Mapping the Molecular Surface of the Analgesic Na_V1.7-Selective Peptide Pn3a Reveals Residues Essential for Membrane and Channel Interactions

Abstract

Compelling human genetic studies have identified the voltage-gated sodium channel Na_V1.7 as a promising therapeutic target for the treatment of pain. The analgesic spider-venom-derived peptide μ-theraphotoxin-Pn3a is an exceptionally potent and selective inhibitor of Na_V1.7; however, little is known about the structure-activity relationships or channel interactions that define this activity. We rationally designed 17 Pn3a analogues and determined their activity at hNa_V1.7 using patch-clamp electrophysiology. The positively charged amino acids K22 and K24 were identified as crucial for Pn3a activity, with molecular modeling identifying interactions of these residues with the S3-S4 loop of domain II of hNa_V1.7. Removal of hydrophobic residues Y4, Y27, and W30 led to a loss of potency (>250-fold), while replacement of negatively charged D1 and D8 residues with a positively charged lysine led to increased potencies (>13-fold), likely through alterations in membrane lipid interactions. Mutating D8 to an asparagine led to the greatest improvement in Pn3a potency at Na_V1.7 (20-fold), while maintaining >100-fold selectivity over the major off-targets Na_V1.4, Na_V1.5, and Na_V1.6. The Pn3a[D8N] mutant retained analgesic activity <i>in vivo</i>, significantly attenuating mechanical allodynia in a clinically relevant mouse model of postsurgical pain at doses 3-fold lower than those with wild-type Pn3a, without causing motor-adverse effects. Results from this study will facilitate future rational design of potent and selective peptidic Na_V1.7 inhibitors for the development of more efficacious and safer analgesics as well as to further investigate the involvement of Na_V1.7 in pain.