Expression studies and pharmacology of neutral amino acid transporter B0AT1 (SLC6A19) - A potential target to treat metabolic diseases
Creators: name: Yadav, Aditya
Publisher: name: The Australian National University
Year: 2023

description: 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. Further characterization of these mice revealed that lack of B0AT1 improved glucose tolerance and enhanced fat metabolism, identifying it as a target to treat type 2 diabetes. More recently, the transporter has also been suggested as a target to treat phenylketonuria. This suggests that pharmacological inhibition of B0AT1 using chemical compounds could lead to new drugs to treat metabolic diseases. To identify novel inhibitors of B0AT1, medicinal chemistry approaches and high throughput screening were used. Results: In this thesis, I evaluated inhibitors arising from high-throughput screening and medicinal chemistry of lead compounds. In addition, I developed novel tools for the pharmacological characterization of B0AT1 inhibitors. Pharmacology: Medicinal chemistry of first-generation lead compounds derived from computational screening yielded diarylmethine compounds, which inhibited B0AT1 with IC50 values ranging from 8-90 uM. A second generation of inhibitors was derived from high-throughput screening and showed higher affinity (IC50 of 1-15 uM) 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 based on competition experiments were likely to bind in the vicinity of the substrate-binding site. A third generation of compounds of B0AT1 was generated by medicinal chemistry of selected lead compounds. These inhibited B0AT1 with higher affinity (IC50 of 0.09-2 uM). Tool development: 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 develop a simplified expression system of B0AT1, which does not require ACE2, I investigated the role of hydrogen bonds for the formation of the B0AT1/ACE2 complex. Conclusions: In this study, I could identify thirteen 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.