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

Abstract

The betaine/GABA transporter 1 (BGT1) is a member of the GABA transporter (GAT) family with still elusive function, largely due to a lack of potent and selective tool compounds. Based on modeling, we here present the design, synthesis and pharmacological evaluation of five novel conformationally restricted cyclic GABA analogs related to the previously reported highly potent and selective BGT1 inhibitor (1S,2S,5R)-5-aminobicyclo[3.1.0]hexane-2-carboxylic acid (bicyclo-GABA). Using [3H]GABA radioligand uptake assays at the four human GATs recombinantly expressed in mammalian cell lines, we identified bicyclo-GABA and its N-methylated analog (2) as the most potent and selective BGT1 inhibitors. Additional pharmacological characterization in a fluorescence-based membrane potential assay showed that bicyclo-GABA and 2 are competitive inhibitors, not substrates, at BGT1, which was validated by a Schild analysis for bicyclo-GABA (pKB value of 6.4). To further elaborate on the selectivity profile both compounds were tested at recombinant α1β2γ2 GABAA receptors. Whereas bicyclo-GABA showed low micromolar agonistic activity, the N-methylated 2 was completely devoid of activity at GABAA receptors. To further reveal the binding mode of bicyclo-GABA and 2 binding hypotheses of the compounds were obtained from in silico-guided mutagenesis studies followed by pharmacological evaluation at selected BGT1 mutants. This identified the non-conserved BGT1 residues Q299 and E52 as the molecular determinants driving BGT1 activity and selectivity. The binding mode of bicyclo-GABA was further validated by the introduction of activity into the corresponding GAT3 mutant L314Q (38 times potency increase cf. wildtype). Altogether, our data reveal the molecular determinants for the activity of bicyclic GABA analogs, that despite their small size act as competitive inhibitors of BGT1. These compounds may serve as valuable tools to selectively and potently target BGT1 in order to decipher its elusive pharmacological role in the brain and periphery such as the liver and kidneys.

Synthesis of γ-Aminobutyric Acid (GABA) Analogues Conformationally Restricted by Bicyclo[3.1.0]hexane/hexene or [4.1.0]Heptane/heptene Backbones as Potent Betaine/GABA Transporter Inhibitors

“…For pharmacological evaluation, compounds 3 – 5 were tested for their ability to inhibit [ 3 H]­GABA uptake at the four human GAT subtypes transiently expressed in tsA201 cells, exactly as described previously . Initially, to investigate subtype selectivity, two concentrations of 10 and 100 μM were probed (Figure a).…”

“…Further conformational restriction of it provided the potent inhibitor bicyclo-GABA ( 2 ), having a chiral bicyclo[3.1.0]­hexane backbone (Figure a) . Bicyclo-GABA is a highly selective BGT1 inhibitor with high nanomolar potency (IC 50 = 590 nM) only reported to date; however, its synthesis required long reaction steps including nonstereoselective and low yield steps (Scheme S1). Due to its potency as a drug lead and a pharmacological tool, development of a new efficient synthesis method for 2 is wanted.…”

“…Docking simulation studies of bicyclo-GABA suggested the bioactive conformation of GABA at BGT1 as a C1,C4- trans /C2,N 4 - trans form (around the C2–C3 bond, and a C1,C4- trans around the C3–C4 bond, and a C2,N 4 - trans conformation of GABA, Figure b). , Thus, we newly designed three other conformationally restricted GABA analogues 3 , 4 , and 5 , having a bicyclo[3.1.0]­hexene, [4.1.0]­heptane, or [4.1.0]­heptene backbone, respectively (Figure c). These three compounds are restricted in the bioactive C1,C4- trans /C2,N 4 - trans form of GABA by their rigid structures, analogous to bicyclo-GABA ( 2 ), as shown by superimposition of their calculated most stable conformations (Figure d).…”

“…Data shown are pooled and normalized to 100% [ 3 H]­GABA uptake ( n = 2–3). (b) Concentration-dependent inhibition of 3 – 5 at BGT1 generated as described in ref . Curves are representative.…”

“…Data analysis yielded micromolar range IC 50 values (Table ). From this, 4 represents an interesting tool compound for further studies, displaying an IC 50 value of 7.3 μM and an improved BGT1/GAT3 selectivity profile compared to the previously reported bicyclo-GABA ( 2 ). , …”