Iain A. Sharpe

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.

Inhibition of the Norepinephrine Transporter by the Venom Peptide χ-MrIA SITE OF ACTION, Na+ DEPENDENCE, AND STRUCTURE-ACTIVITY RELATIONSHIP

χ-Conopeptide MrIA (χ-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 χ-MrIA targeted the other members of the monoamine transporter family and found no effect of the peptide (100 μm) on the activity of the dopamine transporter and the serotonin transporter, indicating a high specificity of action. The binding of the NET inhibitors, [3H]nisoxetine and [3H]mazindol, to the expressed rat and human NET was inhibited by χ-MrIA with the conopeptide displaying a slight preference toward the rat isoform. For both radioligands, saturation binding studies showed that the inhibition by χ-MrIA was competitive in nature. It has previously been demonstrated that χ-MrIA does not compete with norepinephrine, unlike classically described NET inhibitors such as nisoxetine and mazindol that do. This pattern of behavior implies that the binding site for χ-MrIA on the NET overlaps the antidepressant binding site and is wholly distinct from the substrate binding site. The inhibitory effect of χ-MrIA was found to be dependent on Na+ with the conopeptide becoming a less effective blocker of [3H]norepinephrine by the NET under the conditions of reduced extracellular Na+. In this respect, χ-MrIA is similar to the antidepressant inhibitors of the NET. The structure-activity relationship of χ-MrIA was investigated by alanine scanning. Four residues in the first cysteine-bracketed loop of χ-MrIA and a His in loop 2 played a dominant role in the interaction between χ-MrIA and the NET. Hα chemical shift comparisons indicated that side-chain interactions at these key positions were structurally perturbed by the replacement of Gly-6. From these data, we present a model of the structure of χ-MrIA that shows the relative orientation of the key binding residues. This model provides a new molecular caliper for probing the structure of the NET.

χ-Conotoxin and Tricyclic Antidepressant Interactions at the Norepinephrine Transporter Define a New Transporter Model

Monoamine neurotransmitter transporters for norepinephrine (NE), dopamine and serotonin are important targets for antidepressants and analgesics. The conopeptide χ-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 (Leu469 and Glu382) that affected the affinity of χ-MrIA to inhibit [3H]NE uptake through human NET. Residues that increased the Kd of a tricyclic antidepressant (nisoxetine) were also identified (Phe207, Ser225, His296, Thr381, and Asp473). Phe207, Ser225, His296, and Thr381 also affected the rate of NE transport without affecting NE Km. In a new model of NET constructed from the bLeuT crystal structure, χ-MrIA-interacting residues were located at the mouth of the transporter near residues affecting the binding of small molecule inhibitors.

Allosteric α1-Adrenoreceptor antagonism by the conopeptide ρ-TIA

A peptide contained in the venom of the predatory marine snail Conus tulipa, ρ-TIA, has previously been shown to possess α1-adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, ρ-TIA inhibited α1-adrenoreceptor-mediated increases in cytosolic Ca2+ concentration that were triggered by norepinephrine, but did not affect presynaptic α2-adrenoreceptor-mediated responses. In radioligand binding assays using [125I]HEAT, ρ-TIA displayed slightly greater potency at the α1B than at the α1A or α1D subtypes. Moreover, although it did not affect the rate of association for [3H]prazosin binding to the α1B-adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by ρ-TIA. N-terminally truncated analogs of ρ-TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of ρ-TIA (Arg4). An alanine walk of ρ-TIA confirmed the importance of Arg4 for activity and revealed a number of other residues clustered around Arg4 that contribute to the potency of ρ-TIA. The unique allosteric antagonism of ρ-TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive α1-adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective.

Allosteric alpha 1-adrenoreceptor antagonism by the conopeptide rho-TIA.

A peptide contained in the venom of the predatory marine snail Conus tulipa, ρ-TIA, has previously been shown to possess α1-adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, ρ-TIA inhibited α1-adrenoreceptor-mediated increases in cytosolic Ca2+ concentration that were triggered by norepinephrine, but did not affect presynaptic α2-adrenoreceptor-mediated responses. In radioligand binding assays using [125I]HEAT, ρ-TIA displayed slightly greater potency at the α1B than at the α1A or α1D subtypes. Moreover, although it did not affect the rate of association for [3H]prazosin binding to the α1B-adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by ρ-TIA. N-terminally truncated analogs of ρ-TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of ρ-TIA (Arg4). An alanine walk of ρ-TIA confirmed the importance of Arg4 for activity and revealed a number of other residues clustered around Arg4 that contribute to the potency of ρ-TIA. The unique allosteric antagonism of ρ-TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive α1-adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective.

Allosteric α1 adrenoceptor antagonism by the conopeptide ρ-TIA

A peptide contained in the venom of the predatory marine snail Conus tulipa, ρ-TIA, has previously been shown to possess α1-adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, ρ-TIA inhibited α1-adrenoreceptor-mediated increases in cytosolic Ca2+ concentration that were triggered by norepinephrine, but did not affect presynaptic α2-adrenoreceptor-mediated responses. In radioligand binding assays using [125I]HEAT, ρ-TIA displayed slightly greater potency at the α1B than at the α1A or α1D subtypes. Moreover, although it did not affect the rate of association for [3H]prazosin binding to the α1B-adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by ρ-TIA. N-terminally truncated analogs of ρ-TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of ρ-TIA (Arg4). An alanine walk of ρ-TIA confirmed the importance of Arg4 for activity and revealed a number of other residues clustered around Arg4 that contribute to the potency of ρ-TIA. The unique allosteric antagonism of ρ-TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive α1-adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective.

Amezinium and debrisoquine are substrates of uptake1 and potent inhibitors of monoamine oxidase in perfused lungs of rats

Previous studies have resulted in the classification of amezinium as a selective inhibitor of neuronal monoamine oxidase (MAO), because it is a much more potent MAO inhibitor in intact tissues, in which it is accumulated in noradrenergic neurones by uptake1, than in tissue homogenates. In the present study, the effects of amezinium on the deamination of noradrenaline were investigated in intact lungs of rats, since the pulmonary endothelial cells are a site where the catecholamine transporter is non-neuronal uptake1. In addition, another drug that is both a substrate of uptake1 and a MAO inhibitor, debrisoquine, was investigated in the study.The first aim of the study was to show whether amezinium and debrisoquine are substrates of uptake1 in rat lungs. After loading of isolated perfused lungs with 3H-noradrenaline (MAO and catechol-O-methyltransferase (COMT) inhibited), the efflux of 3H-noradrenaline was measured for 30 min. When 1 μmol/l amezinium or 15 μmol/l debrisoquine was added for the last 15 min of efflux, there was a rapid and marked increase in the fractional rate of loss of 3H-noradrenaline, which was reduced by about 70% when 1 μmol/l desipramine was present throughout the efflux period. These results showed that both drugs were substrates for uptake1 in rat lungs. In lungs perfused with 1 nmol/l 3H-noradrenaline (COMT inhibited), 10, 30 and 300 nmol/l amezinium caused 58%, 76% and 74% inhibition of noradrenaline deamination, respectively, and 30, 300 and 3000 nmol/l debrisoquine caused 56%, 89% and 96% inhibition of noradrenaline deamination, respectively. When MAO-B was also inhibited, 10 nmol/l amezinium caused 84% inhibition of the deamination of noradrenaline by MAO-A in the lungs. In contrast, in hearts perfused with 10 nmol/l 3H-noradrenaline under conditions where the amine was accumulated by uptake2 (COMT, uptake1 and vesicular transport inhibited), 10 nmol/l amezinium had no effect and 300 nmol/l amezinium caused only 36% inhibition of deamination of noradrenaline.The results when considered with previous reports in the literature show that amezinium is about 1000 times more potent and debrisoquine is about 20 times more potent for MAO inhibition in rat lungs than in tissue homogenates, and the reason for their high potencies in the intact lungs is transport and accumulation of the drugs in the pulmonary endothelial cells by uptake1. Amezinium is much less potent as a MAO inhibitor in cells with the uptake2 transporter, such as the myocardial cells of the heart. The results also confirmed previous reports that amezinium is highly selective for MAO-A.