Structure–Activity Relationship, Pharmacological Characterization, and Molecular Modeling of Noncompetitive Inhibitors of the Betaine/γ-Aminobutyric Acid Transporter 1 (BGT1)

Abstract

N-(1-Benzyl-4-piperidinyl)-2,4-dichlorobenzamide 5 (BPDBA) is a noncompetitive inhibitor of the betaine/GABA transporter 1 (BGT1). We here report the synthesis and structure-activity relationship of 71 analogues. We identify 26m as a more soluble 2,4-Cl substituted 3-pyridine analogue with retained BGT1 activity and an improved off-target profile compared to 5. We performed radioligand-based uptake studies at chimeric constructs between BGT1 and GAT3, experiments with site-directed mutated transporters, and computational docking in a BGT1 homology model based on the newly determined X-ray crystal structure of the human serotonin transporter (hSERT). On the basis of these experiments, we propose a binding mode involving residues within TM10 in an allosteric site in BGT1 that corresponds to the allosteric binding pocket revealed by the hSERT crystal structure. Our study provides first insights into a proposed allosteric binding pocket in BGT1, which accommodates the binding site for a series of novel noncompetitive inhibitors.

Pharmacological Characterization of a Betaine/GABA Transporter 1 (BGT1) Inhibitor Displaying an Unusual Biphasic Inhibition Profile and Anti-seizure Effects

“…This is in line with recent crystallographic evidence that the antidepressant escitalopram not only binds to the orthosteric pocket of the human SERT, but also to an allosteric pocket in the extracellular vestibule above the orthosteric site [15]. Since hSERT is highly related to hBGT1 (44% sequence identity), both belong to the SLC6A family as well as share the same fold, it is very likely that a similar allosteric binding site exist in BGT1 and the other GATs [13,15,37,46,47].…”

“…tsA201 cells were transfected with DNA constructs (8 μg per 10 cm dish if not otherwise mentioned) using 40 μL PolyFect transfection reagent according to the manufacturer's protocol (Qiagen, Venlo, Netherlands). The DNA constructs encoding hBGT1, hGAT1-3 [36], the mutated constructs hBGT1 Q299 and hGAT3 L314Q described previously [37], and hBGT Y453A were synthesized and sequence validated by Genscript (NJ, USA).…”

“…A hBGT1 homology model based on the hSERT crystal structure PDB ID 5I73 [15] was used from Jørgensen et al [37]. Protein and ligand preparation were performed with PrepWiz [38] and LigPrep [39], respectively, in the Schrödinger Suite 2015-2.…”

“…). 100 SBV2-114 poses per site were generated and scored according to the internal GOLD 5.2.2 ChemPLP scoring function [37] as well as rescored with GBVI/WSA dG and London dG scoring functions…”

“…To address the possibility of two binding sites for SBV2-114 at BGT1, SBV2-114 was docked into the orthosteric and allosteric site of a hBGT1 homology model based on the hSERT crystal structure PDB ID 5I73 co-crystallized with escitalopram in the orthosteric and allosteric site [15,37]. After visually investigating the top scored poses of The compounds were tested for their ability to inhibit uptake of 30 nM [3H]GABA for 3 min.…”