Structure-guided optimization of SLC1A1/EAAT3-selective inhibitors targeting renal cancer metabolism.

Renal cell carcinomas (RCCs) depend on the trimeric sodium-coupled aspartate and glutamate transporter, SLC1A1/EAAT3; however, pharmacologically targeting SLC1A1 is challenging. Here we determined a cryo-EM structure of human SLC1A1 bound to compound 3e, a recently described SLC1A1-selective bicyclic imidazo[1,2 α]pyridine-3-amine (BIA) inhibitor with an unclear mechanism of action. 3e binds a membrane-embedded allosteric pocket accessible only in the apo state, when SLC1A1 is unbound to substrate and sodium, and likely prevents sodium and substrate binding. Moreover, by forming a wedge between the trimerization domain and the substrate-binding transport domain, alongside a cholesterol moiety from the lipid bilayer, 3e blocks SLC1A1's elevator-like movements that support the transport cycle. Mutations in this binding pocket abolish the 3e interaction and counteract 3e's cytotoxicity in RCC cells, confirming on-target activity and explaining SLC1A1 selectivity. The subsequent design of two new SLC1A1-selective BIA derivatives, PBJ1 and PBJ2, was directed by the SLC1A1-3e structures; both inhibited SLC1A1-dependent aspartate, glutamate, and cysteine metabolism and showed enhanced cytotoxicity.