RESUMEN
We previously designed and synthesized a series of cyclopropane-based conformationally restricted analogues of γ-aminobutyric acid (GABA). The study demonstrated that the critical conformation of the analogues that selectively active to betaine/GABA transporter 1 (BGT1) subtype is the trans-syn-form, in which the amino and carboxyl groups are in trans-configuration and the cyclopropane ring and the carboxyl group are in syn-arrangement. In this study, we designed and synthesized cyclopropane-based GABA analogues, which were conformationally restricted in the trans-syn-form by cyclopropylic strain based on the stereochemistry of the carbon adjacent to cyclopropane. Their conformation was confirmed as the syn-form by calculations and NMR studies, and their pharmacological evaluation clarified that compounds 11a and 11d had the BGT1 selectivity, although their inhibitory effects were insufficient.
Asunto(s)
Ciclopropanos/farmacología , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Inhibidores de Recaptación de GABA/farmacología , Ácido gamma-Aminobutírico/análogos & derivados , Ácido gamma-Aminobutírico/farmacología , Animales , Células CHO , Cricetulus , Ciclopropanos/síntesis química , Ciclopropanos/química , Inhibidores de Recaptación de GABA/síntesis química , Inhibidores de Recaptación de GABA/química , Conformación Molecular , Estereoisomerismo , Ácido gamma-Aminobutírico/síntesis químicaRESUMEN
A series of cyclopropane-based conformationally restricted γ-aminobutyric acid (GABA) analogs with stereochemical diversity, that is, the trans- and cis-2,3-methano analogs Ia and Ib and their enantiomers ent-Ia and ent-Ib, and also the trans- and cis-3,4-methano analogs IIa and IIb and their enantiomers ent-IIa and ent-Iib, were synthesized from the chiral cyclopropane units Type-a and Type-b that we developed. These analogs were systematically evaluated with four GABA transporter (GAT) subtypes. The trans-3,4-methano analog IIa had inhibitory effects on GAT3 (IC50=23.9µM) and betaine-GABA transporter1 (5.48µM), indicating its potential as an effective lead compound for the development of potent GAT inhibitors due to its hydrophilic and low molecular weight properties and excellent ligand efficiency.
Asunto(s)
Ciclopropanos/química , Moduladores del GABA/química , Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Ácido gamma-Aminobutírico/química , Animales , Anticonvulsivantes/química , Anticonvulsivantes/metabolismo , Anticonvulsivantes/uso terapéutico , Encéfalo/metabolismo , Moduladores del GABA/metabolismo , Moduladores del GABA/uso terapéutico , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Ligandos , Ratones , Unión Proteica , Ratas , Convulsiones/inducido químicamente , Convulsiones/tratamiento farmacológico , Estereoisomerismo , Relación Estructura-Actividad , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/uso terapéuticoRESUMEN
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 (pK B value of 6.4). To further elaborate on the selectivity profile both compounds were tested at recombinant α1ß2γ2 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.
RESUMEN
On the basis of the three-dimensional diversity-oriented conformational restriction strategy using key chiral cyclopropane units, we previously identified 3 ((2S,3R)-4-amino-3,4-methanobutyric acid) with a chiral trans-cyclopropane structure as a γ-aminobutyric acid (GABA) transporter inhibitor selective for GABA transporter (GAT) subtypes GAT-3 and BGT-1 (betaine/GABA transporter-1). Further conformational restriction of 3 with the rigid bicyclo[3.1.0]hexane backbone led to the successful development of the first highly potent and selective BGT-1 inhibitor 4 (IC50 = 0.59 µM). The bioactive conformation of 3 for BGT-1 was also identified.