Discovery of Novel Antinociceptive α‑Conotoxin Analogues from the Direct In Vivo Screening of a Synthetic Mixture-Based Combinatorial Library
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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 <i>n</i>-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