Metabolomics analysis of SNAT2-deficient cells: Implications for the discovery of selective small-molecule inhibitors of an amino acid transporter

Amino acid uptake by the solute carrier family of transporter proteins is critical to support cell metabolism, and inhibition of transporter activity represents a tractable strategy to restrict nutrient availability to cancer cells. A small-molecule inhibitor of the sodium-coupled neutral amino acid transporter 2 (SNAT2), 3-(N-methyl(4-methylphenyl)sulfonamido)-N-(2-trifluoromethylbenzyl)thiophene-2-carboxamide (MMTC/57E), was recently identified and shown to inhibit cell proliferation when combined with glucose transport inhibitors in breast and pancreatic cancer cell lines. In this study, we use mass spectrometry and a model competitive substrate inhibitor, α-(methylamino)-isobutyric acid (MeAIB), to establish cell-based SNAT2 activity assays and validate target engagement of MMTC/57E. We show that cellular uptake of MeAIB is dependent on SNAT2 or the closely related SNAT1 and is inhibited by the endogenous substrate l-alanine in a dose-dependent manner. We show that SNAT2-KO cells or cells treated with MeAIB exhibit a similar metabolomic signature associated with defects in amino acid availability and other metabolites. Applying these assays, we fail to observe that MMTC/57E inhibits SNAT2 activity. MMTC/57E exhibits poor aqueous solubility that hinders its use as a tool SNAT2 inhibitor. Our results highlight the challenges associated with identifying and validating transporter inhibitors and report robust assays that may be used to identify and evaluate SNAT2 inhibitors in the future.