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_V1.7. 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_V1.7 to include the epilepsy target Na_V1.2 and the pain target Na_V1.3. 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_V1.3 (∼10-fold), while N14D-P27R-rSsp1a enhanced Na_V1.2/1.7 selectivity over Na_V1.3. 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_V1.2, Na_V1.3 and/or Na_V1.7 as research tools and therapeutic leads.