Development of a Multiplex Assay for Studying Functional Selectivity of Human Serotonin 5-HT2A Receptors and Identification of Active Compounds by High-Throughput Screening

作者：Iglesias Alba; Lage Sonia; Isabel Cadavid Maria; Isabel Loza Maria; Brea Jose^*

来源：Journal of Biomolecular Screening, 2016, 21(8): 816-823.

DOI：10.1177/1087057116644162

• 5-HT2A receptor
• functional selectivity
• multiplex
• phospholipase A(2)
• phospholipase C

G protein-coupled receptors (GPCRs) exist as collections of conformations in equilibrium, and the efficacy of drugs has been proposed to be associated with their absolute and relative affinities for these different conformations. The serotonin 2(A) (5-HT2A) receptor regulates multiple physiological functions, is involved in the pathophysiology of schizophrenia, and serves as an important target of atypical antipsychotic drugs. This receptor was one of the first GPCRs for which the functional selectivity phenomenon was observed, with its various ligands exerting differential effects on the phospholipase A(2) (PLA(2)) and phospholipase C (PLC) signaling pathways. We aimed to develop a multiplex functional assay in 96-well plates for the simultaneous measurement of the PLA(2) and PLC pathways coupled to 5-HT2A receptors; this approach enables the detection of either functional selectivity or cooperativity phenomena in early drug screening stages. The suitability of the method for running screening campaigns was tested using the Prestwick Chemical Library, and 22 confirmed hits with activities of more than 90% were identified; 11 of these hits produced statistically significant differences between the two effector pathways. Thus, we have developed a miniaturized multiplex assay in 96-well plates to measure functional selectivity for 5-HT2A receptors in the early stages of the drug discovery process.

• 出版日期2016-9