Pharmacological Identification of a Guanidine-Containing β-Alanine Analogue with Low Micromolar Potency and Selectivity for the Betaine/GABA Transporter 1 (BGT1)

Abstract

The γ-aminobutyric acid (GABA) transporters (GATs) are key membrane transporter proteins involved in the termination of GABAergic signaling at synapses in the mammalian brain and proposed drug targets in neurological disorders such as epilepsy. To date, four different GAT subtypes have been identified: GAT1, GAT2, GAT3 and the betaine/GABA transporter 1 (BGT1). Owing to the lack of potent and subtype selective inhibitors of the non-GAT1 GABA transporters, the physiological role and therapeutic potential of these transporters remain to be fully understood. Based on bioisosteric replacement of the amino group in β-alanine or GABA, a series of compounds was generated, and their pharmacological activity assessed at human GAT subtypes. Using a cell-based [(3)H]GABA uptake assay, several selective inhibitors at human BGT1 were identified. The guanidine-containing compound 9 (2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid hydrochloride) displayed more than 250 times greater potency than the parent compound β-alanine at BGT1 and is thus the most potent inhibitor reported to date for this subtype (IC50 value of 2.5 µM). In addition, compound 9 displayed about 400, 16 and 40 times lower inhibitory potency at GAT1, GAT2 and GAT3, respectively. Compound 9 was shown to be a substrate for BGT1 and to have an overall similar pharmacological profile at the mouse orthologue. Compound 9 constitutes an interesting pharmacological tool for specifically investigating the cellular pharmacology of BGT1 and is the first small-molecule substrate identified with such a high selectivity for BGT1 over the three other GAT subtypes.

Structure activity relationship of selective GABA uptake inhibitors

“…Recently, highly selective BGT-1 inhibitors were reported further demonstrating that high selectivity can be achieved. 30,31 However, these small amino acids are most likely substrates for the transporter, and probably they do not penetrate the BBB. Thus, continued efforts with more lipophilic derivatives could provide highly selective tool compounds that are important to understand the role of GABA transporters in health and diseases, and may point to new therapeutic strategies.…”

GAT3 selective substrate l-isoserine upregulates GAT3 expression and increases functional recovery after a focal ischemic stroke in mice

“…Further, Xenopus laevis oocytes recombinantly expressing related SLC6A members, including GlyT1, GlyT2, TauT and CreaT, were also used to assess the specificity for the GATs. 26 [ 3 H]taurine competition uptake assay was performed at human TauT transiently expressed in tsA201 cells following the protocol for the [ 3 H]GABA competition uptake assay described previously 27,28 with the exception of exchanging [ 3 H]GABA with 22 nM [ 3 H]taurine for 5 min.…”

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

“…1 ), as a template. These compounds were identified as substrate-inhibitors with pronounced selectivity towards BGT1 over the other GATs 38 . Within the series, 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA) and the corresponding amidine analog (6-amino-2,3,4,5-tetrahydropyridine-3-carboxylic acid, 2 (Fig.…”