Abstract A029: Structure-guided design of SLC1A1/EAAT3-selective inhibitors reveals a metabolic vulnerability in kidney cancer

Abstract Background: SLC1A1/EAAT3 is a trimeric Na+-coupled transporter that imports aspartate, glutamate, and, to a lesser extent, cysteine. ccRCC and other cancers, such as hematologic malignancies and lung cancer, are metabolically dependent on these substrates for nucleotide synthesis, TCA cycle anaplerosis, and glutathione production, which drives an oncogenic dependence on SLC1A1. However, the lack of potent, subtype-selective SLC1A1 inhibitors has limited therapeutic targeting. Methods: We determined high-resolution cryo-EM structures of human SLC1A1 bound to the bicyclic imidazo[1,2-a]pyridine-3-amine inhibitor, 3e, and used these data to guide medicinal chemistry. New analogs were prioritized using in silico docking, thermal-shift binding assays, and SLC1A1-dependent cytotoxicity screens in RCC cell lines. On-target activity was tested using SLC1A1 drug-resistant mutants, impedance-based transport assays in inducible SLC1A1/2/3 HEK293 cells, and metabolic rescue with cell-permeable aspartate, glutamate, and glutathione. Results: The 2.5–2.8 Å cryo-EM maps show that 3e occupies a lipid-embedded allosteric cavity between the scaffold and transport domains, accessible only in an inward-facing apo state. Compound 3e, together with a bound sterol, wedges these domains apart, disfavors Na+ binding, and arrests the elevator-like translocation of SLC1A1. The non-conserved residue, F99, and the conserved T402 residue are critical for inhibitor engagement, and SLC1A1 F99M and T402I mutants remain functional but are resistant to 3e and its analogs in RCC cells, confirming on-target inhibition. Structure-activity studies yielded two optimized 3e analogs, PBJ1 and PBJ2, which retain SLC1A1 selectivity over SLC1A2/3, display improved cancer cell killing in multiple RCC lines, and deplete glutathione and TCA intermediates. Supplementation with methyl-aspartate, methyl-glutamate, or glutathione restores cell fitness, linking PBJ1/PBJ2 cytotoxicity to blockade of SLC1A1-mediated amino acid uptake. Conclusions: Our work defines a previously unrecognized, apo-state allosteric pocket in SLC1A1, establishes F99 as one structural determinant of subtype selectivity, and delivers PBJ1 and PBJ2 as next-generation, on-target probes that exploit an aspartate–glutamate dependency in kidney cancer. These findings provide a mechanistic foundation and chemical tools for developing SLC1A1-directed therapies in RCC. Citation Format: Pooneh Koochaki, Biao Qiu, Jesse A. Coker, Alexander Earsley, Nancy S. Wang, Todd Romigh, Christopher M. Goins, Shaun R. Stauffer, Olga Boudker, Abhishek A. Chakraborty. Structure-guided design of SLC1A1/EAAT3-selective inhibitors reveals a metabolic vulnerability in kidney cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_2):Abstract nr A029.