Xu, JunyangHu, ZiweiDai, LuYadav, AdityaJiang, YashanBröer, AngelikaGardiner, Michael G.McLeod, MalcolmYan, RenhongBröer, Stefan2025-05-302025-05-302041-1723PubMed:39174516http://www.scopus.com/inward/record.url?scp=85201821802&partnerID=8YFLogxKhttps://hdl.handle.net/1885/733755519The epithelial neutral amino acid transporter B0AT1 (SLC6A19) is the major transporter for the absorption of neutral amino acids in the intestine and their reabsorption in the kidney. Mouse models have demonstrated that lack of B0AT1 can normalize elevated plasma amino acids in rare disorders of amino acid metabolism such as phenylketonuria and urea-cycle disorders, implying a pharmacological approach for their treatment. Here we employ a medicinal chemistry approach to generate B0AT1 inhibitors with IC50-values of 31-90 nM. High-resolution cryo-EM structures of B0AT1 in the presence of two compounds from this series identified an allosteric binding site in the vestibule of the transporter. Mechanistically, binding of these inhibitors prevents a movement of TM1 and TM6 that is required for the transporter to make a conformational change from an outward open state to the occluded state.The authors thank J\u0119drzej Kuku\u0142owicz for helping with modelling inhibitor binding to B0AT1. We also thank the Cryo-EM Facility of Southern University of Science and Technology (SUSTech) for providing the facility support. We thank Shuman Xu and Lei Zhang at the Cryo-EM Center of SUSTech for technical support in electron microscopy data acquisition. This work was funded by the Shenzhen Medical Research Fund (A2303046 to R.Y.) and by NHMRC grant GNT2020757 to S.B.enPublisher Copyright: © The Author(s) 2024.2024-08-2210.1038/s41467-024-51748-185201821802