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.
Original language | English |
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Article number | 12992 |
Journal | Scientific Reports |
Volume | 10 |
Issue number | 1 |
Number of pages | 15 |
ISSN | 2045-2322 |
DOIs | |
Publication status | Published - 2020 |
Keywords
- GABA TRANSPORTER
- PHARMACOLOGICAL CHARACTERIZATION
- CEREBRAL-CORTEX
- HIGH-AFFINITY
- ANTICONVULSANT ACTION
- BINDING-SITE
- LOCALIZATION
- TARGETS
- IDENTIFICATION
- DIVERSITY