High-throughput screening for the discovery of antidepressants targeting adenosine A_2A receptors

The adenosine A_2A receptor (A_2AR) is a promising therapeutic target for depression, as evidenced by the notable antidepressant-like efficacy of its antagonists. However, conventional screening methods for A_2AR ligands are hampered by low throughput and operational complexity. To address this, we developed a high-throughput screening (HTS) strategy based on the detection of calcium flow fluorescence signals in engineered HEK-ADORA2A cells. After rigorous optimization, this HTS platform was deployed to screen a structurally diverse library of ∼20,784 compounds, leading to the identification of potent candidates. The binding affinity of these hits for A_2AR was confirmed in vitro using bio-layer interferometry (BLI). We evaluated the compounds using a series of behavioral tests, including the tail suspension test (TST), spontaneous activity test (SAT), forced swim test (FST), and open field test (OFT). The results demonstrated that a single administration of either Compound 10 or Compound 21 effectively reversed corticosterone (CORT)-induced depression-like behaviors. Remarkably, these compounds exhibited rapid-onset antidepressant effects within 1 h, without obvious abnormal behaviors or mortality, and their efficacy was comparable to the A_2AR antagonist istradefylline (KW6002). In summary, we have established a robust HTS method for the efficient discovery of A_2AR-targeting compounds. This work has led to the identification of novel antagonist compounds with rapid-acting antidepressant-like potential, thereby providing a solid foundation for the development of new antidepressant therapeutics.