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.