PMID- 37052238 OWN - NLM STAT- MEDLINE DCOM- 20230905 LR - 20230905 IS - 2470-9239 (Electronic) IS - 2470-9239 (Linking) VI - 8 IP - 3 DP - 2023 Sep TI - Discovery of E2730, a novel selective uncompetitive GAT1 inhibitor, as a candidate for anti-seizure medication. PG - 834-845 LID - 10.1002/epi4.12741 [doi] AB - OBJECTIVE: As of 2022, 36 anti-seizure medications (ASMs) have been licensed for the treatment of epilepsy, however, adverse effects (AEs) are commonly reported. Therefore, ASMs with a wide margin between therapeutic effects and AEs are preferred over ASMs that are associated with a narrow margin between efficacy and risk of AEs. E2730 was discovered using in vivo phenotypic screening and characterized as an uncompetitive, yet selective, inhibitor of gamma-aminobutyric acid (GABA) transporter 1 (GAT1). Here, we describe the preclinical characteristics of E2730. METHODS: Anti-seizure effects of E2730 were evaluated in several animal models of epilepsy: corneal kindling, 6 Hz-44 mA psychomotor seizure, amygdala kindling, Fragile X syndrome, and Dravet syndrome models. Effects of E2730 on motor coordination were assessed in accelerating rotarod tests. The mechanism of action of E2730 was explored by [(3) H]E2730 binding assay. The GAT1-selectivity over other GABA transporters was examined by GABA uptake assay of GAT1, GAT2, GAT3, or betaine/GABA transporter 1 (BGT-1) stably expressing HEK293 cells. To further investigate the mechanism for E2730-mediated inhibition of GAT1, in vivo microdialysis and in vitro GABA uptake assays were conducted under conditions of different GABA concentrations. RESULTS: E2730 showed anti-seizure effects in the assessed animal models with an approximately >20-‍fold margin between efficacy and motor incoordination. [(3) H]E2730 binding on brain synaptosomal membrane was abolished in GAT1-deficient mice, and E2730 selectively inhibited GAT1-mediated GABA uptake over other GABA transporters. In addition, results of GABA uptake assays showed that E2730-mediated inhibition of GAT1 positively correlated to the level of ambient GABA in vitro. E2730 also increased extracellular GABA concentration in hyperactivated conditions but not under basal levels in vivo. SIGNIFICANCE: E2730 is a novel, selective, uncompetitive GAT1 inhibitor, which acts selectively under the condition of increasing synaptic activity, contributing to a wide margin between therapeutic effect and motor incoordination. CI - (c) 2023 The Authors. Epilepsia Open published by Wiley Periodicals LLC on behalf of International League Against Epilepsy. FAU - Fukushima, Kazuyuki AU - Fukushima K AD - Deep Human Biology Learning, Eisai Co., Ltd., Tsukuba, Ibaraki, Japan. FAU - Higashiyama, Hiroyuki AU - Higashiyama H AD - Neurology Business Group, Eisai Co., Ltd., Tsukuba, Ibaraki, Japan. FAU - Kazuta, Yuji AU - Kazuta Y AD - Deep Human Biology Learning, Eisai Co., Ltd., Tsukuba, Ibaraki, Japan. FAU - Hashimoto, Keisuke AU - Hashimoto K AD - Deep Human Biology Learning, Eisai Co., Ltd., Tsukuba, Ibaraki, Japan. FAU - Watanabe, Naoto AU - Watanabe N AD - Deep Human Biology Learning, Eisai Co., Ltd., Tsukuba, Ibaraki, Japan. FAU - Furuya, Yoshiaki AU - Furuya Y AD - Deep Human Biology Learning, Eisai Co., Ltd., Tsukuba, Ibaraki, Japan. FAU - Ito, Yoshimasa AU - Ito Y AD - Neurology Business Group, Eisai Co., Ltd., Tsukuba, Ibaraki, Japan. FAU - Wu, Ting AU - Wu T AD - Alzheimer's Disease and Brain Health, Eisai Co., Ltd., Tsukuba, Ibaraki, Japan. FAU - Kosasa, Takashi AU - Kosasa T AD - Neurology Business Group, Eisai Co., Ltd., Tsukuba, Ibaraki, Japan. FAU - Talos, Delia M AU - Talos DM AD - Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. FAU - Song, Yeri AU - Song Y AD - Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. FAU - Roberts, Nicholas S AU - Roberts NS AD - Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. FAU - Jensen, Frances E AU - Jensen FE AD - Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. FAU - Hanada, Takahisa AU - Hanada T AD - Deep Human Biology Learning, Eisai Co., Ltd., Tsukuba, Ibaraki, Japan. FAU - Ido, Katsutoshi AU - Ido K AD - Neurology Business Group, Eisai Co., Ltd., Tsukuba, Ibaraki, Japan. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20230518 PL - United States TA - Epilepsia Open JT - Epilepsia open JID - 101692036 RN - 0 (GABA Plasma Membrane Transport Proteins) RN - 56-12-2 (gamma-Aminobutyric Acid) RN - 0 (Anticonvulsants) SB - IM MH - Animals MH - Humans MH - Mice MH - Ataxia MH - *Epilepsy/drug therapy MH - *GABA Plasma Membrane Transport Proteins/administration & dosage MH - gamma-Aminobutyric Acid/pharmacology/metabolism MH - HEK293 Cells MH - *Anticonvulsants/pharmacology/therapeutic use PMC - PMC10472371 OTO - NOTNLM OT - GABA transporters OT - GAT1 OT - anti-seizure medications OT - uncompetitive inhibition COIS- Kazuyuki Fukushima, Yuji Kazuta, Keisuke Hashimoto, Naoto Watanabe, Yoshiaki Furuya, Ting Wu, Takahisa Hanada, and Katsutoshi Ido are all employees of Eisai Co., Ltd. Hiroyuki Higashiyama, Yoshimasa Ito, and Takashi Kosasa were employees of Eisai Co., Ltd. during these works and now are retired. Delia M. Talos, Yeri Song, Nicholas S. Roberts, and Frances E. Jensen contributed to a part of these studies as sponsored research. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. EDAT- 2023/04/14 06:00 MHDA- 2023/09/04 06:44 PMCR- 2023/05/18 CRDT- 2023/04/13 07:23 PHST- 2022/12/12 00:00 [received] PHST- 2023/04/08 00:00 [accepted] PHST- 2023/09/04 06:44 [medline] PHST- 2023/04/14 06:00 [pubmed] PHST- 2023/04/13 07:23 [entrez] PHST- 2023/05/18 00:00 [pmc-release] AID - EPI412741 [pii] AID - 10.1002/epi4.12741 [doi] PST - ppublish SO - Epilepsia Open. 2023 Sep;8(3):834-845. doi: 10.1002/epi4.12741. Epub 2023 May 18.