Fluorescence-based assay for rapid screening of GABA_A receptor modulating steroid antagonists (GAMSA)

GABA_A receptor modulating steroid antagonists (GAMSA) represent a promising therapeutic class by selectively inhibiting the effects of positive allosteric modulators (PAMs) at GABA_A receptors without affecting GABA-evoked currents alone. This mechanism avoids the risk of overexcitation and seizures associated with direct GABA_A receptor blockers, making GAMSA a safer alternative for conditions linked to elevated levels of endogenous PAMs such as allopregnanolone (ALLO), including compulsive disorders and hepatic encephalopathy. Despite growing interest, a high-throughput screening (HTS) method for the identification of novel GAMSA has been lacking. Here, we introduce a fluorescence-based membrane potential assay in CHO cells stably expressing GABA_A (α1β2γ2) receptors for rapid identification of compounds with GAMSA activity. This robust three-component assay, previously unused in this context, yielded a Z-factor of 0.45 and a minimum significant difference (MSD) of 4.90 %. A library of 108 neurosteroids (10 μM) was subsequently tested in the presence of GABA and ALLO. Isoallopregnanolone (IsoALLO), a known GAMSA, was the only compound that significantly reduced ALLO-potentiated GABA responses (to 42 ± 3 %) without affecting GABA-evoked responses alone. The inhibitory effect of IsoALLO was confirmed using manual patch-clamp recordings in primary rat anterior pituitary cells, which endogenously express GABA_A receptors, where it reduced ALLO-potentiated GABA-induced currents by 32 ± 4 %. This study validates the first cell-based HTS method for GAMSA identification and demonstrates its utility in distinguishing GAMSA from direct channel blockers or negative allosteric modulators (NAMs) using standard laboratory equipment.