Cryo-EM reveals an unprecedented binding site for Na1.7 inhibitors enabling rational design of potent hybrid inhibitors.

The voltage-gated sodium (Na) channel Na1.7 has been identified as a potential novel analgesic target due to its involvement in human pain syndromes. However, clinically available Na channel-blocking drugs are not selective among the nine Na channel subtypes, Na1.1-Na1.9. Moreover, the two currently known classes of Na1.7 subtype-selective inhibitors (aryl- and acylsulfonamides) have undesirable characteristics that may limit their development. To this point understanding of the structure-activity relationships of the acylsulfonamide class of Na1.7 inhibitors, exemplified by the clinical development candidate , has been based solely on a single co-crystal structure of an arylsulfonamide inhibitor bound to voltage-sensing domain 4 (VSD4). To advance inhibitor design targeting the Na1.7 channel, we pursued high-resolution ligand-bound Na1.7-VSD4 structures using cryogenic electron microscopy (cryo-EM). Here, we report that engages the Na1.7-VSD4 through an unexpected binding mode orthogonal to the arylsulfonamide inhibitor class binding pose, which identifies a previously unknown ligand binding site in Na channels. This finding enabled the design of a novel hybrid inhibitor series that bridges the aryl- and acylsulfonamide binding pockets and allows for the generation of molecules with substantially differentiated structures and properties. Overall, our study highlights the power of cryo-EM methods to pursue challenging drug targets using iterative and high-resolution structure-guided inhibitor design. This work also underscores an important role of the membrane bilayer in the optimization of selective Na channel modulators targeting VSD4.