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.

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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 ...

Monoamine neurotransmitter transporters for norepinephrine (NE), dopamine and serotonin are important targets for antidepressants and analgesics. The conopeptide -MrIA is a noncompetitive and highly selective inhibitor of the NE transporter (NET) and is being developed as a novel intrathecal analgesic. We used site-directed mutagenesis to generate a suite of mutated transporters to identify two amino acids ( The monoamine neurotransmitter transporters are part of a larger family of Na ϩ -and Cl Ϫ -dependent transporters found in bacteria through to mammals. Dopamine, serotonin, and norepinephrine transporters (DAT, 3 SERT and NET, respectively) mediate the neuronal reuptake of their cognate neurotransmitter substance, terminating neurotransmission. NET has been implicated in mood states including depression and arousal, as well as in the control of blood pressure and pain (1-5), and is one of the targets of many psychoactive compounds including stimulants and antidepressants. Precisely how these compounds bind to NET is not well understood, but their interactions appear distinct from those of norepinephrine (NE) (6 -9). Unlike NE, tricyclic antidepressants such as desipramine and nisoxetine are not transported and appear to block by occluding the pore of the transporter.-MrIA is a peptide isolated from the venom of the predatory marine snail Conus mamoreus (10). This conopeptide specifically inhibits NE transport by NET without affecting dopamine or serotonin uptake by DAT and SERT, respectively (10, 11) and suppresses neuropathic pain upon intrathecal administration to rodents (5). -MrIA is a non-competitive inhibitor of NE transport but a competitive inhibitor of tricyclic antidepressants binding (11). Since desipramine and nisoxetine competitively inhibit NE transport, it appears that the binding site of -MrIA overlaps the antidepressant but not the NE binding site.There is currently no crystal structure of NET. Hence, structural details have been inferred from hydrophobicity, site-directed mutagenesis (performed mostly on related DAT and SERT proteins), and sequence analysis and subsequent computer homology models based on related bacterial transporters (12-19). NET and other monoamine neurotransmitter transporters are predicted to have 12 membrane-spanning regions, intracellular C and N termini, and a large extracellular loop between transmembrane domains 3 and 4. A more detailed view of monoamine transporters is starting to emerge with the recent crystal structure of a bacterial leucine transporter (bLeuT) (20), the closest functionally related transporter crystallized to date. Like monoamine transporters, bLeuT is Na ϩ -dependent with 12 membrane-spanning regions. bLeuT shares 28% identity with human NET (hNET) (see Fig. 1).In the present study, we used a combination of site-directed mutagenesis and homology modeling to locate residues on the hNET that interact with -MrIA. In the process, we identified a number of new interactions that affect NE transport and small molecule antidepressant binding at hNET....

The chi-conopeptides MrIA and MrIB are 13-residue peptides with two disulfide bonds that inhibit human and rat norepinephrine transporter systems and are of significant interest for the design of novel drugs involved in pain treatment. In the current study we have determined the solution structure of MrIA using NMR spectroscopy. The major element of secondary structure is a beta-hairpin with the two strands connected by an inverse gamma-turn. The residues primarily involved in activity have previously been shown to be located in the turn region (Sharpe, I. A.; Palant, E.; Schroder, C. I.; Kaye, D. M.; Adams, D. J.; Alewood, P. F.; Lewis, R. J. J Biol Chem 2003, 278, 40317-40323), which appears to be more flexible than the beta-strands based on disorder in the ensemble of calculated structures. Analogues of MrIA with N-terminal truncations indicate that the N-terminal residues play a role in defining a stable conformation and the native disulfide connectivity. In particular, noncovalent interactions between Val3 and Hyp12 are likely to be involved in maintaining a stable conformation. The N-terminus also affects activity, as a single N-terminal deletion introduced additional pharmacology at rat vas deferens, while deleting the first two amino acids reduced chi-conopeptide potency.