Exploring the molecular determinants for subtype-selectivity of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid analogs as betaine/GABA transporter 1 (BGT1) substrate-inhibitors

Author(s)
Stefanie Kickinger, Anas Al-Khawaja, Anne Staehr Haugaard, Maria E. K. Lie, Francesco Bavo, Rebekka Loeffler, Maria Damgaard, Gerhard F. Ecker, Bente Frolund, Petrine Wellendorph
Abstract

We have previously identified 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA) as the most potent substrate-inhibitor of the betaine/GABA transporter 1 (BGT1) (IC50 2.5 mu M) reported to date. Herein, we characterize the binding mode of 20 novel analogs and propose the molecular determinants driving BGT1-selectivity. A series of N-1-, exocyclic-N-, and C-4-substituted analogs was synthesized and pharmacologically characterized in radioligand-based uptake assays at the four human GABA transporters (hGATs) recombinantly expressed in mammalian cells. Overall, the analogs retained subtype-selectivity for hBGT1, though with lower inhibitory activities (mid to high micromolar IC50 values) compared to ATPCA. Further characterization of five of these BGT1-active analogs in a fluorescence-based FMP assay revealed that the compounds are substrates for hBGT1, suggesting they interact with the orthosteric site of the transporter. In silico-guided mutagenesis experiments showed that the non-conserved residues Q299 and E52 in hBGT1 as well as the conformational flexibility of the compounds potentially contribute to the subtype-selectivity of ATPCA and its analogs. Overall, this study provides new insights into the molecular interactions governing the subtype-selectivity of BGT1 substrate-inhibitors. The findings may guide the rational design of BGT1-selective pharmacological tool compounds for future drug discovery.

Organisation(s)
External organisation(s)
University of Copenhagen
Journal
Scientific Reports
Volume
10
No. of pages
15
ISSN
2045-2322
DOI
https://doi.org/10.1038/s41598-020-69908-w
Publication date
08-2020
Peer reviewed
Yes
Austrian Fields of Science 2012
301207 Pharmaceutical chemistry
Keywords
Portal url
https://ucris.univie.ac.at/portal/en/publications/exploring-the-molecular-determinants-for-subtypeselectivity-of-2amino1456tetrahydropyrimidine5carboxylic-acid-analogs-as-betainegaba-transporter-1-bgt1-substrateinhibitors(cb73ce37-7d25-44f5-b465-d074e114b463).html