Discovery of a potent and selective α3ß4 nicotinic acetylcholine receptor antagonist from an α-conotoxin synthetic combinatorial library.

α-Conotoxins are disulfide-rich peptide neurotoxins that selectively inhibit neuronal nicotinic acetylcholine receptors (nAChRs). The α3ß4 nAChR subtype has been identified as a novel target for managing nicotine addiction. Using a mixture-based positional-scanning synthetic combinatorial library (PS-SCL) with the α4/4-conotoxin BuIA framework, we discovered a highly potent and selective α3ß4 nAChR antagonist. The initial PS-SCL consisted of a total of 113 379 904 sequences that were screened for α3ß4 nAChR inhibition, which facilitated the design and synthesis of a second generation library of 64 individual α-conotoxin derivatives. Eleven analogues were identified as α3ß4 nAChR antagonists, with TP-2212-59 exhibiting the most potent antagonistic activity and selectivity over the α3ß2 and α4ß2 nAChR subtypes. Final electrophysiological characterization demonstrated that TP-2212-59 inhibited acetylcholine evoked currents in α3ß4 nAChRs heterogeneously expressed in Xenopus laevis oocytes with a calculated IC50 of 2.3 nM and exhibited more than 1000-fold selectivity over the α3ß2 and α7 nAChR subtypes. As such, TP-2212-59 is among the most potent α3ß4 nAChRs antagonists identified to date and further demonstrates the utility of mixture-based combinatorial libraries in the discovery of novel α-conotoxin derivatives with refined pharmacological activity.

Design and synthesis of α-conotoxin GID analogues as selective α4ß2 nicotinic acetylcholine receptor antagonists.

The α4ß2 nicotinic acetylcholine receptor (nAChR) is an important target for currently approved smoking cessation therapeutics. However, the development of highly selective α4ß2 nAChR antagonists remains a significant challenge. α-Conotoxin GID is an antagonist of α4ß2 nAChRs, though it is significantly more potent toward the α3ß2 and α7 subtypes. With the goal of obtaining further insights into α-conotoxin GID/nAChR interactions that could lead to the design of GID analogues with improved affinity for α4ß2 nAChRs, we built a homology model of the GID/α4ß2 complex using an X-ray co-crystal structure of an α-conotoxin/acetylcholine binding protein (AChBP) complex. Several additional interactions that could potentially enhance the affinity of GID for α4ß2 nAChRs were observed in our model, which led to the design and synthesis of 22 GID analogues. Seven analogues displayed inhibitory activity toward α4ß2 nAChRs that was comparable to GID. Significantly, both GID[A10S] and GID[V13I] demonstrated moderately improved selectivity toward α4ß2 over α3ß2 when compared with GID, while GID[V18N] exhibited no measurable inhibitory activity for the α3ß2 subtype, yet retained inhibitory activity for α4ß2. In this regard, GID[V18N] is the most α4ß2 nAChR selective α-conotoxin analogue identified to date.

The chemical synthesis of α-conotoxins and structurally modified analogs with enhanced biological stability.

α-Conotoxins are peptide neurotoxins isolated from the venom ducts of carnivorous marine cone snails that exhibit exquisite pharmacological potency and selectivity for various nicotinic acetylcholine receptor subtypes. As such, they are important research tools and drug leads for treating various diseases of the central nervous system, including pain and tobacco addiction. Despite their therapeutic potential, the chemical synthesis of α-conotoxins for use in structure-activity relationship studies is complicated by the possibility of three disulfide bond isomers, where inefficient folding methods can lead to a poor recovery of the pharmacologically active isomer. In order to achieve higher yields of the native isomer, especially in high-throughput syntheses it is necessary to select appropriate oxidative folding conditions. Moreover, the poor biochemical stability exhibited by α-conotoxins limits their general therapeutic applicability in vivo. Numerous strategies to enhance their stability including the substitution of disulfide bond with diselenide bond and N-to-C cyclization via an oligopeptide spacer have successfully overcome these limitations. This chapter describes methods for performing both selective and nonselective disulfide bond oxidation strategies for controlling the yields and formation of α-conotoxin disulfide bond isomers, as well as methods for the production of highly stable diselenide-containing and N-to-C cyclized conotoxin analogs.

Discovery of novel antinociceptive α-conotoxin analogues from the direct in vivo screening of a synthetic mixture-based combinatorial library.

Marine cone snail venoms consist of large, naturally occurring combinatorial libraries of disulfide-constrained peptide neurotoxins known as conotoxins, which have profound potential in the development of analgesics. In this study, we report a synthetic combinatorial strategy that probes the hypervariable regions of conotoxin frameworks to discover novel analgesic agents by utilizing high diversity mixture-based positional-scanning synthetic combinatorial libraries (PS-SCLs). We hypothesized that the direct in vivo testing of these mixture-based combinatorial library samples during the discovery phase would facilitate the identification of novel individual compounds with desirable antinociceptive profiles while simultaneously eliminating many compounds with poor activity or liabilities of locomotion and respiration. A PS-SCL was designed based on the α-conotoxin RgIA-ΔR n-loop region and consisted of 10,648 compounds systematically arranged into 66 mixture samples. Mixtures were directly screened in vivo using the mouse 55 °C warm-water tail-withdrawal assay, which allowed deconvolution of amino acid residues at each position that confer antinociceptive activity. A second generation library of 36 individual α-conotoxin analogues was synthesized using systematic combinations of amino acids identified from PS-SCL deconvolution and further screened for antinociceptive activity. Six individual analogues exhibited comparable antinociceptive activity to that of the recognized analgesic α-conotoxin RgIA-ΔR, and were selected for further examination of antinociceptive, respiratory, and locomotor effects. Three lead compounds were identified that produced dose-dependent antinociception without significant respiratory depression or decreased locomotor activity. Our results represent a unique approach for rapidly developing novel lead α-conotoxin analogues as low-liability analgesics with promising therapeutic potential.

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

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.