A synthetic combinatorial strategy for developing alpha-conotoxin analogs as potent alpha7 nicotinic acetylcholine receptor antagonists

Abstract

alpha-Conotoxins are peptide neurotoxins isolated from venomous cone snails that display exquisite selectivity for different subtypes of nicotinic acetylcholine receptors (nAChR). They are valuable research tools that have profound implications in the discovery of new drugs for a myriad of neuropharmacological conditions. They are characterized by a conserved two-disulfide bond framework, which gives rise to two intervening loops of extensively mutated amino acids that determine their selectivity for different nAChR subtypes. We have used a multistep synthetic combinatorial approach using alpha-conotoxin ImI to develop potent and selective alpha(7) nAChR antagonists. A positional scan synthetic combinatorial library was constructed based on the three residues of the n-loop of alpha-conotoxin ImI to give a total of 10,648 possible combinations that were screened for functional activity in an alpha(7) nAChR Fluo-4/Ca2+ assay, allowing amino acids that confer antagonistic activity for this receptor to be identified. A second series of individual alpha-conotoxin analogs based on the combinations of defined active amino acid residues from positional scan synthetic combinatorial library screening data were synthesized. Several analogs exhibited significantly improved antagonist activity for the alpha(7) nAChR compared with WT-ImI. Binding interactions between the analogs and the alpha(7) nAChR were explored using a homology model of the amino-terminal domain based on a crystal structure of an acetylcholine-binding protein. Finally, a third series of refined analogs was synthesized based on modeling studies, which led to several analogs with refined pharmacological properties. Of the 96 individual alpha-conotoxin analogs synthesized, three displayed > or =10-fold increases in antagonist potency compared with WT-ImI.

FIGURE 1.

Sequences of selected α-conotoxins containing the SDPR motif in the m-loop. Asterisk denotes a carboxyl-terminal carboxamide. Cysteine residues are shaded in gray, and the native disulfide bond connectivity is indicated.

FIGURE 2.

Functional characterization of α-conotoxin ImI PS-SCL mixtures at the α₇-GH3 cell line in the Fluo-4/Ca²⁺ assay. a, each graph shows the results of one of the three sub-libraries, with bars representing the ratio of the IC₅₀ values of each mixture relative to WT-ImI. The specific IC₅₀ values determined for the mixtures are given in supplemental Table S1. Bars marked with + indicate mixtures with the native amino acids at the O_n-position. Bars shown in gray represent amino acid substitutions that were used to construct a second series of individual analogs. b, concentration-response curves of WT-ImI and selected PS-SCL mixtures using EC₈₀–EC₉₀ concentrations of acetylcholine as agonist. The figures depict data from single representative experiments, and error bars are omitted for reasons of clarity.

FIGURE 3.

Comparative potency of the second (a) and third (b) series of individual analogs at the α₇-GH3 cell line in the Ca²⁺/Fluo-4 assay represented as relative fold differences in WT-ImI activities. Each analog was tested as 3–5 individual experiments performed in duplicate. An assay concentration of 30 μm acetylcholine (EC₈₀–EC₉₀) was used for the experiments. Dark red bars are defined as very high potency analogs; red, high potency; yellow, medium potency; cyan, low potency; and black, inactive.

FIGURE 4.

NMR solution structure of analog 36. a, secondary H^α chemical shifts. b, ribbon representation with disulfide bonds shown in yellow. c, overlay of the structures of WT-ImI (red) and compound 36 (blue).

FIGURE 5.

Binding model of compound 28 into the binding pocket of a homology model of α₇ nAChR. The binding sub-pockets of the three mutated residues of the α-conotoxin are shown, a, O_9; b, O₁₀; and c, O₁₁. In all the structures, conotoxin is shown as ball and stick model of backbone atoms (green carbon) with side chain of each mutated residue (pink carbon). Receptor is shown in slightly transparent cartoon model (colored by chain magenta and cyan). The binding site residues are shown only as stick models (carbon color colored by chain). Only polar hydrogen atoms are shown.

FIGURE 6.

Binding model of compound 36 into the binding pocket of a homology model of α₇ nAChR. The binding site view of the two α₇ nAChR subunits with docked and optimized α-conotoxin (compound 36) is shown. The α-conotoxin is shown as ball and stick model of backbone atoms (green carbon) with side chains shown only for three mutated n-loop residues (pink carbon). Receptor is shown in slightly transparent cartoon model (colored by chain cyan (A) and magenta (B)). The binding site residues are shown only as stick models (carbon color colored by chain). Water molecules are shown as orange spheres, and only polar hydrogen atoms of side chains are shown.

FIGURE 7.

Pairwise structure-activity relationships for amino acids in the O₉–O₁₀ (a), O₉–O₁₁ (b), and O₁₀-O₁₁-positions (c). The relative size of each pie graph is proportional to the number of analogs synthesized and screened. Dark red, very high; red, high; yellow, medium; cyan, low; black, inactive antagonist potency at the α₇ nAChR.