title: 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.
authors.affiliation: Torrey Pines Institute for Molecular Studies, Port St Lucie, Florida 34987, USA.
authors.forename: Reena
authors.initials: R
authors.lastname: Gyanda
authors.forename: Jayati
authors.initials: J
authors.lastname: Banerjee
authors.forename: Yi-Pin
authors.initials: YP
authors.lastname: Chang
authors.forename: Angela M
authors.initials: AM
authors.lastname: Phillips
authors.forename: Lawrence
authors.initials: L
authors.lastname: Toll
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Armishaw
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D020916:Conotoxins / Q000737:chemistry*; D057166:High-Throughput Screening Assays; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D020916:Conotoxins