Oxidative folding and preparation of a-conotoxins for use in high-throughput structure–activity relationship studies Reena Gyanda, Jayati Banerjee, Yi-Pin Chang, Angela M. Phillips, Lawrence Toll and Christopher J. Armishaw* a-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 a-conotoxin analogs with increased potency and selectivity towards specific nicotinic acetylcholine receptor subtypes, the efficient production of diversity-oriented a-conotoxin combinatorial libraries has been limited by inefficient folding and purification procedures. We have investigated the optimized conditions for the reliable folding of a-conotoxins using simplified oxidation procedures for use in the accelerated production of synthetic combinatorial libraries of a-conotoxins. To this end, the effect of co-solvent, redox reagents, pH, and temperature on the proportion of disulfide bond isomers was determined for a-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 a-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 a-conotoxin analogs and permit the rapid identification of active hits directly from high-throughput pharmacological screening assays. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. Keywords: conotoxin folding; disulfide bonds; high-throughput purification; combinatorial chemistry Introduction a-Conotoxins are a class of disulfide-rich peptides isolated from venomous marine cone snails that are selective antagonists of nicotinic acetylcholine receptors (nAChRs) [1]. Their unique selec- tivity for different nAChR subtypes represents an exciting research opportunity to develop novel subtype selective ligands as probes for studying tobacco addiction, neuropathic pain, and other neuropathological conditions. In addition, their implications in the design of new therapeutics for treating such conditions have been recognized [2]. Nonetheless, the preparation of a-conotoxin isomers continues to be a challenging endeavor for high-throughput structure–activity relationship studies. Structurally, a-conotoxins consist of 12–20 amino acid residues within a highly conserved Cys framework where Cys1 and Cys2 are always adjacent, thus giving rise to two loops of amino acids denoted as m- and n- respectively (Figure 1). For a-conotoxins, the disulfide bonds of the pharmacologically active isomer are usually connected in a (Cys1–3), (Cys2–4) ‘globular’ arrangement, which induces a helical barrel conformation that projects pharma- cophoric residues towards the receptor-binding pocket. However, two alternative disulfide bond isomers are also possible, namely the (Cys1–4), (Cys2-3) ‘ribbon’ and (Cys1-2), (Cys3–4) ‘beads’ isomers. Formation of ribbon and beads isomers of a-conotoxins induces structural distortions and decreased conformational stabil- ity that leads to lower biological activity and stability when compared with the globular isomer [3]. However, in certain cases, the greater structural flexibility exhibited by these two isomers can lead to analogs with unique pharmacological properties. For example, the ribbon isomer of a-conotoxin AuIB has been shown to be several times more potent at rat parasympathetic nAChR than the globular isomer [4]. Moreover, the related w/l-conotoxins, which inhibit the norepinephrine transporter, exhibit the ribbon disulfide topology in their pharmacologically active form [5,6]. So far, several different Cys loop spacing frameworks with vary- ing m/n-loop sizes have been identified in native a-conotoxins, including 4/7 (MII, PnIB, and GID), 4/6 (AuIB), 4/4 (BuIA), 4/3 (ImI), and 3/5 (GI) (Figure 1). Apart from the conserved disulfide bonding Cys residues, extensive mutation is observed across the m- and n-loops of all known examples of a-conotoxins, thereby accounting for their exquisite selectivity towards various nAChR subtypes. a-Conotoxins may contain a varying number of amino acid residues in their respective m- and n-loops, although the majority * Correspondence to: Christopher J. Armishaw, Torrey Pines Institute for Molecular Studies, 11350 SW Village Pkwy, Port St. Lucie, FL 34987, USA. E-mail: carmishaw@tpims.org Torrey Pines Institute for Molecular Studies, Port St Lucie, Florida, 34987, USA Abbreviations used: GSH, reduced glutathione; GSSG, oxidized glutathione; IPA, isopropanol; MBHA, 4-methylbenzylhydrylamine; MeBzl, 4-methylbenzyl; nAChR, nicotinic acetylcholine receptor; PAM, phenylacetomidomethyl; SPE, solid-phase extraction. J. Pept. Sci. 2013; 19: 16–24 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. Research Article Received: 28 June 2012 Revised: 27 September 2012 Accepted: 16 October 2012 Published online in Wiley Online Library: 28 November 2012 (wileyonlinelibrary.com) DOI 10.1002/psc.2467 16