Development of a Multiplex Assay for Studying Functional Selectivity of Human Serotonin 5-HT2A Receptors and Identification of Active Compounds by High-Throughput Screening.
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 2A (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 A2 (PLA2) and phospholipase C (PLC) signaling pathways. We aimed to develop a multiplex functional assay in 96-well plates for the simultaneous measurement of the PLA2 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.
BioFarma Research Group, Centro Singular de Investigación en Medicina Molecular y Enfermedades Crónicas (CIMUS), Universidade de Santiago de Compostela, Santiago de Compostela, Spain.
Alba
A
Iglesias
BioFarma Research Group, Centro Singular de Investigación en Medicina Molecular y Enfermedades Crónicas (CIMUS), Universidade de Santiago de Compostela, Santiago de Compostela, Spain.
Sonia
S
Lage
BioFarma Research Group, Centro Singular de Investigación en Medicina Molecular y Enfermedades Crónicas (CIMUS), Universidade de Santiago de Compostela, Santiago de Compostela, Spain.
Maria Isabel
MI
Cadavid
BioFarma Research Group, Centro Singular de Investigación en Medicina Molecular y Enfermedades Crónicas (CIMUS), Universidade de Santiago de Compostela, Santiago de Compostela, Spain.
Maria Isabel
MI
Loza
BioFarma Research Group, Centro Singular de Investigación en Medicina Molecular y Enfermedades Crónicas (CIMUS), Universidade de Santiago de Compostela, Santiago de Compostela, Spain pepo.brea@usc.es.
José
J
Brea
D014150:Antipsychotic Agents / Q000302:isolation & purification* / Q000627:therapeutic use; D001665:Binding Sites; D004353:Drug Evaluation, Preclinical; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008024:Ligands; D054467:Phospholipases A2 / Q000378:metabolism; D044348:Receptors, Serotonin, 5-HT2 / Q000378:metabolism*; D012559:Schizophrenia / Q000188:drug therapy* / Q000378:metabolism; D058830:Serotonin 5-HT2 Receptor Antagonists / Q000302:isolation & purification* / Q000378:metabolism; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism
D014150:Antipsychotic Agents; D008024:Ligands; D044348:Receptors, Serotonin, 5-HT2; D058830:Serotonin 5-HT2 Receptor Antagonists; D054467:Phospholipases A2; D010738:Type C Phospholipases
5-HT2A receptor; functional selectivity; multiplex; phospholipase A2; phospholipase C