A novel ω-conotoxin Bu8 inhibiting N-type voltage-gated calcium channels displays potent analgesic activity

ω-Conotoxins inhibit N-type voltage-gated calcium (CA_V2.2) channels and exhibit efficacy in attenuating neuropathic pain but have a low therapeutic index. Here, we synthesized and characterized a novel ω-conotoxin, Bu8 from Conus bullatus, which consists of 25 amino acid residues and three disulfide bridges. Bu8 selectively and potently inhibits depolarization-activated Ba²⁺ currents mediated by rat CA_V2.2 expressed in HEK293T cells (IC₅₀ = 89 nmol/L). Bu8 is two-fold more potent than ω-conotoxin MVIIA, a ω-conotoxin currently used for the treatment of severe chronic pain. It also displays potent analgesic activity in animal pain models of hot plate and acetic acid writhing but has fewer side effects on mouse motor function and lower toxicity in goldfish. Its lower side effects may be attributed to its faster binding rate and higher recovery ratios. The NMR structure demonstrates that Bu8 contains a small irregular triple β-strand. The structure-activity relationships of Bu8 Ala mutants and Bu8/MVIIA hybrid mutants demonstrate that the binding mode of CA_V2.2 with the amino acid residues in loop 1 and loop 2 of Bu8 is different from that of MVIIA. This study characterizes a novel, more potent ω-conotoxin and provides new insights for designing CA_V2.2 antagonists.

Analgesic activity; Bu8; DIEA, diisopropylethylamine; ESI-MS, electrospray ionization-mass spectroscopy; Fmoc, N-(9-fluorenyl)methyloxy-carbonyl; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; IC50, half-maximal inhibitory concentration; N-type calcium ion channel; RP-HPLC, reversed phase high-performance liquid chromatography; Structure–activity relationship; TFA, trifluoroacetic acid; ω-conotoxin.