α-Conotoxin ImI

alpha-Conotoxin ImI incorporating stable cystathionine bridges maintains full potency and identical three-dimensional structure.[Pubmed:21899353]

J Am Chem Soc. 2011 Oct 12;133(40):15866-9.

The two disulfide bonds of alpha-conotoxin ImI, a peptide antagonist of the alpha7 nicotinic acetylcholine receptor (nAChR), were systematically replaced with isosteric redox-stable cystathionine thioethers. Regioselective thioether formation was accomplished on solid support through substitution of a gamma-chlorohomoalanine by an intramolecular cysteine thiol to produce hybrid thioether/disulfide analogues (2 and 3) as well as a dual cystathionine analogue (4) that were found to be structurally homologous to alpha-conotoxin ImI by (1)H NMR. The antagonistic activity at the alpha7 nAChR of cystathionine analogue 3 (pIC(50) = 6.41 +/- 0.09) was identical to that of alpha-conotoxin ImI (1, pIC(50) = 6.41 +/- 0.09), whereas those of 2 (pIC(50) = 5.96 +/- 0.09) and 4 (pIC(50) = 5.89 +/- 0.09) showed a modest decrease. The effect of oxidation of the thioethers to sulfoxides was also investigated, with significant changes in the biological activities observed ranging from a >30-fold reduction (2S horizontal lineO) to a 3-fold increase (3S horizontal lineO(B)) in potencies.

Neurotoxins from snake venoms and alpha-conotoxin ImI inhibit functionally active ionotropic gamma-aminobutyric acid (GABA) receptors.[Pubmed:26221036]

J Biol Chem. 2015 Sep 11;290(37):22747-58.

Ionotropic receptors of gamma-aminobutyric acid (GABAAR) regulate neuronal inhibition and are targeted by benzodiazepines and general anesthetics. We show that a fluorescent derivative of alpha-cobratoxin (alpha-Ctx), belonging to the family of three-finger toxins from snake venoms, specifically stained the alpha1beta3gamma2 receptor; and at 10 mum alpha-Ctx completely blocked GABA-induced currents in this receptor expressed in Xenopus oocytes (IC50 = 236 nm) and less potently inhibited alpha1beta2gamma2 approximately alpha2beta2gamma2 > alpha5beta2gamma2 > alpha2beta3gamma2 and alpha1beta3delta GABAARs. The alpha1beta3gamma2 receptor was also inhibited by some other three-finger toxins, long alpha-neurotoxin Ls III and nonconventional toxin WTX. alpha-Conotoxin ImI displayed inhibitory activity as well. Electrophysiology experiments showed mixed competitive and noncompetitive alpha-Ctx action. Fluorescent alpha-Ctx, however, could be displaced by muscimol indicating that most of the alpha-Ctx-binding sites overlap with the orthosteric sites at the beta/alpha subunit interface. Modeling and molecular dynamic studies indicated that alpha-Ctx or alpha-bungarotoxin seem to interact with GABAAR in a way similar to their interaction with the acetylcholine-binding protein or the ligand-binding domain of nicotinic receptors. This was supported by mutagenesis studies and experiments with alpha-conotoxin ImI and a chimeric Naja oxiana alpha-neurotoxin indicating that the major role in alpha-Ctx binding to GABAAR is played by the tip of its central loop II accommodating under loop C of the receptors.

Delineation of the unbinding pathway of alpha-conotoxin ImI from the alpha7 nicotinic acetylcholine receptor.[Pubmed:22571488]

J Phys Chem B. 2012 May 31;116(21):6097-105.

alpha-Conotoxins potently and specifically inhibit isoforms of nicotinic acetylcholine receptors (nAChRs) and are used as molecular probes and as drugs or drug leads. Interactions occurring during binding and unbinding events are linked to binding kinetics, and knowledge of these interactions could help in the development of alpha-conotoxins as drugs. Here, the unbinding process for the prototypical alpha-conotoxin ImI/alpha7-nAChR system was investigated theoretically, and three exit routes were identified using random accelerated molecular dynamics simulations. The route involving the smallest conformation perturbation was further divided into three subpathways, which were studied using steered molecular dynamics simulations. Of the three subpathways, two had better experimental support and lower potential of mean force, indicating that they might be sampled more frequently. Additionally, these subpathways were supported by previous experimental studies. Several pairwise interactions, including a cation-pi interaction and charge and hydrogen bond interactions, were identified as potentially playing important roles in the unbinding event.

The use of alpha-conotoxin ImI to actualize the targeted delivery of paclitaxel micelles to alpha7 nAChR-overexpressing breast cancer.[Pubmed:25542793]

Biomaterials. 2015 Feb;42:52-65.

Alpha7 nicotinic acetylcholine receptor (alpha7 nAChR), a ligand-gated ion channel, is increasingly emerging as a new tumor target owing to its expression specificity and significancy for cancer. In an attempt to increase the targeted drug delivery to the alpha7 nAChR-overexpressing tumors, herein, alpha-conotoxin ImI, a disulfide-rich toxin with highly affinity for alpha7 nAChR, was modified on the PEG-DSPE micelles (ImI-PMs) for the first time. The DLS, TEM and HPLC detections showed the spherical nanoparticle morphology about 20 nm with negative charge and high drug encapsulation. The ligand modification did not induce significant differences. The immunofluorescence assay confirmed the expression level of alpha7 nAChR in MCF-7 cells. In vitro and in vivo experiments demonstrated that the alpha7 nAChR-targeted nanomedicines could deliver more specifically and faster into alpha7 nAChR-overexpressing MCF-7 cells. Furthermore, fluo-3/AM fluorescence imaging technique indicated that the increased specificity was attributed to the ligand-receptor interaction, and the inducitivity for intracellular Ca(2+) transient by ImI was still remained after modification. Moreover, paclitaxel, a clinical frequently-used anti-tumor drug for breast cancer, was loaded in ImI-modified nanomedicines to evaluate the targeting efficacy. Besides of exhibiting greater cytotoxicity and inducing more cell apoptosis in vitro, paclitaxel-loaded ImI-PMs displayed stronger anti-tumor efficacy in MCF-7 tumor-bearing nu/nu mice. Finally, the active targeting system showed low systemic toxicity and myelosuppression evidenced by less changes in body weight, white blood cells, neutrophilic granulocyte and platelet counts. In conclusion, alpha7 nAChR is also a promising target for anti-tumor drug delivery and in this case, alpha-conotoxin ImI-modified nanocarrier is a potential delivery system for targeting alpha7 nAChR-overexpressing tumors.

Alpha-conotoxin-ImI: a competitive antagonist at alpha-bungarotoxin-sensitive neuronal nicotinic receptors in hippocampal neurons.[Pubmed:8819535]

J Pharmacol Exp Ther. 1996 Sep;278(3):1472-83.

In the present study, the patch-clamp technique was applied to rat hippocampal neurons or myoballs in culture to study the actions of alpha-conotoxin-ImI on the native alpha-bungarotoxin-sensitive, presumably alpha 7-bearing, neuronal nicotinic receptor and on other ligand-gated channels. Preexposure of the neurons for 5 min to alpha-conotoxin-ImI decreased the peak amplitude of alpha-BGT-sensitive currents (referred to as type IA currents) in a concentration-dependent fashion. Several lines of evidence revealed that the inhibitory effect of alpha-conotoxin-ImI was competitive with respect to the agonist (IC50 approximately 85 nM) and reversible by washing. At 300 nM, alpha-conotoxin-ImI decreased by only 15% the peak amplitude of ACh-evoked currents in rat myoballs, did not affect the activation of currents gated by gamma-aminobutyric acid, glycine, N-methyl-D-aspartate, kainate, or quisqualate in hippocampal neurons, but reduced to approximately 60% the peak amplitude and shortened the decay phase of curare-sensitive, serotonin-gated currents in these neurons. The competitive and reversible nature of the alpha-conotoxin-ImI-induced inhibition of native alpha 7-bearing neuronal nicotinic receptors makes this peptide a valuable new tool for the functional and structural characterization of these receptors in the central nervous system.

alpha-Conotoxin ImI exhibits subtype-specific nicotinic acetylcholine receptor blockade: preferential inhibition of homomeric alpha 7 and alpha 9 receptors.[Pubmed:7651351]

Mol Pharmacol. 1995 Aug;48(2):194-9.

Through a study of cloned nicotinic receptors expressed in Xenopus oocytes, we provide evidence that alpha-conotoxin ImI, a peptide marine snail toxin that induces seizures in rodents, selectively blocks subtypes of nicotinic acetylcholine receptors. alpha-Conotoxin ImI blocks homomeric alpha 7 nicotinic receptors with the highest apparent affinity and homomeric alpha 9 receptors with 8-fold lower affinity. This toxin has no effect on receptors composed of alpha 2 beta 2, alpha 3 beta 2, alpha 4 beta 2, alpha 2 beta 4, alpha 3 beta 4, or alpha 4 beta 4 subunit combinations. In contrast to alpha-bungarotoxin, which has high affinity for alpha 7, alpha 9, and alpha 1 beta 1 gamma delta receptors, alpha-conotoxin ImI has low affinity for the muscle nAChR. Related Conus peptides, alpha-conotoxins MI and GI, exhibit a distinct specificity, strictly targeting the muscle subtype receptor but not alpha 7 or alpha 9 receptors. alpha-Conotoxins thus represent selective tools for the study of neuronal nicotinic acetylcholine receptors.

A nicotinic acetylcholine receptor ligand of unique specificity, alpha-conotoxin ImI.[Pubmed:8206995]

J Biol Chem. 1994 Jun 17;269(24):16733-9.

We report the isolation, characterization, and total synthesis of a small peptide ligand for nicotinic acetylcholine receptors (nAChRs). It is highly active against the neuromuscular receptor in frog but not in mice. In contrast, it induces seizures when injected centrally in mice and rats, suggesting that it may target neuronal nAChRs in mammals. Although such receptors may be important in both normal cognition and the pathophysiology of several neuropsychiatric disorders, there are few ligands to discriminate between the multiple receptor subtypes. The new peptide is a highly divergent alpha-conotoxin from the snail Conus imperialis, which preys on polychaete worms. In this article, the purification, structural analysis, synthesis, and preliminary physiological characterization of alpha-conotoxin ImI (alpha-CTx-ImI) are reported. The sequence of the peptide is: Gly-Cys-Cys-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Cys-NH2. The peptide shows striking sequence differences from all alpha-conotoxins of fish-hunting Conus, but its disulfide-bridging is similar: [2-8; 3-12]. We suggest that cone venoms may provide an array of ligands with selectivity for various neuronal nAChR subtypes.