Discovery of allosteric modulators for GABA(A) receptors by ligand-directed chemistry

摘要

The fast inhibitory actions of gamma-aminobutyric acid (GABA) are mainly mediated by GABA(A) receptors (GABA(A)Rs) 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 GABA(A)Rs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABA(A)R 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 GABA(A)Rs. 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 GABA(A)R-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins.