Abstracts

Rationale: Although a large number of anti-seizure medications (ASMs) are available, they have multiple side effects causing a decline in the quality of life of patients with epilepsy. In order to create an ASM that has a wider margin between therapeutic effect and adverse effect than that of existing ASMs, E2730 was discovered by in vivo phenotypic screening, then the mechanism of action was identified as an uncompetitive and selective inhibitor of γ-aminobutyric acid transporter 1 (GAT-1). Here, we describe the preclinical in vitro characteristics of E2730.

Methods: To determine whether the binding site of E2730 exists in brain, the amount of binding and the binding affinity of [³H]E2730 to rat or human brain synaptosomal membranes were evaluated. Competitive displacement of [³H]E2730 binding by various ASMs was examined in the rat brain synaptosomal membranes to determine whether the binding site of E2730 is different from those of clinically available ASMs. [³H]E2730 binding in brain synaptosomal membranes of GAT1-deficient mice (homozygous and heterozygous) and their wild-type littermates was assessed to determine whether GAT1 is the target protein of E2730. To investigate subtype selectivity among GATs, effects of E2730 on [³H]γ-aminobutyric acid (GABA) uptake in HEK293 cells stably expressing human GATs (hGAT1, hGAT2, hGAT3, or human betaine/GABA transporter 1 [hBGT-1]) were examined. GABA-concentration dependency of hGAT1 inhibition by E2730 or tiagabine was evaluated to investigate the mode of inhibition.

Results: In rat syaptosomal membranes, the maximum binding (B_max) and binding affinity (K_D) values were 3419 fmol/mg protein and 553.4 nmol/L, respectively. In human symaptosomal membranes, the B_max and K_D values were 2503 fmol/mg protein and 709.9 nmol/L, respectively. In the competitive displacement assay, [³H]E2730 binding was not displaced by any of the clinically available ASMs tested. [³H]E2730 binding was significantly diminished to 8.0% and 54.0% in the homozygous and heterozygous GAT1-deficient mouse brain, respectively, relative to that in the wild-type mouse brain. In the [³H]GABA uptake assay, E2730 selectively inhibited hGAT1-mediated GABA uptake. The 50% inhibitory concentration (IC₅₀) values were 1.1, >1000, >1000, and 890 μmol/L for hGAT1, hGAT2, hGAT3, and hBGT-1, respectively. In addition, E2730 showed inhibitory effects on hGAT1-mediated GABA uptake to a greater degree with increasing concentrations of GABA than at lower concentrations of GABA. On the other hand, the inhibitory activity of tiagabine against hGAT1-mediated GABA uptake showed no GABA concentration dependency.

Conclusions: Among four distinct GAT subtypes, E2730 selectively inhibits hGAT1, which is predominantly expressed on presynaptic neurons. The inhibitory activity of E2730 against hGAT1-mediated GABA uptake is more potent with increasing GABA concentration. This is the characteristic of an uncompetitive inhibition mode. In contrast, tiagabine has noncompetitive mode of inhibition against hGAT1. Taken together, E2730 is a novel selective uncompetitive GAT-1 inhibitor.

Funding: Please list any funding that was received in support of this abstract.: Eisai Co., Ltd.