Inhibition of human Nav1.4 by D-amino acid modified μ-CnIIIC.

Voltage-gated sodium channel Nav1.4 is a key target for neuromuscular disorders; however, existing peptide inhibitors face stability challenges. We systematically investigated the effect of D-amino acid modification on the inhibition of the human voltage-gated sodium channel NaV1.4 by μ-CnIIIC. The N-terminal pyroglutamic acid (pGlu) of wild-type μ-CnIIIC was replaced with D-arginine (Dar), D-lysine (Dly), or D-histidine (Dhi) via solid-phase peptide synthesis, yielding the derivatives dR-μ-CnIIIC, dK-μ-CnIIIC, and dH-μ-CnIIIC, respectively. Voltage patch clamp experiments demonstrated that dR-μ-CnIIIC, which inhibited NaV1.4 in its resting state, exhibited superior inhibitory activity, with an IC50 valueof 54.14 ± 6.26 nM (P < 0.01) compared to 96.37 ± 10.68 nM for the wild-type peptide. Molecular docking and kinetic simulations revealed that dR-μ-CnIIIC formed strong electrostatic interactions and hydrogen-bonding networks with the hNaV1.4 protein α-subunits Glu352, Asp1514, and Asp1515, as well as the β-subunit Glu48, thereby enhancing its binding affinity. Serum stability assays further demonstrated that D-amino acid modification substantially enhanced the resistance to protease degradation, with dR-μ-CnIIIC retaining 31.7% activity after 480 min. This study provides a rational design strategy for the development of efficient and stable sodium channel-targeted therapeutic agents. Moreover, this D-amino acid substitution strategy may be generalizable to other conotoxin-based therapeutics.