Roger Drinkwater

Novel omega -Conotoxins from Conus catus Discriminate among Neuronal Calcium Channel Subtypes

ω-Conotoxins selective for N-type calcium channels are useful in the management of severe pain. In an attempt to expand the therapeutic potential of this class, four new ω-conotoxins (CVIA–D) have been discovered in the venom of the piscivorous cone snail, Conus catus, using assay-guided fractionation and gene cloning. Compared with other ω-conotoxins, CVID has a novel loop 4 sequence and the highest selectivity for N-type over P/Q-type calcium channels in radioligand binding assays. CVIA−D also inhibited contractions of electrically stimulated rat vas deferens. In electrophysiological studies, ω-conotoxins CVID and MVIIA had similar potencies to inhibit current through central (α1B-d) and peripheral (α1B-b) splice variants of the rat N-type calcium channels when coexpressed with rat β3 in Xenopus oocytes. However, the potency of CVID and MVIIA increased when α1B-d and α1B-b were expressed in the absence of rat β3, an effect most pronounced for CVID at α1B-d (up to 540-fold) and least pronounced for MVIIA at α1B-d (3-fold). The novel selectivity of CVID may have therapeutic implications. 1H NMR studies reveal that CVID possesses a combination of unique structural features, including two hydrogen bonds that stabilize loop 2 and place loop 2 proximal to loop 4, creating a globular surface that is rigid and well defined.

μO-conotoxin MrVIB selectively blocks Nav1.8 sensory neuron specific sodium channels and chronic pain behavior without motor deficits

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

Conotoxins and Their Potential Pharmaceutical Applications

The neurotoxins isolated from cone shell venoms are a diverse group of small, disulfide‐rich peptides. Most of the active peptides isolated to date have been shown to specifically target various components of neural transmission, and have generally demonstrated high specificities for ion channel and receptor types and subtypes. The specificity of conotoxins is one of the attributes that make them valuable diagnostic tools in the characterisation of neural pathways, as therapeutic agents in medicine, and potentially as biodegradable toxic agents in agroveterinary applications. The number of novel, active peptides within the numerous Conus species is considered to be enormous. Currently, however, relatively few peptides have been characterised. In this article, we review current research on conotoxins with a focus on drug potential being developed at the University of Queensland, Australia. Drug Dev. Res. 46:219–234, 1999.

Anti-allodynic efficacy of the χ-conopeptide, Xen2174, in rats with neuropathic pain

&NA; Xen2174 is a structural analogue of Mr1A, a χ‐conopeptide recently isolated from the venom of the marine cone snail, Conus marmoreus. Although both χ‐conopeptides are highly selective inhibitors of the norepinephrine transporter (NET), Xen2174 has superior chemical stability relative to Mr1A. It is well‐known that tricyclic antidepressants (TCAs) are also potent NET inhibitors, but their poor selectivity relative to other monoamine transporters and various G‐protein‐coupled receptors, results in dose‐limiting side‐effects in vivo. As TCAs and the α2‐adrenoceptor agonist, clonidine, have established efficacy for the relief of neuropathic pain, this study examined whether intrathecal (i.t.) Xen2174 alleviated mechanical allodynia in rats with either a chronic constriction injury of the sciatic nerve (CCI‐rats) or an L5/L6 spinal‐nerve injury. The anti‐allodynic responses of i.t. Mr1A and i.t. morphine were also investigated in CCI‐rats. Paw withdrawal thresholds were assessed using calibrated von Frey filaments. Bolus doses of i.t. Xen2174 produced dose‐dependent relief of mechanical allodynia in CCI‐rats and in spinal nerve‐ligated rats. Dose‐dependent anti‐allodynic effects were also produced by i.t. bolus doses of Mr1A and morphine in CCI‐rats, but a pronounced ‘ceiling’ effect was observed for i.t. morphine. The side‐effect profiles were mild for both χ‐conopeptides with an absence of sedation. Confirming the noradrenergic mechanism of action, i.t. co‐administration of yohimbine (100 nmol) with Xen2174 (10 nmol) abolished Xen2174 s anti‐allodynic actions. Xen2174 appears to be a promising candidate for development as a novel therapeutic for i.t. administration to patients with persistent neuropathic pain.

Isolation and Structure-Activity of μ-Conotoxin TIIIA, A Potent Inhibitor of Tetrodotoxin-Sensitive Voltage-Gated Sodium Channels

μ-Conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels during prey capture. Using polymerase chain reaction techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new μ-conotoxin-TIIIA (TIIIA). A 125I-TIIIA binding assay was established to isolate native TIIIA from the crude venom of Conus striatus. The isolated peptide had three post-translational modifications, including two hydroxyproline residues and C-terminal amidation, and <35% homology to other μ-conotoxins. TIIIA potently displaced [3H]saxitoxin and 125I-TIIIA from rat brain (Nav1.2) and skeletal muscle (Nav1.4) membranes. Alanine and glutamine scans of TIIIA revealed several residues, including Arg14, that were critical for high-affinity binding to tetrodotoxin (TTX)-sensitive Na+ channels. We were surprised to find that [E15A]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC50, 15 vs. 148 pM at rat brain membrane). TIIIA was selective for Nav1.2 and -1.4 over Nav1.3, -1.5, -1.7, and -1.8 expressed in Xenopus laevis oocytes and had no effect on rat dorsal root ganglion neuron Na+ current. 1H NMR studies revealed that TIIIA adopted a single conformation in solution that was similar to the major conformation described previously for μ-conotoxin PIIIA. TIIIA and analogs provide new biochemical probes as well as insights into the structure-activity of μ-conotoxins.

χ-Conopeptide Pharmacophore Development: Toward a Novel Class of Norepinephrine Transporter Inhibitor (Xen2174) for Pain

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.

Analgesic omega-conotoxins CVIE and CVIF selectively and voltage dependently block recombinant and native N-type calcium channels

Neuronal (N)-type Ca2+ channel-selective ω-conotoxins have emerged as potential new drugs for the treatment of chronic pain. In this study, two new ω-conotoxins, CVIE and CVIF, were discovered from a Conus catus cDNA library. Both conopeptides potently displaced 125I-GVIA binding to rat brain membranes. In Xenopus laevis oocytes, CVIE and CVIF potently and selectively inhibited depolarization-activated Ba2+ currents through recombinant N-type (α1B-b/α2δ1/β3) Ca2+ 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 ω-conotoxin action was investigated by creating molecular diversity in β subunits: N-type channels with β2a subunits almost completely recovered from CVIE or CVIF block, whereas those with β3 subunits exhibited weak recovery, suggesting that reversibility of the ω-conotoxin block may depend on the type of β-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 Ca2+ 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 ω-conotoxin/voltage-gated Ca2+ 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 Ca2+ channel-selective ω-conotoxins are therefore useful as neurophysiological tools and as potential therapeutic agents to inhibit nociceptive pain pathways.