Effects of 4(1H)-quinolinone derivative, a novel non-nucleotide allosteric purinergic P2Y2 agonist, on cardiomyocytes in neonatal rats

Kensuke Sakuma,Shota Ikeda,Tatsuo Oikawa,Hideyuki Nakagawa,Masakuni Noda

doi:10.1038/s41598-017-06481-9

Abstract

Purinergic P2Y2 receptors, G-protein coupled receptors that primarily couple with Gαq/11-proteins, are activated equipotently by adenosine-5′-triphosphate (ATP) and uridine-5′-triphosphate. Evidence suggests that P2Y2 agonists make potential drug candidates for the treatment of cardiovascular diseases. However, selective non-nucleotide, small-molecule P2Y2 agonists have yet to be developed. In this report, we discuss Compound 89, a novel non-nucleotide allosteric P2Y2 agonist that was active in signal transduction and gene induction, and in our in vitro cardiac hypertrophy model. Compound 89 exhibited selective P2Y2 agonistic activity and potentiated responses to the endogenous agonist ATP, while exhibiting no agonistic activities for four other Gαq/11-coupled human P2Y (hP2Y) receptors and one representative Gαi/o-coupled hP2Y12 receptor. Its P2Y2 agonistic effect on mouse P2Y2 receptors suggested non-species-specific activity. Compound 89 acted as a pure positive allosteric modulator in a Ca2+ mobilization assay of neonatal rat cardiomyocytes; it potentiated ATP-induced expression of genes in the nuclear receptor 4A family (negative regulators of hypertrophic stimuli in cardiomyocytes). Additionally, Compound 89 attenuated isoproterenol-induced cardiac hypertrophy, presumably through dose-dependent interaction with pericellular ATP. These results indicate that Compound 89 is potentially efficacious against cardiomyocytes and therefore a good proof-of-concept tool for elucidating the therapeutic potential of P2Y2 activation in various cardiovascular diseases.

Highlights

Compound 89 attenuated isoproterenol-induced cardiac hypertrophy, presumably through dose-dependent interaction with pericellular ATP
We studied the effects of Compound 89 on Ca2+ signalling, nuclear receptor 4A (NR4A) gene induction, and isoproterenol-induced cardiac hypertrophy in neonatal rat cardiomyocytes (NRCMs)
A FLIPR Ca2+ mobilization assay of hP2Y2–1321N1 cells revealed that Compound 89 had agonistic activity against human P2Y2, it was less potent than ATP (Fig. 1b)

Summary

Introduction

Compound 89 attenuated isoproterenol-induced cardiac hypertrophy, presumably through dose-dependent interaction with pericellular ATP. Increasing evidence indicates that P2Y2 receptors could function as cell-surface ‘mechano-sensors’ for the autocrine or paracrine release of adenosine-5′-triphosphate (ATP) and/or uridine-5′-triphosphate (UTP) from different cellular sources[6,7,8,9] in order to primarily mediate Gαq/11 signalling, resulting in downstream transcriptional regulation via nuclear inositol 1,4,5-trisphosphate receptor-induced local Ca2+ signalling in muscle fibres and cardiac myocytes[10,11,12] Such regulation of transcriptional activity through local Ca2+ signal transduction is known as excitation-transcription coupling, as opposed to the excitation-contraction coupling that links global increases in intracellular Ca2+ more closely with muscle contractions. While the recent rapid advances in GPCR and ion channel crystallography have enhanced the practical understanding of purinergic receptors such as A2AAR, P2X4, P2Y1, and P2Y1229–34, a structure-based approach to ligand design that is capable of predicting ligand selectivity and functionality remains under development[35]

Methods

Results

Conclusion