BSB PhD Exit Seminar: Expression studies and pharmacology of neutral amino acid transporter B0AT1 (SLC6A19) - A potential target to treat metabolic diseases

Aditya Yadav, Broer Group, BSB, RSB

Background

B0AT1 (Slc6a19) is a sodium-dependent neutral amino acid transporter catalyzing the secondary active transport of neutral amino acids across the brush border membrane of the kidney and intestine. The surface expression of B0AT1 requires either Collectrin or angiotensin-converting enzyme 2 (ACE2) in the kidney and intestine, respectively. A Slc6a19 KO mouse showed neutral aminoaciduria in urine as observed in Hartnup disorder, a benign medical condition caused by a mutation in the Slc6a19 gene. Further characterization of these mice revealed that lack of B0AT1 improved glucose tolerance and enhanced fat metabolism. This suggests that pharmacological inhibition of B0AT1 using chemical compounds could lead to new drugs to treat type 2 diabetes (T2DM). More recently, the transporter has also been suggested as a target to treat phenylketonuria. To identify novel inhibitors of B0AT1, medicinal chemistry approaches and high throughput screening were used.

Results

Modified diarylmethine compounds inhibited B0AT1 with IC50 values ranging from 8–90 µM. The second generation of inhibitors was derived from high-throughput screening and showed higher affinity (IC50 of 1–15 µM) and strong selectivity against amino acid transporters with similar substrate specificities, such as ASCT2 (SLC1A5) and LAT1 (SLC7A5). All compounds were unrelated to B0AT1 substrates but were likely to bind in the vicinity of the substrate-binding site. The third generation of compounds of B0AT1 was generated by medicinal chemistry of selected lead compounds. These inhibited B0AT1 with higher affinity (IC50 of 0.7–2 µM).

To obtain a structural understanding of B0AT1 inhibition, I performed expression studies to purify the heterodimeric complex of ACE2 and B0AT1. I evaluated different techniques and expression systems such as Xenopus oocytes, CHO (Chinese Hamster Ovary) cells, and Drosophila S2 cells. A CHO-based cell line was generated, stably expressing B0AT1 and ACE2 using a Flip-In transfection system. Using this cell line and membrane solubilization with SMALPs (Styrene maleic Acid Lipid Nanoparticles), I was able to purify B0AT1 alone but was not able to purify the ACE2-B0AT1 complex.

To improve the understanding of the ACE2-B0AT1 interaction, I investigated the role of the TM7-extension of B0AT1, which contains critical residues for the complex formation.

Conclusions

In this study, I could identify five compounds that are highly potent and specific inhibitors of B0AT1, and the structure-activity relationships derived from using medicinal chemistry can help the further development of the compounds. The expression studies and investigation of the role of TM7 in B0AT1 improve our understanding of the B0AT1-ACE2 interaction.

 

Vanessa Howieson will present her talk before Aditya, please find more details here.