Structure-guided development of a potent human B0AT1 inhibitor effective in a mouse model of phenylketonuria.

B0AT1 (SLC6A19) is a neutral amino acid transporter mediating intestinal absorption and renal reuptake of amino acids, including phenylalanine (Phe). Inhibiting B0AT1 enhances Phe excretion, offering a therapeutic strategy for phenylketonuria (PKU). Using cryo-EM, we determined human B0AT1 structures in outward- and inward-open states, revealing an allosteric pocket ~17 Å from the substrate site that is present in the outward-open conformation and has been previously reported. Structure-guided inhibitor design targeting this pocket produced a potent B0AT1 inhibitor that locks the transporter in an outward-occluded state and blocks transport. The higher-resolution structures reveal detailed interactions at the binding site, including water-mediated coordination and conformational changes around Leu52. This inhibitor exhibited submicromolar IC50 against both human and mouse B0AT1, and oral administration in PKU model mice increased urinary Phe and reduced plasma Phe levels. These findings provide structural insight into allosteric inhibition of B0AT1 and establish a framework for the rational optimization of inhibitors targeting conformationally dynamic allosteric sites in SLC6-family transporters.