De novo protein design targeting voltage-gated sodium channel 1.7 using RFdiffusion and AlphaFold2

Voltage-gated sodium (NaV) channels—pore-forming transmembrane proteins regulating sodium flux across cell membranes—have been genetically and pre-clinically identified as a nociceptive drug target with limited addiction potential. In human sensory neurons, NaV subtypes hNaV1.7, hNaV1.8, and hNaV1.9 have been genetically and pre-clinically identified as promising pain targets. Small molecules typically bind non-selectively to other NaV subtypes and ion channel families, potentially leading to cardiac arrest, paralysis, and sedation. Natural peptide toxins targeting NaV channels have limited contacts with subtype-specific residues, resulting in limited selectivity. We have previously redesigned protoxin-II to improve selectivity, but there are structural limitations with peptide toxin topologies to take full advantage of all possible hNaV1.7-selective residues. Thus, we aim to apply Rosetta protein design methods to the design of de novo protein topologies that could prompt greater hNaV1.7 selectivity while prioritizing known peptide toxin - channel interactions to maintain potency. We describe our design process and preliminary results using RFDiffusion and AlphaFold2 as the primary methodology for targeting hNaV1.7 voltage-sensing domain II. De novo designed proteins targeting hNaV1.7 channels will be useful as molecular probes to study Nav channel activity and as therapeutics to treat pain.