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

BACKGROUND AND PURPOSEThe neutral amino acid transporter B 0 AT1 (SLC6A19) has recently been identified as a possible target to treat type 2 diabetes and related disorders. B 0 AT1 mediates the Na + -dependent uptake of all neutral amino acids. For surface expression and catalytic activity, B 0 AT1 requires coexpression of collectrin (TMEM27). In this study, we established tools to identify and evaluate novel inhibitors of B 0 AT1. EXPERIMENTAL APPROACHA CHO-based cell line was generated, stably expressing collectrin and B 0 AT1. 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 B 0 AT1. In parallel to these functional assays, we ran a computational compound screen using AutoDock4 and a homology model of B 0 AT1 based on the high-resolution structure of the highly homologous Drosophila dopamine transporter. KEY RESULTSWe characterized a series of novel inhibitors of the B 0 AT1 transporter. Benztropine was identified as a competitive inhibitor of the transporter showing an IC 50 of 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 IMPLICATIONSThe 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. AbbreviationsCHO-BC, CHO cells stably transfected with B 0 AT1 and collectrin; FGF21, fibroblast growth factor 21; GIP, gastric inhibitory peptide; GLP-1, glucagon-like peptide 1; MW, molecular weight; NMDG, N-methyl-D-glucamine

Dietary methionine restriction modulates renal response and attenuates kidney injury in mice

“…Alternatively, low urine SAAs could be due to increased activity by the renal amino acid transporter Slc6a19 , which could promote reabsorption. Of interest, Slc6a19 was shown to have high affinity toward methionine (32, 33). Conversely, hyperhomocysteinemia could be a physiologic response to the effects of MR on the trans‐sulfuration pathway (12, 25) that is unrelated to kidney function.…”

Novel Chemical Scaffolds to Inhibit the Neutral Amino Acid Transporter B0AT1 (SLC6A19), a Potential Target to Treat Metabolic Diseases

“…Nimesulide (IC 50 23 mM) and related compounds were identified as inhibitors of B 0 AT1 using native transporter purified from rat b r u s h -b o r d e r m e m b r a n e s a n d r e c o n s t i t u t e d i n t o proteoliposomes (Pochini et al, 2014). In order to develop inhibitors of the human B 0 AT1, recombinant expression was optimized in CHO cells (Cheng et al, 2017). Recombinant expression of B 0 AT1 requires co-expression with its accessory proteins collectrin (Danilczyk et al, 2006;Malakauskas et al, 2007) or angiotensin-converting enzyme 2 (ACE2) (Kowalczuk et al, 2008).…”

“…These proteins are required for surface expression in kidney (collectrin) and intestine (ACE2), but are also required for catalytical activity (Fairweather et al, 2015). Notably, functional expression of B 0 AT1 in mammalian cells requires high expression levels of collectrin (Danilczyk et al, 2006;Cheng et al, 2017), while only minute amounts are required in Xenopus laevis oocytes (Fairweather et al, 2015). The functional protein mediates the symport of 1Na + together with any neutral amino acid, resulting in inward currents.…”

“…This knowledge was used to generate a membrane potential assay with a fluorescence readout (FLIPR), which can be applied to high throughput screens (Cheng et al, 2017;Danthi et al, 2019). High-resolution structures of B 0 AT1 homologues, such as LeuT (Yamashita et al, 2005), the Drosophila dopamine transporter (Penmatsa et al, 2013), and the human serotonin transporter (Coleman et al, 2016) have also allowed to generate homology models useful for in silico screening (Cheng et al, 2017). HTS and in silico screening have resulted in additional inhibitors, such as benztropine (IC 50 44 mM) (Cheng et al, 2017) and cinromide (IC 50 0.3 mM).…”

“…High-resolution structures of B 0 AT1 homologues, such as LeuT (Yamashita et al, 2005), the Drosophila dopamine transporter (Penmatsa et al, 2013), and the human serotonin transporter (Coleman et al, 2016) have also allowed to generate homology models useful for in silico screening (Cheng et al, 2017). HTS and in silico screening have resulted in additional inhibitors, such as benztropine (IC 50 44 mM) (Cheng et al, 2017) and cinromide (IC 50 0.3 mM). Two of the established B 0 AT1 inhibitors were initially developed for other targets, such as COX-2 (Nimesulide) and muscarinic acetylcholine receptor (Benztropine).…”