Abstract
Compelling human genetic studies have identified the voltage-gated sodium channel Na(V)1.7 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(V)1.7; 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(V)1.7 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(V)1.7. 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(V)1.7 (20-fold), while maintaining >100-fold selectivity over the major off-targets Na(V)1.4, Na(V)1.5, and Na(V)1.6. The Pn3a[D8N] mutant retained analgesic activity in vivo, 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(V)1.7 inhibitors for the development of more efficacious and safer analgesics as well as to further investigate the involvement of Na(V)1.7 in pain.