Oxidative folding and preparation of α‐conotoxins for use in high‐throughput structure–activity relationship studies

Abstract

α-Conotoxins are peptide neurotoxins that selectively inhibit various subtypes of nicotinic acetylcholine receptors. They are important research tools for studying numerous pharmacological disorders, with profound potential for developing drug leads for treating pain, tobacco addiction, and other conditions. They are characterized by the presence of two disulfide bonds connected in a globular arrangement, which stabilizes a bioactive helical conformation. Despite extensive structure-activity relationship studies that have produced α-conotoxin analogs with increased potency and selectivity towards specific nicotinic acetylcholine receptor subtypes, the efficient production of diversity-oriented α-conotoxin combinatorial libraries has been limited by inefficient folding and purification procedures. We have investigated the optimized conditions for the reliable folding of α-conotoxins using simplified oxidation procedures for use in the accelerated production of synthetic combinatorial libraries of α-conotoxins. To this end, the effect of co-solvent, redox reagents, pH, and temperature on the proportion of disulfide bond isomers was determined for α-conotoxins exhibiting commonly known Cys loop spacing frameworks. In addition, we have developed high-throughput 'semi-purification' methods for the quick and efficient parallel preparation of α-conotoxin libraries for use in accelerated structure-activity relationship studies. Our simplified procedures represent an effective strategy for the preparation of large arrays of correctly folded α-conotoxin analogs and permit the rapid identification of active hits directly from high-throughput pharmacological screening assays.

Optimal Cleavage and Oxidative Folding of α-Conotoxin TxIB as a Therapeutic Candidate Peptide

“…We also tested the effect of co-solvents, IPA and DMSO, on folding reactions. We observed that, in contrast to other studies [22], IPA addition significantly decreased the yield of globular isomer (runs 41–48). IPA addition improved the yield of ribbon isomer yield, suggesting that IPA is a key factor for oxidative folding of the ribbon isomer.…”

“…As observed by other researchers for α-conotoxin folding, temperature is not a significant factor for globular isomer yield, while the pH plays a key role [22]. Overall, our results of temperature and pH experiments are consistent with prior studies for α-conotoxins.…”

Discovery of a Potent and Selective α3β4 Nicotinic Acetylcholine Receptor Antagonist from an α-Conotoxin Synthetic Combinatorial Library

“…28 The formation of disulfide bonds was achieved by cosolvent assisted oxidative folding (50% isopropanol in aqueous ammonium bicarbonate buffer at pH 8.2), which was previously shown to maximize the accumulation of the native globular isomer of α-conotoxin BuIA. 29 A simplified desalting procedure that employed disposable solid-phase extraction (SPE) columns was used for the rapid and efficient preparation of library samples in parallel prior to initial pharmacological screening. 29 Note that the globular and ribbon isomers of selected second generation individual analogues were later synthesized separately to confirm their identity and were purified by RP-HPLC prior to formal functional characterization.…”

“…29 A simplified desalting procedure that employed disposable solid-phase extraction (SPE) columns was used for the rapid and efficient preparation of library samples in parallel prior to initial pharmacological screening. 29 Note that the globular and ribbon isomers of selected second generation individual analogues were later synthesized separately to confirm their identity and were purified by RP-HPLC prior to formal functional characterization.…”

“…29 Following evaporation of the isopropyl alcohol in vaccuo (Genevac Rocket, Ipswich, U.K.), samples were isolated in parallel with solid-phase extraction (SPE) cartridges using a 24-sample vacuum manifold. 29 For SPE cartridge semipurification, the sample was loaded by passing through an Oasis; 3 cc, 540 mg SPE cartridge (Waters) pre-equilibrated with buffer A and washed with a further 10 mL of buffer A to remove hydrophilic impurities (i.e., buffer salts). The α-conotoxin product was then eluted with 65% aqueous acetonitrile/0.1% TFA (buffer E, 10 mL), collected in a clean tube, and lyophilized.…”