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

B0AT1 is a neutral amino acid transporter responsible for the (re)absorption in the intestine and kidney. Here, we developed small-molecule inhibitors of B0AT1 as a therapeutic strategy to a transient pocket, located [~]17 [A] away from the substrate-binding site and unique to the outward-open conformation, and stabilize an outward-occluded conformation that prevents the conformational transitions required for transport. Guided by structural insights, we optimized an initial inhibitor (Cinromide) to improve potency and cross-species activity, yielding compound 3, which inhibits both human and mouse B0AT1 with submicromolar IC_50 values. In a PKU mouse model (Pahenu2), oral administration of compound 3 increased urinary Phe excretion and significantly reduced plasma Phe. Our findings identify a druggable allosteric site in B0AT1, demonstrate its utility for achieving potent and selective inhibition in vivo, and establish allosteric blockade as a therapeutic approach for PKU and other SLC6-family transporters.