Pharmacodynamic analyses of LAT1 inhibitors in vitro and in vivo by targeted metabolomics reveal target-independent effects.

L-type amino acid transporters LAT1 (SLC7A5) and LAT2 (SLC7A8) facilitate the bidirectional transport of branched and aromatic amino acids (AAs) across the plasma membrane. LAT1 has emerged as a key therapeutic target in cancer due to its upregulation in different tumor types. We generated and characterized LAT1- and LAT2-expressing cells using the human MDST8 cell line lacking these transporters to evaluate the specificity and selectivity of the clinical candidate JPH203 and novel LAT1 inhibitors. Both LAT1 and LAT2 increased the expression of 4F2hc, a heavy chain protein essential for LAT1 functioning and AA transport. We show for the first time that two potent nanomolar LAT1 inhibitors, JPH203 and JX-078, can enter cells through LAT1-independent mechanisms and inhibit LAT1-mediated L-leucine efflux, giving rise to differential cell type and time-dependent effects on the AA metabolome and metabolic activity in cancer cells and human PBMCs. Intriguingly, the antiproliferative effects of JPH203 and JX-078 on MDST8 cells were LAT1-independent, showing also micromolar IC50 values in HT-29 and U937 cells that overexpress LAT1. We found notable differences in the bioavailability of LAT1 inhibitors in mice. Oral administration of JX-078 efficiently penetrated tissues and crossed the blood-brain barrier, leading to increased levels of inhibitory neurotransmitters glycine and GABA in the brain. This study demonstrates the utility of employing targeted metabolomics to interrogate LAT1/2 inhibitor selectivity in different physiological matrices in vitro, ex vivo and in vivo. Overall, our findings reveal LAT1-dependent and previously unrecognized LAT-independent effects of inhibitors believed to act specifically on LAT1.