Design, synthesis and mechanism of action of novel μ-conotoxin KIIIA analogs for inhibition of the voltage-gated sodium channel Na v 1.7.

μ-Conotoxin KIIIA, a selective blocker of sodium channels, has strong inhibitory activity against several Nav isoforms, including Nav 1.7, and has potent analgesic effects, but it contains three pairs of disulfide bonds, making structural modification difficult and synthesis complex. To circumvent these difficulties, we designed and synthesized three KIIIA analogs with one disulfide bond deleted. The most active analog, KIIIA-1, was further analyzed and its binding pattern to hNav 1.7 was determined by molecular dynamic (MD) simulations. Guided by the MD computational model, we designed and tested 32 2nd-generation and 6 3rd-generation analogs of KIIIA-1 on hNav 1.7 expressed in HEK293 cells. Several analogs showed significantly improved inhibitory activity on the hNav 1.7, and the most potent peptide, 37, was approximately 4-fold more potent than KIIIA Isomer I and 8-fold more potent than the wild-type (WT) KIIIA in inhibiting hNav 1.7 current. Intraperitoneally injected 37 exhibited potent in vivo analgesic activity in a formalin-induced inflammatory pain model, with activity reaching ∼350-fold of the positive control drug morphine. Overall, peptide 37 has a simplified disulfide-bond framework and exhibits potent in vivo analgesic effects, and has promising potential for development as a pain therapy in the future.