Abstract

Postpartum depression (PPD) is an extreme form of the “baby blues” with serious consequences and an unmet need for treatments. γ-aminobutyric acid type A receptors (GABA_ARs) are implicated in PPD pathophysiology, very relevant given the recent approval of Brexanolone, a proprietary formulation of the neurosteroid allopregnanolone, a GABA_AR positive allosteric modulator (PAM). δ-GABA_ARs are more sensitive to the hormonal changes of pregnancy, being downregulated to counteract enhanced GABA_AR activity that presumably occurs due to increased allopregnanolone production. We propose that drugs which enhance the function of the δ subunit-containing GABA_ARs (δ-GABA_ARs) will be efficacious in PPD, without side-effects (unconsciousness and sedation) that accompany Brexanolone. The Medicines Discovery Institute is working to identify such compounds. This work describes the development of a functional assay to serve as a primary screen for the effect of novel compounds on GABA_ARs. Using Fluorescent Imaging Plate Reader (FLIPR) technology, an in-house assay was developed using Red Membrane Potential Explorer Dye (Molecular Devices Inc.) to characterise compounds against GABA_AR over-expressing HEK293 cells. Receptor activation was measured by change in fluorescence, and validated using known δ-GABA_AR PAMs. Statistical analysis were conducted on normalised data using a nonlinear regression model to determine EC50 values (GraphPad Prism 9.3.1). Known PAMs of δ-GABA_ARs, consistently produced data in-line with literature. Our data supports the concept of a δ-GABA_AR PAM, which can be used to generate optimised compounds. The results support the validity of the in-house FLIPR assay and are comparable to electrophysiological data. Novel compounds can therefore be screened and considered for the next stage of the drug discovery process.