Identification and characterization of potent and selective inhibitors for the B0AT2/SLC6A15 amino acid transporter

Abstract

1.The gene SLC6A15 encodes the protein B0AT2, a transporter for neutral amino acids. It is highly expressed in the brain and has been associated with depression but little is otherwise know about its function. In this study, we identified the first inhibitors of this protein to pharmacologically investigate its function. The transporter activity was evaluated using a cellular uptake of the substrate 3H-proline. A miniaturized assay was developed and used for a High Throughput Screening (HTS) of 200,000 compounds. Hits were tested for cell toxicity and selectivity versus related transporters of the SLC6 family. The most promising inhibitors were validated by proline uptake and neurite outgrowth in primary hippocampal neurons. Of the 10 chemical scaffolds identified, a 1,5-benzodiazepine series had the most promising selectivity and structure-activity relationship (SAR) profile. The best compounds showed drug-like properties and inhibited B0AT2 with an IC50 of 250 nM both in SLC6A15-overexpressing HEK293 cells and primary neurons, with no detectable inhibition (> 80{micro}M) of SERT, DAT, GAT1, or NTT4/SLC6A17. These compounds also dose-dependently stimulated neurite outgrowth in primary neurons. The identified compounds are the first inhibitors of the amino acids transporter B0AT2/SLC6A15. Their potency, selectivity and physicochemical properties allow to target the transporter in relevant biological systems and to initiate new studies to understand its role and implication in diseases.