title: Discovery of allosteric modulators for GABAA receptors by ligand-directed chemistry.
abstract: The fast inhibitory actions of γ-aminobutyric acid (GABA) are mainly mediated by GABAA receptors (GABAARs) in the brain. The existence of multiple ligand-binding sites and a lack of structural information have hampered the efficient screening of drugs capable of acting on GABAARs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABAAR ligands on live cells via coupling of affinity-based chemical labeling reagents to a bimolecular fluorescence quenching and recovery system. These biosensors were amenable to the high-throughput screening of a chemical library, leading to the discovery of new small molecules capable of interacting with GABAARs. Electrophysiological measurements revealed that one hit, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), was a novel negative allosteric modulator capable of strongly suppressing GABA-induced chloride currents. Thus, these semisynthetic biosensors represent versatile platforms for screening drugs to treat GABAAR-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Yamaura
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, Japan.|Department of Technology and Ecology, Hall of Global Environmental Studies, Kyoto University, Kyoto, Japan.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Kiyonaka
authors.affiliation: Department of Physiology, School of Medicine, Fukuoka University, Jonan-ku, Fukuoka, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Numata
authors.affiliation: Department of Physiology, School of Medicine, Fukuoka University, Jonan-ku, Fukuoka, Japan.
authors.forename: Ryuji
authors.initials: R
authors.lastname: Inoue
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, Japan.|Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kawaguchi, Saitama, Japan.
authors.forename: Itaru
authors.initials: I
authors.lastname: Hamachi
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D015374:Biosensing Techniques; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D010636:Phenols / Q000737:chemistry / Q000494:pharmacology*; D011720:Pyrazoles / Q000737:chemistry / Q000494:pharmacology*; D011963:Receptors, GABA-A / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D008024:Ligands; D010636:Phenols; D011720:Pyrazoles; D011963:Receptors, GABA-A; D054852:Small Molecule Libraries; C486184:4,4',4''-(4-propyl-((1)H)-pyrazole-1,3,5-triyl) tris-phenol