Dataset Information

Identification of novel inhibitors of the amino acid transporter B⁰ AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes.

ABSTRACT:

Background and purpose

The neutral amino acid transporter B⁰ AT1 (SLC6A19) has recently been identified as a possible target to treat type 2 diabetes and related disorders. B⁰ AT1 mediates the Na⁺ -dependent uptake of all neutral amino acids. For surface expression and catalytic activity, B⁰ AT1 requires coexpression of collectrin (TMEM27). In this study, we established tools to identify and evaluate novel inhibitors of B⁰ AT1.

Experimental approach

A CHO-based cell line was generated, stably expressing collectrin and B⁰ 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 B⁰ AT1. In parallel to these functional assays, we ran a computational compound screen using AutoDock4 and a homology model of B⁰ AT1 based on the high-resolution structure of the highly homologous Drosophila dopamine transporter.

Key results

We characterized a series of novel inhibitors of the B⁰ AT1 transporter. Benztropine was identified as a competitive inhibitor of the transporter showing an IC₅₀ 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 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.

SUBMITTER: Cheng Q

PROVIDER: S-EPMC5323516 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Publications

Identification of novel inhibitors of the amino acid transporter B<sup>0</sup> AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes.

Cheng Qi Q Shah Nishank N Bröer Angelika A Fairweather Stephen S Jiang Yang Y Schmoll Dieter D Corry Ben B Bröer Stefan S

British journal of pharmacology 20170214 6

<h4>Background and purpose</h4>The neutral amino acid transporter B<sup>0</sup> AT1 (SLC6A19) has recently been identified as a possible target to treat type 2 diabetes and related disorders. B<sup>0</sup> AT1 mediates the Na<sup>+</sup> -dependent uptake of all neutral amino acids. For surface expression and catalytic activity, B<sup>0</sup> AT1 requires coexpression of collectrin (TMEM27). In this study, we established tools to identify and evaluate novel inhibitors of B<sup>0</sup> AT1.<h4>Ex ...[more]

PMID: 28176326

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Project description:Recent studies have established that dietary protein restriction improves metabolic health and glucose homeostasis. SLC6A19 (B⁰AT1) is the major neutral amino acid transporter in the intestine and carries out the bulk of amino acid absorption from the diet. Mice lacking SLC6A19 show signs of protein restriction, have improved glucose tolerance, and are protected from diet-induced obesity. Pharmacological blockage of this transporter could be used to induce protein restriction and to treat metabolic diseases such as type 2 diabetes. A few novel inhibitors of SLC6A19 have recently been identified using in vitro compound screening, but it remains unclear whether these compounds block the transporter in vivo. To evaluate the efficacy of SLC6A19 inhibitors biomarkers are required that can reliably detect successful inhibition of the transporter in mice. A gas chromatography mass spectrometry (GC-MS)-based untargeted metabolomics approach was used to discriminate global metabolite profiles in plasma, urine and faecal samples from SLC6A19ko and wt mice. Due to inefficient absorption in the intestine and lack of reabsorption in the kidney, significantly elevated amino acids levels were observed in urine and faecal samples. By contrast, a few neutral amino acids were reduced in the plasma of male SLC6A19ko mice as compared to other biological samples. Metabolites of bacterial protein fermentation such as p-cresol glucuronide and 3-indole-propionic acid were more abundant in SLC6A19ko mice, indicating protein malabsorption of dietary amino acids. Consistently, plasma appearance rates of [14C]-labelled neutral amino acids were delayed in SLC6A19ko mice as compared to wt after intra-gastric administration of a mixture of amino acids. Receiver operating characteristic (ROC) curve analysis was used to validate the potential use of these metabolites as biomarkers. These findings provide putative metabolite biomarkers that can be used to detect protein malabsorption and the inhibition of this transporter in intestine and kidney.

Project description:B0AT1 (SLC6A19) is a major sodium-coupled neutral amino acid transporter that relies on angiotensin converting enzyme 2 (ACE2) or collectrin for membrane trafficking. Despite its significant role in disorders associated with amino acid metabolism, there is a deficit of comprehensive structure-function understanding of B0AT1 in lipid environment. Herein, we have employed molecular dynamics (MD) simulations to explore the architectural characteristics of B0AT1 in two distinct environments: a simplified POPC bilayer and a complex lipid system replicating the native membrane composition. Notably, our B0AT1 analysis in terms of structural stability and regions of maximum flexibility shows consistency in both the systems with enhanced structural features in the case of complex lipid system. Our findings suggest that diacylglycerol phospholipids significantly alter the pore radius, hydrophobic index, and surface charge distribution of B0AT1, thereby affecting the flexibility of transmembrane helices TM7, TM12, and loop connecting TM7-TM8, crucial for ACE2-B0AT1 interaction. Pro41, Ser190, Arg214, Arg240, Ser413, Pro414, Cys463, and Val582 are among the most prominent lipid binding residues that might influence B0AT1 functionality. We also perceive notable lipid mediated deviation in the degree of tilt and loss of helicity in TM1 and TM6 which might affect the substrate binding sites S1 and S2 in B0AT1. Considerably, destabilization in the structure of B0AT1 in lipid environment was evident upon mutation in TM domain, associated with Hartnup disorder through various structure-based protein stability tools. Our two-tiered approach allowed us to validate the use of POPC as a baseline for initial analyses of SLC transporters. Altogether, our all-atoms MD study provides a platform for future investigations into the structure-function mechanism of B0AT1 in realistic lipid mimetic bilayers and offers a framework for developing new therapeutic agents targeting this transporter.

Dataset Information

Identification of novel inhibitors of the amino acid transporter B⁰ AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes.

Background and purpose

Experimental approach

Key results

Conclusion and implications

Publications

Identification of novel inhibitors of the amino acid transporter B<sup>0</sup> AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Identification of novel inhibitors of the amino acid transporter B0 AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes.

Background and purpose

Experimental approach

Key results

Conclusion and implications

Publications

Identification of novel inhibitors of the amino acid transporter B<sup>0</sup> AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Identification of novel inhibitors of the amino acid transporter B⁰ AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes.