Identification of novel inhibitors of the amino acid transporter B⁰AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes

Background and Purpose: The neutral amino acid transporter B⁰AT1 (SLC6A19) has recently been identified as a possible target to treat type 2 diabetes and related disorders. B⁰AT1 mediates the Na⁺-dependent uptake of all neutral amino acids. For surface expression and catalytic activity, B⁰AT1 requires coexpression of collectrin (TMEM27). In this study, we established tools to identify and evaluate novel inhibitors of B⁰AT1. Experimental Approach: A CHO-based cell line was generated, stably expressing collectrin and B⁰AT1. Using this cell line, a high-throughput screening assay was developed, which uses a fluorescent dye to detect depolarisation of the cell membrane during amino acid uptake via B⁰AT1. In parallel to these functional assays, we ran a computational compound screen using AutoDock4 and a homology model of B⁰AT1 based on the high-resolution structure of the highly homologous Drosophila dopamine transporter. Key Results: We characterized a series of novel inhibitors of the B⁰AT1 transporter. Benztropine was identified as a competitive inhibitor of the transporter showing an IC₅₀of 44 ± 9 μM. The compound was selective with regard to related transporters and blocked neutral amino acid uptake in inverted sections of mouse intestine. Conclusion And Implications: The tools established in this study can be widely used to identify new transport inhibitors. Using these tools, we were able to identify compounds that can be used to study epithelial transport, to induce protein restriction, or be developed further through medicinal chemistry.