Cone snails use venom containing a cocktail of peptides ('conopeptides') to capture their prey. Many of these peptides also target mammalian receptors, often with exquisite selectivity. Here we report the discovery of two new classes of conopeptides. One class targets alpha1-adrenoceptors (rho-TIA from the fish-hunting Conus tulipa), and the second class targets the neuronal noradrenaline transporter (chi-MrIA and chi-MrIB from the mollusk-hunting C. marmoreus). rho-TIA and chi-MrIA selectively modulate these important membrane-bound proteins. Both peptides act as reversible non-competitive inhibitors and provide alternative avenues for the identification of inhibitor drugs.

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.

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

A peptide contained in the venom of the predatory marine snail Conus tulipa, -TIA, has previously been shown to possess ␣ 1 -adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, -TIA inhibited ␣ 1 -adrenoreceptor-mediated increases in cytosolic Ca 2؉ concentration that were triggered by norepinephrine, but did not affect presynaptic ␣ 2 -adrenoreceptor-mediated responses. In radioligand binding assays using [125 I]HEAT, -TIA displayed slightly greater potency at the ␣ 1B than at the ␣ 1A or ␣ 1D subtypes. Moreover, although it did not affect the rate of association for [ 3 H]prazosin binding to the ␣ 1B -adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by -TIA. N-terminally truncated analogs of -TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of -TIA (Arg 4 ). An alanine walk of -TIA confirmed the importance of Arg 4 for activity and revealed a number of other residues clustered around Arg 4 that contribute to the potency of -TIA. The unique allosteric antagonism of -TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive ␣ 1 -adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective.␣ 1 -Adrenoceptors, members of the G protein-coupled receptor superfamily, are the predominant mediators of the response to norepinephrine released from the sympathetic nerves that innervate resistance vessels (1). Norepinephrine release modulates vascular tone and, as such, ␣ 1 -adrenoreceptors are critically involved in circulatory homeostasis. Several ␣ 1 -adrenoreceptor antagonists, such as the quinazoline derivative, prazosin, are widely used for the treatment of hypertension. ␣ 1 -Adrenoreceptor antagonists are also used to treat bladder outlet obstruction in benign prostatic hyperplasia (for review, see Ref.2) because of their ability to relax smooth muscle.Nevertheless, the ␣ 1 -adrenoreceptor ligands developed to date interact largely with residues of the transmembrane segments that are homologous between the various receptor subtypes, rather than with residues forming the framework regions (the intra-and extracellular loops). It is not surprising, therefore, that available agonists, and also antagonists, show limited subtype selectivity (affinities differing by 50-fold or less between the various subtypes). For this reason, we sought to identify novel ligands that are likely to interact allosterically and, thus, more likely with the framework residues that are distinct between the three ␣ 1 -adrenoreceptor subtypes (␣ 1A , ␣ 1B , and ␣ 1D ).The venoms of cone snails (marine gastropods of the genus Conus) contain bioactive peptides that disrupt neurotransmission. These compounds are referred to generically as "conopeptides" or "conotoxins." Individual ...

-Conopeptide MrIA (-MrIA) is a 13-residue peptide contained in the venom of the predatory marine snail Conus marmoreus that has been found to inhibit the norepinephrine transporter (NET). We investigated whether -MrIA targeted the other members of the monoamine transporter family and found no effect of the peptide (100 M) on the activity of the dopamine transporter and the serotonin transporter, indicating a high specificity of action. The binding of the NET inhibitors, [ Because of its poor lipid solubility and degree of ionization at physiological pH, norepinephrine crosses cell membranes poorly by diffusion (1) and so relies on the operation of the norepinephrine transporter (NET) 1 for uptake into cells. Clearance by this integral membrane protein constitutes the major mechanism for the termination of action of this neurotransmitter at noradrenergic synapses (2), and disturbances in the functioning of the NET are associated with pathological states including depression (3), congestive heart failure (4), and orthostatic intolerance, and tachycardia (5). Known inhibitors of the NET include antidepressants (e.g. desipramine and nisoxetine), the appetite suppressant mazindol, and the abused drug cocaine (for review, see Ref. 6). The NET, together with the dopamine transporter (DAT) and the serotonin transporter (SERT), forms a family of Na ϩ -and Cl