2,2′-Pyridylisatogen tosylate antagonizes P2Y1 receptor signaling without affecting nucleotide binding

Abstract

The effect of 2,2′-pyridylisatogen tosylate (PIT) on the human P2Y 1 receptor and on other recombinant P2Y receptors has been studied. We first examined the modulation by PIT of the agonist-induced accumulation of inositol phosphates. PIT blocked 2-methylthio-ADP (2-MeSADP)-induced accumulation of inositol phosphates in 1321N1 astrocytoma cells stably expressing human P2Y 1 receptors in a non-competitive and concentration-dependent manner. The IC 50 for reduction of the maximal agonist effect was 0.14 μM. In contrast, MRS2179, a competitive P2Y 1 receptor antagonist, parallel-shifted the agonist concentration-response curve to the right. PIT also concentration-dependently blocked the P2Y 1 receptor signaling induced by the endogenous agonists, ADP and ATP. A simple structural analogue of PIT was synthesized and found to be inactive as a P2Y 1 receptor antagonist, suggesting that the nitroxyl group of PIT is a necessary structural component for P2Y 1 receptor antagonism. We next examined the possible modulation of the binding of the newly available antagonist radioligand for the P2Y 1 receptor, [ 3 H] MRS2279. It was found that PIT (0.01-10 μM) did not inhibit [ 3 H] MRS2279 binding to the human P2Y 1 receptor. PIT (10 μM) had no effect on the competition for [ 3 H] MRS2279 binding by agonists, ADP and ATP, suggesting that its antagonism of the P2Y 1 receptor may be allosteric. PIT had no significant effect on agonist activation of other P2Y receptors, including P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 and P2Y 12 receptors. Thus, PIT selectively and non-competitively blocked P2Y 1 receptor signaling without affecting nucleotide binding.

Targeting Platelet G-Protein Coupled Receptors (GPCRs): Looking Beyond Conventional GPCR Antagonism

“…2-2'-Pyridylisatogen tosylate (PIT) is an allosteric modulator of P2Y 1 [149]. It inhibits P2Y 1 receptor-mediated signaling induced by either ATP or ADP in astrocytoma cells without affecting nucleotide binding to P2Y 1 [150]. PIT has been used in megakaryocytes to study P2Y 1 receptor-evoked Ca 2+ mobilization [151], but its effect on P2Y 1 receptormediated platelet activation is unknown.…”

Ticagrelor binds to human P2Y12 independently from ADP but antagonizes ADP‐induced receptor signaling and platelet aggregation

“…Examples have been reported of non-competitive inhibition at the related human receptors P2Y 1 [7], P2Y 13 [24], and adenosine A 1 [25], suggesting that a conserved allosteric site may be present on this subfamily of GPCRs. These examples also raise the interesting question of whether the presence of such a site is a coincidence or the result of evolutionary selection through which additional endogenous control mechanisms for receptor activity may function.…”

“…In recent years, a number of allosteric interactions with family A GPCRs have been identified, such as those at adenosine [3], muscarinic [4], serotonergic [5], cannabinoid [6] and P2 purinergic receptors [7]. Common to all these interactions is the binding of synthetic ligands and the absence of endogenous ligands acting at allosteric sites.…”

CysLT1 leukotriene receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors

“…The quantity of inositol phosphates was measured by a modification of the method of Gao et al [35]. Agonists and antagonists were dissolved as stock solutions in PBS buffer (pH 7.4) and stored at −20 °C.…”

“…P2Y 1 receptor binding experiments were performed as previously described [35,37] nM at the P2Y 1 receptor. Binding reactions were terminated by filtration through Whatman GF/B glass-fiber filters under reduced pressure with a MT-24 cell harvester (Brandel, Gaithersburg, MD, USA), and radioactivity was determined with a 1414 liquid scintillation counter (Wallac, Win Spectral, Perkin-Elmer Life Sciences, Downers Grove, IL, USA).…”