Discovery of Novel Antinociceptive α-Conotoxin Analogues from the Direct In Vivo Screening of a Synthetic Mixture-Based Combinatorial Library

Abstract

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.

Design and synthesis of α‐conotoxin GID analogues as selective α4β2 nicotinic acetylcholine receptor antagonists

“…As part of our continued efforts to investigate α‐conotoxins as molecular probes and therapeutic agents for studying pain and nicotine addiction, we have developed a homology model of the GID/α4β2 nAChR complex to provide further insights regarding the interactions of GID within the receptor binding pocket. Here we report the design and synthesis of a series of α‐conotoxin GID analogues based on our homology model that exhibit increased selectivity for α4β2 nAChRs over the α3β2 subtype.…”

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

“…The PS-SCL consisted of a total of 113 379 904 α-conotoxin sequences, which expands immensely upon the diversity of conotoxin libraries previously reported by our group. 36,37 …”

α-Conotoxin Peptidomimetics: Probing the Minimal Binding Motif for Effective Analgesia

“…ImI, from Conus imperialis, contains 12 amino acid residues with two disulfide bridges linking the backbone in a globular CysI-III, CysII-IV framework (Table 1, Figure 2) [80,81]. Interestingly, it is a selective inhibitor of the α7 nAChR but was found to be devoid of analgesic activity when tested in a warm water tail withdrawal assay [82]. Diselenide substitution in either Loop I or Loop II, or replacement of both, provides only a marginal increase in potency at the α7 nAChR, which is attributed to increased hydrophobic interactions between the diselenide bridge and the receptor [79].…”