Compelling human genetic studies have identified the
voltage-gated sodium channel NaV1.7 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 NaV1.7; 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 hNaV1.7 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 hNaV1.7. 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 NaV1.7 (20-fold), while maintaining >100-fold selectivity over the major off-targets NaV1.4, NaV1.5, and NaV1.6. 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 NaV1.7 inhibitors for the development of more efficacious and safer
analgesics as well as to further investigate the involvement of NaV1.7 in
pain.