title: 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 0.14microM. 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, [3H] MRS2279. It was found that PIT (0.01-10microM) did not inhibit [3H] MRS2279 binding to the human P2Y(1) receptor. PIT (10microM) had no effect on the competition for [3H] 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.
authors.affiliation: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0810, USA.
authors.forename: Zhan-Guo
authors.initials: ZG
authors.lastname: Gao
authors.forename: Liaman
authors.initials: L
authors.lastname: Mamedova
authors.forename: Susanna
authors.initials: S
authors.lastname: Tchilibon
authors.forename: Ariel S
authors.initials: AS
authors.lastname: Gross
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Jacobson
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D001254:Astrocytoma / Q000473:pathology; D001665:Binding Sites; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007510:Isatin / Q000031:analogs & derivatives / Q000494:pharmacology*; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*; D058919:Purinergic P2 Receptor Antagonists; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D058472:Receptors, Purinergic P2Y1; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D014316:Tritium; D014407:Tumor Cells, Cultured
chemical_list: C465253:2-chloro-N6-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate; D007295:Inositol Phosphates; D009711:Nucleotides; C546382:P2RY1 protein, human; D058919:Purinergic P2 Receptor Antagonists; D043562:Receptors, G-Protein-Coupled; D058472:Receptors, Purinergic P2Y1; D014316:Tritium; C009128:2,2'-pyridylisatogen tosylate; D000244:Adenosine Diphosphate; D007510:Isatin; D000255:Adenosine Triphosphate