Structure-function and rational design of a spider toxin Ssp1a at human voltage-gated sodium channel subtypes

The structure-function and optimization studies of Na(V)-inhibiting spider toxins have focused on developing selective inhibitors for peripheral pain-sensing Na(V)1.7. With several Na(V) subtypes emerging as potential therapeutic targets, structure-function analysis of Na(V)-inhibiting spider toxins at such subtypes is warranted. Using the recently discovered spider toxin Ssp1a, this study extends the structure-function relationships of Na(V)-inhibiting spider toxins beyond Na(V)1.7 to include the epilepsy target Na(V)1.2 and the pain target Na(V)1.3. Based on these results and docking studies, we designed analogues for improved potency and/or subtype-selectivity, with S7R-E18K-rSsp1a and N14D-P27R-rSsp1a identified as promising leads. S7R-E18K-rSsp1a increased the rSsp1a potency at these three Na(V) subtypes, especially at Na(V)1.3 (∼10-fold), while N14D-P27R-rSsp1a enhanced Na(V)1.2/1.7 selectivity over Na(V)1.3. This study highlights the challenge of developing subtype-selective spider toxin inhibitors across multiple Na(V) subtypes that might offer a more effective therapeutic approach. The findings of this study provide a basis for further rational design of Ssp1a and related NaSpTx1 homologs targeting Na(V)1.2, Na(V)1.3 and/or Na(V)1.7 as research tools and therapeutic leads.