several A-conopeptides from other Conus species recorded in GenBank, we date the major duplication events after the divergence between fish-hunting and non-fish-hunting species. Furthermore, likelihood approaches revealed strong positive selection; the magnitude depends on which A-conopeptide lineage and amino-acid locus is analyzed. The four major A-conopeptide clades defined are consistent with the current division of the superfamily into families and subfamilies based on the Cys pattern. The function of three of these clades (the kappaA-family, the alpha4/7-subfamily, and alpha3/5-subfamily) has previously been characterized. The function of the remaining clade, corresponding to the alpha4/4-subfamily, has not been elucidated. This subfamily is also found in several other fish-hunting species clades within Conus. The analysis revealed a surprisingly diverse origin of alpha4/4 conopeptides from a single species, Conus bullatus. This phylogenetic approach that defines different genetic lineages of Conus venom peptides provides a guidepost for identifying conopeptides with potentially novel functions.

Conoserver

Kaas Q, Westermann JC, Craik DJ. (2010) Conopeptide characterization and classifications: An analysis using ConoServer. Toxicon. 55: 1491-1509

The University of Queensland, Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, Brisbane, 4072 QLD Australia.

Abstract: Cone snails are carnivorous marine gastropods that have evolved potent venoms to capture their prey. These venoms comprise a rich and diverse cocktail of peptide toxins, or conopeptides, whose high diversity has arisen from an efficient hypermutation mechanism, combined with a high frequency of post-translational modifications. Conopeptides bind with high specificity to distinct membrane receptors, ion channels, and transporters of the central and muscular nervous system. As well as serving their natural function in prey capture, conopeptides have been utilized as versatile tools in neuroscience and have proven valuable as drug leads that target the nervous system in humans. This paper examines current knowledge on conopeptide sequences based on an analysis of gene and peptide sequences in ConoServer (http://www.conoserver.org), a specialized database of conopeptide sequences and three-dimensional structures. We describe updates to the content and organization of ConoServer and discuss correlations between gene superfamilies, cysteine frameworks, pharmacological families targeted by conopeptides, and the phylogeny, habitat, and diet of cone snails. The study identifies gaps in current knowledge of conopeptides and points to potential directions for future research. Copyright © 2010 Elsevier Ltd. All rights reserved.

kappaM-conotoxin RIIJ from Conus radiatus

Chen P, Dendorfer A, Finol-Urdaneta RK, Terlau H, Olivera BM. (2010) Biochemical characterization of kM-RIIIJ, a KV1.2 channel blocker: Evaluation of cardioprotective effects of kM-conotoxins. J Biol Chem. 285:14882-14889;

University of Utah, United States;

Abstract: Conus snail (Conus) venoms are a valuable source of pharmacologically active compounds; some of the peptide toxin families from the snail venoms are known to interact with K channels. We report the purification, synthesis and characterization of kappaM-conotoxin RIIIJ from the venom of a fish-hunting species, Conus radiatus. This conopeptide, like a previously characterized peptide in the same family, kappaM-RIIIK, inhibits the homotetrameric human Kv1.2 channels. When tested in Xenopus oocytes, kappaM-RIIIJ has an order of magnitude higher affinity (IC50 = 33 nM) to Kv1.2 than kappaM-RIIIK (IC50 = 352 nM). Chimeras of RIIIK and RIIIJ tested on the human Kv1.2 channels revealed that Lys-9 from kappaM-RIIIJ is a determinant of its higher potency against hKv1.2. However, when compared in a model of ischemia/reperfusion, kappaM-RIIIK (100 mug/kg b.w.), administered just before reperfusion, significantly reduces the infarct size in rat hearts in vivo without influencing hemodynamics, providing a potential compound for cardioprotective therapeutics. In contrast, kappaM-RIIIJ does not exert any detectable cardioprotective effect. kappaM-RIIIJ shows more potency for Kv1.2-Kv1.5 and Kv1.2-Kv1.6 heterodimers than kappaM-RIIIK; whereas the affinity of kappaM-RIIIK to Kv1.2-Kv1.7 heterodimeric channels is higher (IC50 = 680 nM) than that of kappaM-RIIIJ (IC50 = 3.15 muM). Thus, the cardioprotection seems to correlate to antagonism to heteromultimeric channels, involving the Kv1.2 alpha-subunit, rather than antagonism to Kv1.2 homotetramers. Furthermore, kappaM-RIIIK and kappaM-RIIIJ provide a valuable set of probes for understanding the underlying mechanism of cardioprotection.

Treatment of trigeminal neuropathy with Ziconotide

Lux EA. (2010) Case report: successful treatment of a patient with trigeminal neuropathy using ziconotide Anesth Analg. 110: 1195-1197.

Clinic of Pain and Palliative Medicine, St.-Marien-Hospital, Lünen, Germany. drlux@web.de

Abstract:A 50-year-old female patient with chronic neuropathic pain in the distribution of the second branch of the trigeminal nerve was unsuccessfully treated over several years. Intrathecal therapy with ziconotide was administered at an initial dose of 0.33 microg/d, which was gradually increased by 0.7 microg/d. Subjective pain on the numeric rating scale was reduced from 9/10 to 3-4/10 at a dose of 6.3 microg/d. Long-term treatment was continued at a ziconotide concentration of 12.5 microg/mL and a daily dose of 3.9 microg/d by a SynchroMed Infusion System (Medtronic, Minneapolis, MN). Pain reduction was maintained in the follow-up observation period over 5 months.

6 April 2010

Defining a clade for Conus praecellens

Biggs JS, Watkins M, Cornell PS and Olivera BM (2010). Defining a clade by morphological, molecular, and toxinological criteria: distinctive forms related to Conus praecellens (Gastropoda: Conidae): The Nautilus 124: 1-19

21 February 2010

Cone shells from Victoria, Australia

Conus anemone: A common species in dredgings at Western Port and living on the shore in crevices of rocks exhibiting much variation in colour. A pure white form invested with a creamy epidermis appears in life on the rocks at Back Beach Portsea and nearby frequently seen on the shores. This is merely an albino of the above and is sometimes known under the name of C. singletoni.

Conus segravei: A rare species in Western Port dredgings and occasionally found in the shores at Shoreham and Flinders. This is a distinct form named by the late Mr. J.H. Gatliff. It is beaurifully coloured and may be separated from anemone by its smoothness and roseate interior.

Conus rutilus: A small charming little species about 1/2 inch. Dredged in Western Port and easily recognized by its pyriform shape and brilliant colouring.

Source: "Forty Years' Dredging in Victorian Waters" An address by Mr. C.J. Gabriel, Hon. Assoc. Conchologist, National Museum of Victoria to the Malacological Club of Victoria on the 30th of March 1953. Reprinted in The Malacological Society of Australasia Inc. Victorian Branch Bulletin, VIC. BR. BULL. NO. 252 pp.4-8, 2010.

Selenium-directed synthesis of potent alpha-conotoxins

Muttenthaler M, Nevin ST, Grishin AA, Ngo ST, Choy PT, Daly NL, Hu SH, Armishaw CJ, Wang CI, Lewis RJ, Martin JL, Noakes PG, Craik DJ, Adams DJ, Alewood PF. (2010) Solving the alpha-Conotoxin Folding Problem: Efficient Selenium-Directed On-Resin Generation of More Potent and Stable Nicotinic Acetylcholine Receptor Antagonists. J Am Chem Soc. 132: 3514-3522

Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, The University of Queensland, Brisbane, Queensland 4072, Australia; Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia; and School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia.

Abstract: alpha-Conotoxins are tightly folded miniproteins that antagonize nicotinic acetylcholine receptors (nAChR) with high specificity for diverse subtypes. Here we report the use of selenocysteine in a supported phase method to direct native folding and produce alpha-conotoxins efficiently with improved biophysical properties. By replacing complementary cysteine pairs with selenocysteine pairs on an amphiphilic resin, we were able to chemically direct all five structural subclasses of alpha-conotoxins exclusively into their native folds. X-ray analysis at 1.4 A resolution of alpha-selenoconotoxin PnIA confirmed the isosteric character of the diselenide bond and the integrity of the alpha-conotoxin fold. The alpha-selenoconotoxins exhibited similar or improved potency at rat diaphragm muscle and alpha3beta4, alpha7, and alpha1beta1deltagamma nAChRs expressed in Xenopus oocytes plus improved disulfide bond scrambling stability in plasma. Together, these results underpin the development of more stable and potent nicotinic antagonists suitable for new drug therapies, and highlight the application of selenocysteine technology more broadly to disulfide-bonded peptides and proteins.

Harvesting conoidean venoms

Seronay RA, Fedosov AE, Astilla MA, Watkins M, Saguil N, Heralde FM 3rd, Tagaro S, Poppe GT, Aliño PM, Oliverio M, Kantor YI, Concepcion GP, Olivera BM. (2010) Accessing novel conoidean venoms: Biodiverse lumun-lumun marine communities, an untapped biological and toxinological resource. Toxicon. 2009 Dec 11. [Epub ahead of print]

Marine Science Institute, University of the Philippines, Diliman, Quezon City, 1101, Philippines; Northern Mindanao State Institute of Science and Technology (NORMISIST), Ampayon, Butuan City, Philippines.

Abstract: Cone snail venoms have yielded pharmacologically active natural products of exceptional scientific interest. However, cone snails are a small minority of venomous molluscan biodiversity, the vast majority being tiny venomous morphospecies in the family Turridae. A novel method called lumun-lumun opens access to these micromolluscs and their venoms. Old fishing nets are anchored to the sea bottom for a period of 1-6months and marine biotas rich in small molluscs are established. In a single lumun-lumun community, we found a remarkable gastropod biodiversity (155 morphospecies). Venomous predators belonging to the superfamily Conoidea (36 morphospecies) were the largest group, the majority being micromolluscs in the family Turridae. We carried out an initial analysis of the most abundant of the turrid morphospecies recovered, Clathurella (Lienardia) cincta (Dunker, 1871). In contrast to all cDNA clones characterized from cone snail venom ducts, one of the C. cincta clones identified encoded two different peptide precursors presumably translated from a single mRNA. The prospect of easily accessing so many different morphospecies of venomous marine snails raises intriguing toxinological possibilities: the 36 conoidean morphospecies in this one net alone have the potential to yield thousands of novel pharmacologically active compounds.

20 February 2010

Conus dorotheae

Visaya Vol.2 No. 6. has been published

Featuring an article by Eric Monnier and Loïc Limpalaër on a new species of cone shell, Conus dorotheae n. sp. Monnier & Limpalaër, 2010

Successful treatment with Ziconotide

Lux EA (2010) Successful Treatment of a Patient with Trigeminal Neuropathy Using Ziconotide. Anesth Analg. 110: 1195-1197.

The St.-Marien-Hospital, Lünen, Germany.

Abstract: A 50-year-old female patient with chronic neuropathic pain in the distribution of the second branch of the trigeminal nerve was unsuccessfully treated over several years. Intrathecal therapy with ziconotide was administered at an initial dose of 0.33 mug/d, which was gradually increased by 0.7 mug/d. Subjective pain on the numeric rating scale was reduced from 9/10 to 3-4/10 at a dose of 6.3 mug/d. Long-term treatment was continued at a ziconotide concentration of 12.5 mug/mL and a daily dose of 3.9 mug/d by a SynchroMed(R) Infusion System (Medtronic, Minneapolis, MN). Pain reduction was maintained in the follow-up observation period over 5 months. PMID: 20142352 [PubMed - as supplied by publisher]

Methodology for trialing Ziconotide

Burton AW, Deer TR, Wallace MS, Rauck RL, Grigsby E.(2010) Considerations and methodology for trialing ziconotide.Pain Physician. 13: 23-33

University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. awburton@mdanderson.org

Abstract: BACKGROUND: Before long-term intrathecal analgesic therapy is initiated, patients often undergo a spinal analgesia trial. Ziconotide is a nonopioid intrathecal analgesic used to manage severe chronic pain, and a variety of methods have been used to trial ziconotide. OBJECTIVES: The purpose of this review is to compare and discuss the different methods of ziconotide trialing. METHODS: Various databases (i.e., PubMed, Excerpta Medica, Cumulative Index to Nursing and Allied Health Literature, Biological Abstracts, Cochrane Database of Systematic Reviews, EMBASE, International Pharmaceutical Abstracts, and Google Scholar) and association meeting abstracts were searched with the use of the terms ziconotide, Prialt, trial, and trialing. In addition, a search was conducted for abstracts/posters presented at a variety of association meetings. RESULTS: Nine sources, including one expert opinion piece, were identified. Three methods of ziconotide trialing were discovered: continuous infusion, limited-duration infusion, and bolus injection. Results indicate that patients often achieve analgesia during trialing, regardless of the trialing method. Adverse events reported during ziconotide trialing studies were similar to those reported during ziconotide clinical trials. Preliminary evidence suggests that both effectiveness and safety may be dose-related. In 3 studies the value of ziconotide trialing in predicting long-term patient response to ziconotide therapy was investigated; however, the results were preliminary. The expert opinion piece from 2008 recommended trialing ziconotide via continuous infusion, using a starting dose of 1.2 mcg/d and dose increases of 1.2 mcg/d every 12 to 24 hours, for up to 3 days; the trial may be extended in some cases. LIMITATIONS: Given the small samples size and lack of controlled ziconotide trialing studies, it is currently not possible to determine the relative safety and effectiveness of different methods of ziconotide trialing, nor is it possible to determine if trialing is predictive of patient response to long-term ziconotide therapy. CONCLUSIONS: All 3 methods of ziconotide trialing appear to be viable options, and no method can be considered superior on the basis of the evidence presented in this review. Controlled studies comparing ziconotide trialing methods may be warranted.

Novel peptides from Conus marmoreus

Zhang L, Shao X, Chi C, Wang C. (2010) Two short D-Phe-containing cysteine-free conopeptides from Conus marmoreus. Peptides.31:177-179.

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

Abstract: The cysteine-free conopeptides are naturally occurring components of the venom of cone snails and have been relatively less investigated than the cysteine-containing conopeptides. In this work, we used thiol-exchange chromatography to isolate cysteine-free conopeptides from the venom of Conus marmoreus. The full-length previously reported conomarphin and two novel shortened forms of it were found in the cysteine-free conopeptide fraction. The two shortened conomarphins also contain a D-Phe and a hydroxylated proline residue, suggesting complex post-translational modifications of these conopeptides. In particular, the shorter conomarphin fragment shares significant sequence similarity with conophans. This work demonstrated that thiol-exchange chromatography is a useful approach to isolate cysteine-free conopeptides from venom cocktail. The newly identified conopeptides further revealed the complexity of post-translational processing and the high diversity of conotoxins.

12 February 2010

Oxidative folding of conotoxins

Safavi-Hemami H, Bulaj G, Olivera BM, Williamson NA, Purcell AW.(2010) Identification of Conus peptidyl Prolyl cis-trans isomerases (PPIases) and assessment of their role in the oxidative folding of conotoxins. J Biol Chem. 285: 12735-12746.

University of Melbourne, Australia;

Abstract: Peptidyl prolyl cis-trans isomerases (PPIases) are ubiquitous proteins that catalyse the cis-trans isomerisation of prolines. A number of proteins have been identified as endogenous substrates for PPIases such as Drosophila rhodopsin and the human immunodeficiency viral protein HIV-1 Gag, however very little is known about the interaction of PPIases with small, disulfide-rich peptides. Marine cone snails synthesise a wide array of cysteine-rich peptides, called conotoxins, many of which contain one or more prolines or hydroxyprolines. To identify whether PPIase-associated cis-trans isomerisation of these residues affects the oxidative folding of conotoxins we identified, sequenced and expressed three functionally active isoforms of PPIase from the venom gland of Conus novaehollandiae and characterised their ability to facilitate oxidative folding of conotoxins in vitro. Three conotoxins, namely mu-GIIIA, mu-SIIIA and omega-MVIIC, derived from two distinct toxin gene families were assayed. Conus PPIase significantly increased the rate of appearance of the native form of mu-GIIIA, a peptide containing three hydroxyprolines. In contrast, the presence of PPIase had no effect on the folding of mu-SIIIA and omega-MVIIC, peptides containing no or one proline residue respectively. We further show that an ER-resident PPIase isoform facilitates folding of mu-GIIIA more efficiently than two cytosolic isoforms. This is the first study to demonstrate PPIase-assisted folding of conotoxins, small disulfide-rich peptides with unique structural properties.

alpha-Conotoxin Lt IA from Conus litteratus

Luo S, Bharati AK, Zhangsun D, Wu Y, Zhu X, Hu Y, Christensen S, Dowell C, Daly NL, Craik DJ, Wang CI, Lewis RJ, Alewood PF, McIntosh JM. (2010) The atypical alpha-conotoxin LtIA from Conus litteratus targets a novel microsite of the alpha3beta2 nicotinic receptor. J Biol Chem. 285: 12355-12366

Hainan University, China;

Abstract: Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states including Parkinson's and nicotine addiction. Alpha-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function and receptor interaction of alpha-conotoxins may serve as a platform for development of new therapies. Previously characterized alpha-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. The current study characterized the novel alpha-conotoxin LtIA, which lacks this highly conserved motif but potently blocked alpha3beta2 nAChRs with a 9.8 nM IC50. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing alpha-conotoxin MII. Nevertheless, pre-block of alpha3beta2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR beta subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for alpha3beta2 vs. alpha3beta4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC50 on alpha3beta2(F119Q) versus wildtype nAChRs, whereas T59K and V111I beta2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the alpha3beta2 nAChR that includes beta2 K79. The K79A mutant disrupted LtIA binding, but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.

Evolution of Conus Peptide Genes

Puillandre N, Watkins M, Olivera BM.(2010) Evolution of Conus Peptide Genes: Duplication and Positive Selection in the A-Superfamily. J Mol Evol. 2010 Feb 9. [Epub ahead of print]

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

Abstract: A remarkable diversity of venom peptides is expressed in the genus Conus (known as "conotoxins" or "conopeptides"). Between 50 and 200 different venom peptides can be found in a single Conus species, each having its own complement of peptides. Conopeptides are encoded by a few gene superfamilies; here we analyze the evolution of the A-superfamily in a fish-hunting species clade, Pionoconus. More than 90 conopeptide sequences from 11 different Conus species were used to build a phylogenetic tree. Comparison with a species tree based on standard genes reveals multiple gene duplication events, some of which took place before the Pionoconus radiation. By analysing several A-conopeptides from other Conus species recorded in GenBank, we date the major duplication events after the divergence between fish-hunting and non-fish-hunting species. Furthermore, likelihood approaches revealed strong positive selection; the magnitude depends on which A-conopeptide lineage and amino-acid locus is analyzed. The four major A-conopeptide clades defined are consistent with the current division of the superfamily into families and subfamilies based on the Cys pattern. The function of three of these clades (the kappaA-family, the alpha4/7-subfamily, and alpha3/5-subfamily) has previously been characterized. The function of the remaining clade, corresponding to the alpha4/4-subfamily, has not been elucidated. This subfamily is also found in several other fish-hunting species clades within Conus. The analysis revealed a surprisingly diverse origin of alpha4/4 conopeptides from a single species, Conus bullatus. This phylogenetic approach that defines different genetic lineages of Conus venom peptides provides a guidepost for identifying conopeptides with potentially novel functions.

Conotoxin therapeutics: A Review

Lewis RJ.(2009) Conotoxin venom Peptide therapeutics. Adv Exp Med Biol. 655: 44-48.

Xenome Ltd, Indooroopilly 4068 and Institute for Molecular Biosciences, The University of Queensland, 4072, Brisbane, Australia, r.lewis@imb.uq.edu.au.

Abstract: Venom peptides offer enormous opportunity for the discovery of peptide drug leads. This review focusses on the potential of cone snails that have developed arrays of small peptides as part of highly evolved venoms used for prey capture and defence. Many of these peptides selectively modulate ion channels and transporters, making them a valuable source of new ligands for studying the role these targets play in normal and disease physiology. A number of these conopeptides reduce pain in animals models and several are now in preclinical and clinical development for the treatment of severe pain often associated with diseases such as cancer.

chi-conopeptide MrIA (XEN2174), a NET inhibitor for pain

Brust A, Palant E, Croker DE, Colless B, Drinkwater R, Patterson B, Schroeder CI, Wilson D, Nielsen CK, Smith MT, Alewood D, Alewood PF, Lewis RJ (2010). chi-Conopeptide pharmacophore development: toward a novel class of norepinephrine transporter inhibitor (Xen2174) for pain. J Med Chem. 52:6991-7002.

Xenome Ltd., 120 Meiers Road, Indooroopilly, Queensland, Australia 4068. andreas.brust@xenome.com

Abstract: Norepinephrine (NE) amplifies the strength of descending pain inhibition, giving inhibitors of spinal NET clinical utility in the management of pain. chi-MrIA isolated from the venom of a predatory marine snail noncompetitively inhibits NET and reverses allodynia in rat models of neuropathic pain. An analogue of chi-MrIA has been found to be a suitable drug candidate. On the basis of the NMR solution structure of this related peptide, Xen2174 (3), and structure-activity relationships of analogues, a pharmacophore model for the allosteric binding of 3 to NET is proposed. It is shown that 3 interacts with NET predominantly through amino acids in the first loop, forming a tight inverse turn presenting amino acids Tyr7, Lys8, and Leu9 in an orientation allowing for high affinity interaction with NET. The second loop interacts with a large hydrophobic pocket within the transporter. Analogues based on the pharmacophore demonstrated activities that support the proposed model. On the basis of improved chemical stability and a wide therapeutic index, 3 was selected for further development and is currently in phase II clinical trials.
This publication is dedicated to Dianne Alewood: gone to early and dearly missed by us all

Control of disulfide bond isomers in conotoxin ImI

Armishaw CJ, Dutton JL, Craik DJ, Alewood PF.(2010) Establishing regiocontrol of disulfide bond isomers of alpha-conotoxin ImI via the synthesis of N-to-C cyclic analogues. Biopolymers 94(3):307-313

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

Abstract: alpha-Conotoxins are multiple disulfide bond containing peptides that are isolated from venomous marine cone snails. They display remarkable selectivity for different subtypes of nicotinic acetylcholine receptors (nAChRs). While alpha-conotoxins display poor resistance to in vivo degradation by proteases which limits their use as drug leads, N-to-C cyclization via an oligopeptide spacer unit has been previously shown to improve stability. However the effect of N-to-C cyclisation on the formation of the disulfide bond framework is not fully understood. Four N-to-C cyclic analogues of alpha-conotoxin ImI; cImI-A, cImI-betaA, cImI-AG and cImI-AGG were synthesised to evaluate the effect of oligopeptide spacer length on disulfide bond selectivity and stability to proteolysis. Different ratios of disulfide bond isomers were obtained for each analogue using a non-selective random disulfide bond forming strategy, which was dependent on the length of the spacer. In order to identify each isomer obtained using the random strategy, and to gain access to disulfide bond isomers otherwise unattainable using the random strategy, both the native (globular) and ribbon isomers were synthesised in good yield and purity using a selective orthogonal cysteine protecting group strategy. As such, a random oxidation strategy showed a clear preference for the ribbon isomer in cImI-A. The cyclic globular isomers showed a high resistance to enzymatic degradation compared to the ribbon isomers, with the cImI-A and cImI-AG globular isomers demonstrating the highest stability. These results suggest that cyclisation can improve the biochemical stability of conotoxins with potential applications in the development of drugs.

More new material will be added here soon, updating from 31 October 2009

Analgesic omega-conotoxins from Conus catus

Berecki G, Motin L, Haythornthwaite A, Vink S, Bansal P, Drinkwater R, Wang CI, Moretta M, Lewis RJ, Alewood PF, Christie MJ, Adams DJ (2009). Analgesic{omega}-conotoxins CVIE and CVIF selectively and voltage dependently block recombinant and native N-type calcium channels. Mol Pharmacol. 77:139-148.

1 University of Queensland;

Abstract: N-type Ca(2+) channel-selective omega-conotoxins have emerged as potential new drugs for the treatment of chronic pain. In this study, two new omega-conotoxins, CVIE and CVIF, were discovered from a Conus catus cDNA library. Both conopeptides potently displaced (125)I-GVIA binding to rat brain membranes. In Xenopus oocytes, CVIE and CVIF potently and selectively inhibited depolarization-activated Ba(2+) currents through recombinant N-type (alpha(1B-b)/alpha(2)delta1/beta(3)) Ca(2+) channels. Recovery from block increased with membrane hyperpolarization, indicating that CVIE and CVIF have a higher affinity for channels in the inactivated state. The link between inactivation and the reversibility of omega-conotoxin action was investigated by creating molecular diversity in beta subunits: N-type channels with beta(2a) subunits almost completely recovered from CVIE or CVIF block, whereas those with beta(3) subunits exhibited weak recovery, suggesting that reversibility of the omega-conotoxin block may depend on the type of beta subunit isoform. In rat dorsal root ganglion sensory neurons, neither peptide had an effect on low voltage-activated (T-type) channels, but potently and selectively inhibited high voltage-activated N-type Ca(2+) channels in a voltage-dependent manner. In rat spinal cord slices, both peptides reversibly inhibited excitatory monosynaptic transmission between primary afferents and dorsal horn superficial lamina neurons. Homology models of CVIE and CVIF suggest that omega-conotoxin/voltage-gated Ca(2+) channel interaction is dominated by ionic/electrostatic interactions. In the rat partial sciatic nerve ligation model of neuropathic pain, CVIE and CVIF (1 nM) significantly reduced allodynic behavior. These N-type Ca(2+) channel-selective omega-conotoxins are therefore useful as neurophysiological tools and as potential therapeutic agents to inhibit nociceptive pain pathways.

Conotoxins Vc1.1 and RgIA are analgesic in alpha9 KO mice

Callaghan, B and Adams, DJ (2010) Analgesic alpha-conotoxins Vc1.1 and RgIA inhibit N-type calcium channels in sensory neurons of alpha9 nicotinic receptor knockout mice. Channels 4: 1-4. (This is an Addendum to Callaghan et al 2008, see below)

Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia.

Abstract: Alpha-conotoxins Vc1.1 and RgIA are peptides from the venom of marine Conus snails that are currently in development as a treatment for neuropathic pain. We have reported previously that the alpha9alpha10 nicotinic acetylcholine receptor (nAChR) selective-conotoxins Vc1.1 and RgIA potently and selectively inhibit high voltage-activated (HVA) N-type calcium channel currents in dissociated neurons from rat dorsal root ganglia (DRG). Our data indicated that Vc1.1 does not interact directly with N-type Ca(2+) channels but inhibits them via GABA(B) receptor activation. The present study investigated Vc1.1 and RgIA inhibition of N-type Ca(2+) channels currents in DRG neurons of wild-type and alpha9 knockout (KO) mice to determine if the alpha9 nAChR was necessary for inhibition of the Ca(2+) channel current. Application of Vc1.1 (100 nM) inhibited N-type Ca(2+) channel currents to 69.2 +/- 3.5% of control in DRG neurons isolated from wild-type mice. In >70% of DRG neurons isolated from the alpha9 KO mice, both Vc1.1 and RgIA selectively inhibited N-type Ca(2+) channel currents with an IC(50) of 24.6 nM and 22.4 nM, respectively. The GABA(B) receptor antagonist CGP55845 (1 microM) antagonized the effect of Vc1.1 and RgIA on the N-type calcium channels in alpha9 KO mice. RT-PCR and western blot analysis confirmed the absence of the alpha9 nAChR in mice carrying a null mutation for the nAChR alpha9 gene. These results demonstrate that the inhibition of N-type Ca(2+) channel channels by Vc1.1 and RgIA is not mediated by the expression of alpha9alpha10 nAChRs in DRG neurons.

Omega conotoxins as drugs: A Review

Bingham JP, Mitsunaga E, Bergeron ZL (2010). Drugs from slugs--past, present and Future perspectives of omega-conotoxin research. Chem Biol Interact. 183: 1-18.

Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, HI 96822, USA. jbingham@hawaii.edu

Abstract: Peptides from the venom of carnivorous cone shells have provided six decades of intense research, which has led to the discovery and development of novel analgesic peptide therapeutics. Our understanding of this unique natural marine resource is however somewhat limited. Given the past pharmacological record, future investigations into the toxinology of these highly venomous tropical marine snails will undoubtedly yield other highly selective ion channel inhibitors and modulators. With over a thousand conotoxin-derived sequences identified to date, those identified as ion channel inhibitors represent only a small fraction of the total. Here we discuss our present understanding of conotoxins, focusing on the omega-conotoxin peptide family, and illustrate how such a seemingly simple snail has yielded a highly effective clinical drug.

An omega conotoxin given intravenously is effective against neuropathic pain

Kolosov A, Goodchild CS, Cooke I. (2010) CNSB004 (Leconotide) Causes Antihyperalgesia Without Side Effects When Given Intravenously: A Comparison with Ziconotide in a Rat Model of Diabetic Neuropathic Pain. Pain Med. 11: 262-273..

Centre for Pain Medicine and Palliative Care, Monash Institute of Medical Research, Monash University, Clayton, Victoria.

Abstract: Leconotide is an omega-conotoxin that blocks neuronal voltage sensitive calcium channels. This study compared the antihyperalgesic potencies of leconotide and ziconotide given intravenously alone and in combinations with a potassium channel modulator flupirtine, given intraperitoneally, in a rat model of diabetic neuropathic pain. Design. Rats were given streptozotocin (150 mg/kg ip) to induce diabetic neuropathy and hyperalgesia. Experiments were performed on diabetic rats with >/=30% hyperalgesia to noxious heat. Rats were given each conopeptide alone and with flupirtine. Open field activity monitoring and non-invasive blood pressure measurements were used to define the maximum doses and combinations that caused no side effects. Doses in a range up to maximum no side effect doses were tested for antihyperalgesic effects in rats with hyperalgesia. Results. The maximum no side effect dose of leconotide (2 mg/kg intravenously) caused 51.7% reversal of hyperalgesia compared with 0.4% for the highest no side effect dose of ziconotide (0.02 mg/kg; P < 0.001, one-way anova). Leconotide caused dose-related antihyperalgesic effects that were potentiated by coadministration with flupirtine at doses that were ineffective when given alone. Leconotide (0.02 mg/kg) and flupirtine (5 mg/kg) caused 25.3 +/- 7.6 and -6 +/- 9.5% reversal of hyperalgesia, respectively when given alone but in combination they caused 84.1 +/- 7.2% reversal of hyperalgesia (P < 0.01; one-way anova). No such interaction occurred with ziconotide. Conclusion. Leconotide could have wider clinical applications than ziconotide. Unlike ziconotide, powerful antihyperalgesia without side effects can be achieved by intravenous administration of leconotide thus avoiding the need for an intrathecal injection.

1 January, 2010

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