Identification of Novel Inhibitors of the Amino Acid Transporter B0AT1 (SLC6A19), a Potential Target to Induce Protein Restriction and to Treat Type 2 Diabetes

Abstract

Apical broad-spectrum neutral (0) amino acid transporter B0AT1 (encoded by the SLC6A19 gene) is mainly expressed in small intestine and kidney, mediating the transport of all neutral amino acids in a Na+-dependent manner. Its ancillary proteins, carboxypeptidase angiotensin-converting enzyme 2 (ACE2) and the homolog collectrin (TMEM27, transmembrane protein 27), are critical for the surface expression and catalytic transport function in small intestine and kidney, respectively. Slc6a19 nullizygous mice have improved insulin sensitivity and show reduced weight gain on a HFD (high fat diet) compared to wild-type mice. Moreover, reduced insulin secretion could protect β-cells from endoplasmic reticulum (ER) stress and long-term failure in insulin secretion. Hence, pharmacological inhibition of B0AT1 using chemical compounds could lead to new drugs to treat type 2 diabetes (T2DM) and its related metabolic disorders. A CHO (Chinese Hamster Ovary) - based cell line was generated, stably expressing B0AT1 and collectrin with the aid of a Flp-In™ transfection system. Using this cell line, a high-throughput screening (HTS) assay was developed, which uses a fluorescent dye to detect depolarisation of the cell membrane during amino acid uptake via B0AT1. A radio-labelled substrate uptake assay was used to determine the potency (IC50) and mechanism (competitive or non-competitive) of inhibition, as well as the specificity of B0AT1 inhibitors. Potential novel inhibitors were derived from three sources, first commercially available substrate analogues, second medium-scale virtual screening, and third high-throughput screening of chemical libraries. Selected inhibitors were further tested in in-vitro (Xenopus laevis oocytes), ex-vivo (mouse small intestine) and in-vivo (C57BL/6NCrl wild-type mice) systems. I characterised a series of novel inhibitors of the B0AT1 transporter in more detail. Benztropine (NSC63912) was identified as a competitive inhibitor showing an IC50 of 44±9µM; whereas 2-benzyl-1-(3-phenylpropyl)piperidine (NSC22789) was identified as a non-competitive inhibitor with an IC50 of 90±21µM. These two compounds were selective with regard to related transporters (system L, ASCT2), and they blocked substrate (leucine) uptake in both Xenopus oocytes overexpressing B0AT1 and collectrin, and inverted mouse small intestine. Two more potent B0AT1 inhibitors with IC50 below 10µM were further identified from the HTS of a small molecule compound library. 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 transporters or be developed further through medicinal chemistry. Show more