JPH203 inhibitor arrests LAT1 in the inwards-open conformation by displacing relevant water molecules

L-type amino acid transporter 1 (LAT1) delivers amino acids and aa-mimicking drugs across blood–brain barrier and is a key underexplored target against cancer. We investigated molecular triggers of LAT1 conformational changes upon ligand binding performing molecular dynamics simulations on LAT1-substrates and inhibitor. We realized LAT1 conformational change occurs via a two-step expansion/contraction of the mid-section and water flow propels substrate translocation. Expansion allows water flow from the extracellular H6/H10-pocket facilitating ligand flipping, while contraction promotes ligand movement toward H8 and water flow toward the intracellular region. Large substrates (cpd1) leave H6/H10 sub-pocket, toward H10–H3 and flipping mid-section for water flow, allowing H10 rotation and intracellular passage. Furthermore, benzoxazole tail of JPH203, a clinically investigated LAT1 inhibitor, can rotate downward during expansion phase, arresting LAT1 in the inward-open conformation by displacing unfavorable water molecules. This finding extends JPH203’s original mechanism, showing that it can block LAT1 not only in outward-facing conformation but by stabilizing the inward-open state.