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.