Disulfide-rich animal venom peptides targeting either the voltage-sensing domain or the pore domain of voltage-gated sodium channel 1.7 (Na_V1.7) have been widely studied as drug leads and pharmacological probes for the treatment of chronic pain. However, despite intensive research efforts, the full potential of Na_V1.7 as a therapeutic target is yet to be realized. In this study, using evolved sortase A, we enzymatically ligated two known Na_V1.7 inhibitors–PaurTx3, a spider-derived peptide toxin that modifies the gating mechanism of the channel through interaction with the voltage-sensing domain, and KIIIA, a small cone snail-derived peptide inhibitor of the pore domain–with the aim of creating a bivalent inhibitor which could interact simultaneously with two noncompeting binding sites. Using electrophysiology, we determined the activity at Na_V1.7, and to maximize potency, we systematically evaluated the optimal linker length, which was nine amino acids. Our optimized synthetic bivalent peptide showed improved channel affinity and potency at Na_V1.7 compared to either PaurTx3 or KIIIA individually. This work shows that novel and improved Na_V1.7 inhibitors can be designed by combining a pore blocker toxin and a gating modifier toxin to confer desired pharmacological properties from both the voltage-sensing domain and the pore domain.

Disulfide-rich animal venom peptides targeting either the voltage-sensing domain or the pore domain of voltage-gated sodium channel 1.7 (Na_V1.7) have been widely studied as drug leads and pharmacological probes for the treatment of chronic pain. However, despite intensive research efforts, the full potential of Na_V1.7 as a therapeutic target is yet to be realized. In this study, using evolved sortase A, we enzymatically ligated two known Na_V1.7 inhibitors–PaurTx3, a spider-derived peptide toxin that modifies the gating mechanism of the channel through interaction with the voltage-sensing domain, and KIIIA, a small cone snail-derived peptide inhibitor of the pore domain–with the aim of creating a bivalent inhibitor which could interact simultaneously with two noncompeting binding sites. Using electrophysiology, we determined the activity at Na_V1.7, and to maximize potency, we systematically evaluated the optimal linker length, which was nine amino acids. Our optimized synthetic bivalent peptide showed improved channel affinity and potency at Na_V1.7 compared to either PaurTx3 or KIIIA individually. This work shows that novel and improved Na_V1.7 inhibitors can be designed by combining a pore blocker toxin and a gating modifier toxin to confer desired pharmacological properties from both the voltage-sensing domain and the pore domain.