Dataset Information

Construction of a Fluorescent Screening System of Allosteric Modulators for the GABAA Receptor Using a Turn-On Probe.

ABSTRACT: ?-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. The fast inhibitory actions of GABA are mainly mediated by GABAA receptors (GABAARs), which are widely recognized as clinically relevant drug targets. However, it remains difficult to create screening systems for drug candidates that act on GABAARs because of the existence of multiple ligand-binding sites and the delicate pentameric structures of GABAARs. We here developed the first turn-on fluorescent imaging probe for GABAARs, which can be used to quantitatively evaluate ligand-receptor interactions under live cell conditions. Using noncovalent labeling of GABAARs with this turn-on probe, a new imaging-based ligand assay system, which allows discovery of positive allosteric modulators (PAMs) for the GABAAR, was successfully constructed. Our system is applicable to high-throughput ligand screening, and we discovered new small molecules that function as PAMs for GABAARs. These results highlight the power of the use of a turn-on fluorescent probe to screen drugs for complicated membrane proteins that cannot be addressed by conventional methods.

SUBMITTER: Sakamoto S

PROVIDER: S-EPMC6764212 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Construction of a Fluorescent Screening System of Allosteric Modulators for the GABAA Receptor Using a Turn-On Probe.

Sakamoto Seiji S Yamaura Kei K Numata Tomohiro T Harada Fumio F Amaike Kazuma K Inoue Ryuji R Kiyonaka Shigeki S Hamachi Itaru I

ACS central science 20190816 9

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. The fast inhibitory actions of GABA are mainly mediated by GABAA receptors (GABAARs), which are widely recognized as clinically relevant drug targets. However, it remains difficult to create screening systems for drug candidates that act on GABAARs because of the existence of multiple ligand-binding sites and the delicate pentameric structures of GABAARs. ...[more]

PMID: 31572781

Similar Datasets

Project description:BackgroundThe current standard of care for insomnia includes gamma-aminobutyric acid receptor A (GABAA) activators, which promote sleep as well as general central nervous system depression. Dual orexin receptor antagonists (DORAs) represent an alternative mechanism for insomnia treatment that induces somnolence by blocking the wake-promoting effects of orexin neuropeptides. The current study compares the role and interdependence of these two mechanisms on their ability to influence sleep architecture and quantitative electroencephalography (qEEG) spectral profiles across preclinical species.ResultsActive-phase dosing of DORA-22 induced consistent effects on sleep architecture in mice, rats, dogs, and rhesus monkeys; attenuation of active wake was accompanied by increases in both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. Eszopiclone, a representative GABAA receptor modulator, promoted sleep in rats and rhesus monkeys that was marked by REM sleep suppression, but had inconsistent effects in mice and paradoxically promoted wakefulness in dogs. Active-phase treatment of rats with DORA-12 similarly promoted NREM and REM sleep to magnitudes nearly identical to those seen during normal resting-phase sleep following vehicle treatment, whereas eszopiclone suppressed REM even to levels below those seen during the active phase. The qEEG changes induced by DORA-12 in rats also resembled normal resting-phase patterns, whereas eszopiclone induced changes distinct from normal active- or inactive-phase spectra. Co-dosing experiments, as well as studies in transgenic rats lacking orexin neurons, indicated partial overlap in the mechanism of sleep promotion by orexin and GABA modulation with the exception of the REM suppression exclusive to GABAA receptor modulation. Following REM deprivation in mice, eszopiclone further suppressed REM sleep while DORA-22 facilitated recovery including increased REM sleep.ConclusionDORAs promote NREM and importantly REM sleep that is similar in proportion and magnitude to that seen during the normal resting phase across mammalian animal models. While limited overlap exists between therapeutic mechanisms, orexin signaling does not appear involved in the REM suppression exhibited by GABAA receptor modulators. The ability of DORAs to promote proportional NREM and REM sleep following sleep deprivation suggests that this mechanism may be effective in alleviating recovery from sleep disturbance.

Dataset Information

Construction of a Fluorescent Screening System of Allosteric Modulators for the GABAA Receptor Using a Turn-On Probe.

Publications

Construction of a Fluorescent Screening System of Allosteric Modulators for the GABA<sub>A</sub> Receptor Using a Turn-On Probe.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets