Abstract 6911: Investigating pharmacological targeting of the SLC1A1 aspartate/glutamate transporter

Abstract Background: SLC1A1/EAAT3 functions as a trimeric, Na+-dependent dicarboxylic amino acids (glutamate and aspartate) transporter. Recent studies have identified SLC1A1/EAAT3 as an oncogenic dependency in several cancers, including clear cell renal cell carcinoma (ccRCC), blood-borne tumors, and lung cancer. Unfortunately, despite this transporter’s potential as a therapeutic target, pharmacological inhibition of SLC1A1 has remained elusive due to the absence of potent and selective inhibitors. In earlier studies, we investigated a previously described bicyclic imidazopyridine aniline SLC1A1 blocker (which we synthesized in-house, Compd 3e). Compd 3e bound SLC1A1 directly and promoted its thermal stability. Furthermore, Compd 3e treatment replicated the effects of genetic SLC1A1 blockade in terms of both metabolic changes and cytotoxicity of ccRCCs. Importantly, these fitness defects were rescued by supplementation of cell-permeable Asp and Glu. Results: Here, we resolved the Cryo-EM structure of Compd 3e-bound SLC1A1. These studies showed that Compd 3e operates through a dual inhibitory mechanism. First, Compd 3e occupies a previously uncharacterized 'hidden' cholesterol-enriched pocket within the transporter, competitively disrupting Na+ binding that is critical for SLC1A1’s transport cycle. Second, Compd 3e wedges itself between the trimeric domains, thereby non-competitively arresting the 'elevator' movements necessary for substrate translocation across the membrane. Compd 3e makes several direct contacts with SLC1A1, including with the F99 residue that is not conserved in the other SLC1A family proteins. Mutating this residue into the SLC1A2/1A3-like (F99M) or the SLC1A5-like (F99L) conferred resistance to Compd 3e. These studies explain the mechanistic basis of Compd 3e’s selectivity towards SLC1A1. Compd 3e has poor pharmacological properties (solubility, bioavailability, etc.). In silico molecular docking enabled a structure-activity relationship (SAR) campaign to optimize the Compd 3e scaffold. Our biochemical, structural, and functional studies have identified improved SLC1A1 inhibitors. Conclusion: Our study provides an understanding of the dual mechanisms by which Compd 3e inhibits SLC1A1. These mechanistic discoveries uncover a novel therapeutic avenue for targeting ccRCC (and other SLC1A1-dependent cancers). More broadly, this work highlights the allosteric targeting of solute carriers outside their substrate-binding pocket, which can be leveraged for several other oncogenic SLCs. Citation Format: Pooneh Koochaki, Jesse A. Coker, Biao Qiu, Nancy Wang, Todd Romigh, Shaun R. Stauffer, Olga Boudker, Abhishek A. Chakraborty. Investigating pharmacological targeting of the SLC1A1 aspartate/glutamate transporter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6911.