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Synthetic α-conotoxin mutants as probes for studying nicotinic acetylcholine receptors and in the development of novel drug leads.

ABSTRACT: α-Conotoxins are peptide neurotoxins isolated from venomous marine cone snails that are potent and selective antagonists for different subtypes of nicotinic acetylcholine receptors (nAChRs). As such, they are valuable probes for dissecting the role that nAChRs play in nervous system function. In recent years, extensive insight into the binding mechanisms of α-conotoxins with nAChRs at the molecular level has aided in the design of synthetic analogs with improved pharmacological properties. This review examines the structure-activity relationship studies involving α-conotoxins as research tools for studying nAChRs in the central and peripheral nervous systems and their use towards the development of novel therapeutics.

SUBMITTER: Armishaw CJ

PROVIDER: S-EPMC3153239 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Synthetic α-conotoxin mutants as probes for studying nicotinic acetylcholine receptors and in the development of novel drug leads.

Armishaw Christopher J CJ

Toxins 20100614 6

α-Conotoxins are peptide neurotoxins isolated from venomous marine cone snails that are potent and selective antagonists for different subtypes of nicotinic acetylcholine receptors (nAChRs). As such, they are valuable probes for dissecting the role that nAChRs play in nervous system function. In recent years, extensive insight into the binding mechanisms of α-conotoxins with nAChRs at the molecular level has aided in the design of synthetic analogs with improved pharmacological properties. This ...[more]

PMID: 22069647

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Molecular determinants of α-conotoxin potency for inhibition of human and rat α6β4 nicotinic acetylcholine receptors.

Project description:Nicotinic acetylcholine receptors (nAChRs) containing α6 and β4 subunits are expressed by dorsal root ganglion neurons and have been implicated in neuropathic pain. Rodent models are often used to evaluate the efficacy of analgesic compounds, but species differences may affect the activity of some nAChR ligands. A previous candidate α-conotoxin-based therapeutic yielded promising results in rodent models, but failed in human clinical trials, emphasizing the importance of understanding species differences in ligand activity. Here, we show that human and rat α6/α3β4 nAChRs expressed in Xenopus laevis oocytes exhibit differential sensitivity to α-conotoxins. Sequence homology comparisons of human and rat α6β4 nAChR subunits indicated that α6 residues forming the ligand-binding pocket are highly conserved between the two species, but several residues of β4 differed, including a Leu-Gln difference at position 119. X-ray crystallography of α-conotoxin PeIA complexed with the Aplysia californica acetylcholine-binding protein (AChBP) revealed that binding of PeIA orients Pro13 in close proximity to residue 119 of the AChBP complementary subunit. Site-directed mutagenesis studies revealed that Leu119 of human β4 contributes to higher sensitivity of human α6/α3β4 nAChRs to α-conotoxins, and structure-activity studies indicated that PeIA Pro13 is critical for high potency. Human and rat α6/α3β4 nAChRs displayed differential sensitivities to perturbations of the interaction between PeIA Pro13 and residue 119 of the β4 subunit. These results highlight the potential significance of species differences in α6β4 nAChR pharmacology that should be taken into consideration when evaluating the activity of candidate human therapeutics in rodent models.

| S-EPMC6240866 | biostudies-literature

Identifying key amino acid residues that affect α-conotoxin AuIB inhibition of α3β4 nicotinic acetylcholine receptors.

Project description:α-Conotoxin AuIB is a selective α3β4 nicotinic acetylcholine receptor (nAChR) subtype inhibitor. Its analgesic properties are believed to result from it activating GABAB receptors and subsequently inhibiting CaV2.2 voltage-gated calcium channels. The structural determinants that mediate diverging AuIB activity at these targets are unknown. We performed alanine scanning mutagenesis of AuIB and α3β4 nAChR, homology modeling, and molecular dynamics simulations to identify the structural determinants of the AuIB·α3β4 nAChR interaction. Two alanine-substituted AuIB analogues, [P6A]AuIB and [F9A]AuIB, did not inhibit the α3β4 nAChR. NMR and CD spectroscopy studies demonstrated that [F9A]AuIB retains its native globular structure, so its activity loss is probably due to loss of specific toxin-receptor residue pairwise contacts. Compared with AuIB, the concentration-response curve for inhibition of α3β4 by [F9A]AuIB shifted rightward more than 10-fold, and its subtype selectivity profile changed. Homology modeling and molecular dynamics simulations suggest that Phe-9 of AuIB interacts with a two-residue binding pocket on the β4 nAChR subunit. This hypothesis was confirmed by site-directed mutagenesis of the β4-Trp-59 and β4-Lys-61 residues of loop D, which form a putative binding pocket. AuIB analogues with Phe-9 substitutions corroborated the finding of a binding pocket on the β4 subunit and gave further insight into how AuIB Phe-9 interacts with the β4 subunit. In summary, we identified critical residues that mediate interactions between AuIB and its cognate nAChR subtype. These findings might help improve the design of analgesic conopeptides that selectively "avoid" nAChR receptors while targeting receptors involved with nociception.

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α-Conotoxin PeIA[S9H,V10A,E14N] potently and selectively blocks α6β2β3 versus α6β4 nicotinic acetylcholine receptors.

Project description:Nicotinic acetylcholine receptors (nAChRs) containing α6 and β2 subunits modulate dopamine release in the basal ganglia and are therapeutically relevant targets for treatment of neurological and psychiatric disorders including Parkinson's disease and nicotine dependence. However, the expression profile of β2 and β4 subunits overlap in a variety of tissues including locus ceruleus, retina, hippocampus, dorsal root ganglia, and adrenal chromaffin cells. Ligands that bind α6β2 nAChRs also potently bind the closely related α6β4 subtype. To distinguish between these two subtypes, we synthesized novel analogs of a recently described α-conotoxin, PeIA. PeIA is a peptide antagonist that blocks several nAChR subtypes, including α6/α3β2β3 and α6/α3β4 nAChRs, with low nanomolar potency. We systematically mutated PeIA and evaluated the resulting analogs for enhanced potency and/or selectivity for α6/α3β2β3 nAChRs expressed in Xenopus oocytes (α6/α3 is a subunit chimera that contains the N-terminal ligand-binding domain of the α6 subunit). On the basis of these results, second-generation analogs were then synthesized. The final analog, PeIA[S9H,V10A,E14N], potently blocked acetylcholine-gated currents mediated by α6/α3β2β3 and α6/α3β4 nAChRs with IC(50) values of 223 pM and 65 nM, respectively, yielding a >290-fold separation between the two subtypes. Kinetic studies of ligand binding to α6/α3β2β3 nAChRs yielded a k(off) of 0.096 ± 0.001 min(-1) and a k(on) of 0.23 ± 0.019 min(-1) M(-9). The synthesis of PeIA[S9H,V10A,E14N] demonstrates that ligands can be developed to discriminate between α6β2 and α6β4 nAChRs.

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