[Establishment and application of a high-throughput screening method for drugs targeting the 5-hydroxytryptamine 2A receptor based on cellular calcium flux signals].

A high-throughput screening method for drugs targeting the 5-HT2A receptor was developed using calcium flux signals. The method, optimized with 10,000 cells per well, 0.2% DMSO, and 2-hour Calcium 6 dye incubation, showed excellent specificity and precision (inter-batch precision below 10% for 5-HT detection). Testing four known compounds (DOI, 5-MeO-DMT, LSD, and MDL100907) yielded Z'-factors above 0.85 and signal window values over 0.91 at EC80, with EC50 values in the nanomolar range. Applied to 38 known active compounds, it efficiently identified agonists and antagonists without responding to non-target compounds, confirming its reliability for screening antidepressants targeting the 5-HT2A receptor.

Study at a glance

Abstract

The 5-hydroxytryptamine 2A receptor (5-HT2AR) is one of the key targets in the development of novel antidepressants. To develop new antidepressants targeting the 5-HT2A receptor, this study established a high-throughput screening method for drugs targeting the 5-HT2A receptor based on the principle of detecting calcium flux signals. The immunofluorescence assay and western blotting were employed to evaluate receptor expression levels in the 5-HT2AR-CHO cell line. The reaction system parameters, including cell seeding density, DMSO concentration, and dye incubation time, were optimized with Z'-factor and signal window values as evaluation indicators. The specificity, precision, stability, and applicability of the method were assessed. Results indicated that the 5-HT2AR-CHO cell line stably expressed high levels of the 5-HT2A receptor. The optimized screening method involved a reaction system with 10 000 cells/well, 0.2% DMSO, and 2 h incubation with Calcium 6 dye. The method demonstrated excellent specificity, with inter-batch precision below 10% for the detection of 5-hydroxytryptamine (5-HT) at low, medium, and high concentrations. Testing four compounds that target the 5-HT2A receptor- agonists 2,5-dimethoxy-4-iodoamphetamine (DOI), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), and lysergic acid diethylamide (LSD), along with the antagonist MDL100907-yielded Z'-factors (at EC80) greater than 0.85 and signal window values over 0.91. The EC50 values of these compounds were in the nanomolar range, and their potency rank order aligned with previously reported data, confirming the reliability of the established method. When being applied to the detection of 38 known active compounds, the method efficiently identified 5-HT2A receptor agonists and antagonists while showing no response to non-target compounds. In conclusion, this study successfully constructs a high-throughput screening approach for 5-HT2A receptor-targeting drugs based on calcium flux signals. The method possesses strong specificity, high sensitivity, and robust stability, being suitable for screening antidepressants targeting the 5-HT2A receptor.