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-inhibiting spider toxins have focused on developing selective inhibitors for peripheral pain-sensing Na1.7. With several Na subtypes emerging as potential therapeutic targets, structure-function analysis of Na-inhibiting spider toxins at such subtypes is warranted. Using the recently discovered spider toxin Ssp1a, this study extends the structure-function relationships of Na-inhibiting spider toxins beyond Na1.7 to include the epilepsy target Na1.2 and the pain target Na1.3. 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 subtypes, especially at Na1.3 (∼10-fold), while N14D-P27R-rSsp1a enhanced Na1.2/1.7 selectivity over Na1.3. This study highlights the challenge of developing subtype-selective spider toxin inhibitors across multiple Na 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 Na1.2, Na1.3 and/or Na1.7 as research tools and therapeutic leads.