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 imidazo1,2 αpyridine-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. SLC1A1 (also known as EAAT3) is a transporter that controls aspartate and glutamate uptake in neuronal synapses and kidney proximal tubules and, when dysregulated, causes kidney cancer growth. This study uses structural data to reveal the mode of action of a recently described SLC1A1 inhibitor, 3e, and rationally design more potent 3e analogs that block critical renal cancer metabolism programs. Cryo-EM structures enable the rational design of novel SLC1A1-targeting cancer compounds with increased cytotoxicity.