As a member of the GABAergic system, the GABA transporters (GATs) play a critical role in the regulation and termination of the GABA-mediated signaling as they function as key proteins in neurotransmitter uptake. 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) reported to date. In order to characterize the molecular basis of GABA-transporter subtype selectivity, a series of ATPCA analogues was synthesized and pharmacologically characterized in radioligand-based uptake assays at the four human GABA transporters (hGATs) recombinantly expressed in mammalian cells. Overall, the analogues retained subtype-selectivity for hBGT1, though with lower inhibitory activities compared to ATPCA. Further characterization of five BGT1-active analogues in a fluorescence-based FMP assay revealed that the compounds are substrates for hBGT1, suggesting that they interact with the orthosteric site of the transporter. <i>In silico</i>-guided mutagenesis experiments showed that the non-conserved residues Q299 and E52 in hBGT1 potentially contribute to the subtype-selectivity of ATPCA and its analogues. Computational docking studies and molecular dynamics simulations suggested that these residues form stable hydrogen bonds with the guanidine or amidine moieties of ATPCA and its derivatives. Overall, this study provides new insights into the molecular interactions governing the subtype-selectivity of BGT1 substrate-inhibitors.

Synthesis, pharmacological activity and molecular modeling studies of a series of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid analogues as betaine/GABA transporter 1 (BGT1) substrate-inhibitors

256th National Meeting (2018)

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