Development of a multiplex assay for studying functional selectivity of human serotonin 5-HT2A receptors.

A Iglesias, MI Cadavid, MI Loza, J Brea. University of Santiago de Compostela, Pharmacology 15782, Spain

G protein-coupled receptors (GPCR) exist as collections of conformations in equilibrium, and it has been proposed that the affinity and efficacy of drugs is related to their absolute and relative affinities for the conformational populations of a receptor (Kenakin T. Curr Opin Pharmacol, 2010). The current challenge in receptor pharmacology is to use maximal number of methods for detecting conformational changes (Kenakin T. Br J Pharmacol, 2012). 5-HT2A are class A GPCRs regulating multiple physiological functions and is involved in the pathophysiology of schizophrenia. These receptors show functional selectivity among the phospholipase C (PLC) and phospholipase A2 (PLA2) signaling pathways. Moreover, recent studies showed that these receptors may exist either as homodimers (Brea J. Mol Pharmacol, 2009) or as 5-HT2A-D2 heterodimers (Lukasiewicz S. Biochim BioPhys, 2010; Borroto-Escuela D. Biochem Biophys Ress Commun, 2010; Albizu L. Neuropharmacology, 2011) which is conditioning their intracelular signaling.

Our aim in this work was to develop a miniaturized assay for studying simultaneously the PLC and PLA2 signaling pathways at 5-HT2A receptors; this would allow evaluating the modulation of either functional selectivity or cooperativity phenomena in early stages of drug screening.

Functional assays were performed in 96-wells plates where CHO cells stably expressing the human 5-HT2A receptor were seeded 48 hours before assay. Cells were labeled with 1µCi/ml myo-[3H] inositol for 24 hours and 0.1μCi/ml [14C] arachidonic acid for 4 hours at 37° C. The tested compounds were incubated for 20 min at 37 °C.

Under these conditions we obtained EC50 values for 5-HT of 135.99 ± 23.92 nM at PLC and 140.27 ± 53.94 nM at PLA2 which were compatibles with those obtained by using a non-miniaturized methodology (346.19 ± 271.1 and 279.71 ± 154.69 nM, of PLC and PLA2 respectively). We evaluated the capability of clozapine to inhibit 5-HT 1µM induced stimulation and we obtained IC50 values of 44.89 ± 9.92 nM at PLC and IC50High 5.35 ± 2.13 nM , IC50Low 3067.31 ± 1811.99 nM PLA2 also compatibles with those obtained by using a non-miniaturized methodology (89.12 ± 3.10 at PLC 2.04 ± 0.5 , 4176 ± 1458 nM at PLA2).

We observed that clozapine shows a particular profile in the pathway signaling useful to differentiate cascades for antipsychotics.

The results obtained validate this methodology as a useful approach for studying functional selectivity and cooperativity phenomenon in early drug discovery screening process.

Grants: This work was supported by Spanish Ministry of Science and Innovation (SAF2009-13609-C04-01).