Design, Synthesis and Mechanism of Action of Novel Μ-Conotoxin KIIIA Analogs for Inhibition of the Voltage-Gated Sodium Channel Nav1.7.

mu-Conotoxin KIIIA, a selective blocker of sodium channels, has strong inhibitory activity against several Nav isoforms, including Nav1.7, and has potent analgesic effects, but it con-tains three pairs of disulfide bonds, making structural modifi- cation difficult and synthesis complex. To circumvent these difficulties, we designed and synthesized three KIIIA analogues with one disulfide bond deleted. The most active analogue, KIIIA-1, was further analyzed, and its binding pattern to hNav1.7 was determined by molecular dynamics simulations. Guided by the molecular dynamics computational model, we designed and tested 32 second-generation and 6 third -generation analogues of KIIIA-1 on hNav1.7 expressed in HEK293 cells. Several analogues showed significantly improved inhibitory activity on hNav1.7, and the most potent peptide, 37, was approximately 4-fold more potent than the KIIIA Isomer I and 8-fold more potent than the wildtype (WT) KIIIA in inhibiting hNav1.7 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 ex-hibits potent in vivo analgesic effects and has promising po-tential for development as a pain therapy in the future.