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

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) (IC 50 2.5 µM) 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 IC 50 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.

Molecular Determinants and Pharmacological Analysis for a Class of Competitive Non-transported Bicyclic Inhibitors of the Betaine/GABA Transporter BGT1

“…The increase is especially remarkable for bicyclo-GABA approaching a potency of 40 nM. This finding is in line with previous findings, making this a consistent phenomenon among different compound classes ( Kickinger et al, 2020 ). Even though we do not understand the underlying nature of this phenomenon, we speculate that it could be related to the size of the residue (bulky glutamine/tyrosine versus small alanine).…”

“…Even though we do not understand the underlying nature of this phenomenon, we speculate that it could be related to the size of the residue (bulky glutamine/tyrosine versus small alanine). However, previous MD simulations of the E52A mutant bound to the GABA analog ATPCA did not reveal additional insights into this ( Kickinger et al, 2020 ). Most likely the used homology model does not provide the necessary accuracy to draw such conclusions.…”

“…This assay detects changes in membrane potential for an average cell population through changes in fluorescence of a membrane pre-equilibrated fluorescent blue dye. It therefore provides an indirect measure of activity with the advantage of being able to discern between GAT substrates and inhibitors ( Kragholm et al, 2013 ; Al-Khawaja et al, 2014 ; Kickinger et al, 2020 ).…”

“…Furthermore, we wanted to test the double mutant E52Y + Q299L. However, in a previous study this mutant was proven non-functional ( Kickinger et al, 2020 ). Instead, we generated the double mutant E52A + Q299L as well as the single mutant E52A.…”

“…This could be due to the fact that 2 can undergo one hydrogen bond less than bicyclo-GABA, which can also explain why 2 is less subtype selective and shows some GAT3 activity ( Table 2 ). In a previous study, we have already identified Q299 and E52 in BGT1 as part of the molecular determinants driving activity and selectivity of a series of cyclic 2-amino-tetrahydropyrimidine/pyridine compounds ( Kickinger et al, 2020 ). We therefore conclude that the interaction between the amino moiety of different BGT1 compound classes with Q299 and E52 is a general key feature for selective BGT1 inhibition ( Jørgensen et al, 2017 ; Kickinger et al, 2020 ; Lie et al, 2020 ).…”