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Image-Based Marker-Free Screening of GABA_A Agonists, Antagonists, and Modulators.

ABSTRACT: The ionotropic GABA_A receptors represent the main target for different groups of widely used drugs having hypnotic and anxiolytic effects. So far, most approaches used to assess GABA activity involve invasive low -throughput electrophysiological techniques or rely on fluorescent dyes, preventing the ability to conduct noninvasive and thus nonperturbing screens. To address this limitation, we have developed an automated marker-free cell imaging method, based on digital holographic microscopy (DHM). This technology allows the automatically screening of compounds in multiple plates without having to label the cells or use special plates. This methodological approach was first validated by screening the GABA_A receptor expressed in HEK cells using a selection of active compounds in agonist, antagonist, and modulator modes. Then, in a second blind screen of a library of 3041 compounds (mostly composed of natural products), 5 compounds having a specific agonist action on the GABA_A receptor were identified. The hits validated from this unbiased screen were the natural products muscimol, neurosteroid alphaxalone, and three compounds belonging to the avermectin family, all known for having an agonistic effect on the GABA_A receptor. The results obtained were exempt from false negatives (structurally similar unassigned hits), and false-positive hits were detected and discarded without the need for performing electrophysiological measurements. The outcome of the screen demonstrates the applicability of our screening by imaging method for the discovery of new chemical structures, particularly regarding chemicals interacting with the ionotropic GABA_A receptor and more generally with any ligand-gated ion channels and transporters.

SUBMITTER: Rappaz B

PROVIDER: S-EPMC7243081 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:γ-aminobutyric acid type A receptors (GABAARs) are the major mediators of synaptic inhibition in the brain. Aberrant GABAAR activity or regulation is observed in various neurodevelopmental disorders, neurodegenerative diseases and mental illnesses, including epilepsy, Alzheimer's and schizophrenia. Benzodiazepines, anesthetics and other pharmaceutics targeting these receptors find broad clinical use, but their inherent lack of receptor subtype specificity causes unavoidable side effects, raising a need for new or adjuvant medications. In this review article, we introduce a new strategy to modulate GABAeric signaling: targeting the intracellular protein interactors of GABAARs. Of special interest are scaffolding, anchoring and supporting proteins that display high GABAAR subtype specificity. Recent efforts to target gephyrin, the major intracellular integrator of GABAergic signaling, confirm that GABAAR-associated proteins can be successfully targeted through diverse molecules, including recombinant proteins, intrabodies, peptide-based probes and small molecules. Small-molecule artemisinins and peptides derived from endogenous interactors, that specifically target the universal receptor binding site of gephyrin, acutely affect synaptic GABAAR numbers and clustering, modifying neuronal transmission. Interference with GABAAR trafficking provides another way to modulate inhibitory signaling. Peptides blocking the binding site of GABAAR to AP2 increase the surface concentration of GABAAR clusters and enhance GABAergic signaling. Engineering of gephyrin binding peptides delivered superior means to interrogate neuronal structure and function. Fluorescent peptides, designed from gephyrin binders, enable live neuronal staining and visualization of gephyrin in the post synaptic sites with submicron resolution. We anticipate that in the future, novel fluorescent probes, with improved size and binding efficiency, may find wide application in super resolution microscopy studies, enlightening the nanoscale architecture of the inhibitory synapse. Broader studies on GABAAR accessory proteins and the identification of the exact molecular binding interfaces and affinities will advance the development of novel GABAAR modulators and following in vivo studies will reveal their clinical potential as adjuvant or stand-alone drugs.

Project description:We report the modification of MIDD0301, an imidazodiazepine GABAA receptor (GABAAR) ligand, using two alkyl substituents. We developed PI310 with a 6-(4-phenylbutoxy)hexyl chain as used in the long-acting β2-agonist salmeterol and PI320 with a poly(ethylene glycol) chain as used to improve the brain:plasma ratio of naloxegol, a naloxone analogue. Both imidazodiazepines showed affinity toward the GABAAR binding site of clonazepam, with IC50 values of 576 and 242 nM, respectively. Molecular docking analysis, using the available α1β3γ2 GABAAR structural data, suggests binding of the diazepine core between the α1+/γ2- interface, whereas alkyl substituents are located outside the binding site and thus interact with the protein surface and solvent molecules. The physicochemical properties of these compounds are very different. The solubility of PI310 is low in water. PEGylation of PI320 significantly improves aqueous solubility and cell permeability. Neither compound is toxic in HEK293 cells following exposure at >300 μM for 18 h. Ex vivo studies using guinea pig tracheal rings showed that PI310 was unable to relax the constricted airway smooth muscle. In contrast, PI320 induced muscle relaxation at organ bath concentrations as low as 5 μM, with rapid onset (15 min) at 25 μM. PI320 also reduced airway hyper-responsiveness in vivo in a mouse model of steroid-resistant lung inflammation induced by intratracheal challenge with INFγ and lipopolysaccharide (LPS). At nebulized doses of 7.2 mg/kg, PI320 and albuterol were equally effective in reducing airway hyper-responsiveness. Ten minutes after nebulization, the lung concentration of PI320 was 50-fold that of PI310, indicating superior availability of PI320 when nebulized as an aqueous solution. Overall, PI320 is a promising inhaled drug candidate to quickly relax airway smooth muscle in bronchoconstrictive disorders, such as asthma. Future studies will evaluate the pharmacokinetic/pharmacodynamic properties of PI320 when administered orally.

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Image-Based Marker-Free Screening of GABA_A Agonists, Antagonists, and Modulators.

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Dataset Information

Image-Based Marker-Free Screening of GABAA Agonists, Antagonists, and Modulators.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Image-Based Marker-Free Screening of GABA_A Agonists, Antagonists, and Modulators.