Design, Synthesis, and Structure–Activity Relationship Study of μ-Conotoxin KIIIA toward Na _V 1.4

Na_V1.4 expressed in skeletal muscle, is a validated target for treating neuromuscular disorders. While the μ-conotoxin KIIIA is the one of the smallest conopeptide inhibitors of Na_V1.4, its therapeutic potential is limited by potency and synthetic complexity. Using a computational model, we designed analogues with added positive charges, yielding [K7R, A15R]KIIIA-1 (IC₅₀ = 20.5 nM). Further optimization produced [S5R]KA with high potency (IC₅₀ = 5.1 nM) but poor enzymatic stability (<i>t</i>_1/2 < 4 h). To improve stability, we replaced Cys4 with penicillamine (Pen), generating C4Pen. This analogue maintained excellent activity (IC₅₀ = 11.1 nM) and significantly improved stability (<i>t</i>_1/2 > 8 h), and both analogues preferentially inhibited Na_V1.4 with moderate activity at Na_V1.2 and Na_V1.7 and exhibited no detectable inhibition of Na_V1.5. <i>In vivo</i> studies confirmed that both [S5R]KA and C4Pen induce potent muscle relaxation. This structure-based optimization combines electrostatic mutagenesis with Pen-mediated stabilization, yielding a promising peptide therapeutic candidate.