Mutant cycle analysis with modified saxitoxins reveals specific interactions critical to attaining high-affinity inhibition of hNa V 1.7

Chronic pain plagues at least 50 million Americans and has an estimated annual cost in excess of $200 billion. Consequently, there exists significant interest in developing effective, nonnarcotic analgesics. Drugs that target individual voltage-gated sodium channel (Na V ) subtypes have appreciable therapeutic potential as pain medicines. Our work uses small-molecule design and protein mutagenesis to gain insight into the molecular architecture of the ion conduction pore of Na V . These studies have revealed structural differences in the outer mouth of the channel that potentiate the binding of guanidinium toxins. Such findings are helping advance the preparation of isoform-selective Na V antagonists. Improper function of voltage-gated sodium channels (Na V s), obligatory membrane proteins for bioelectrical signaling, has been linked to a number of human pathologies. Small-molecule agents that target Na V s hold considerable promise for treatment of chronic disease. Absent a comprehensive understanding of channel structure, the challenge of designing selective agents to modulate the activity of Na V subtypes is formidable. We have endeavored to gain insight into the 3D architecture of the outer vestibule of Na V through a systematic structure–activity relationship (SAR) study involving the bis-guanidinium toxin saxitoxin (STX), modified saxitoxins, and protein mutagenesis. Mutant cycle analysis has led to the identification of an acetylated variant of STX with unprecedented, low-nanomolar affinity for human Na V 1.7 (hNa V 1.7), a channel subtype that has been implicated in pain perception. A revised toxin-receptor binding model is presented, which is consistent with the large body of SAR data that we have obtained. This new model is expected to facilitate subsequent efforts to design isoform-selective Na V inhibitors.