title: Inhibition of Ecto-Apyrase and Ecto-ATPase by Pyridoxal Phosphate-Related Compounds.
abstract: Studies of nucleotide receptors (P2-receptors) in cells and tissues are complicated by cleavage of phosphate groups from nucleotide agonist ligands by ecto-nucleotidases. Some P2 receptor antagonists may also inhibit ecto-nucleotidases, making these studies even more complex. In order to systematically approach this problem, we investigated structure-activity relationships of pyridoxal-5'-phosphate-6-azophenyl-2,4-disulfonate (PPADS) and 14 derivatives, many potent as antagonists at P2 receptors, as inhibitors of ecto-nucleotidases. The compounds were tested for their ability to inhibit enzymatic nucleotide breakdown by CHO cells stably transfected with plasmids containing the cDNA for rat ecto-apyrase (NTPDase1) and rat ecto-ATPase (NTPDase2). All inhibitors were tested at a concentration of 100 µM and ATP hydrolysis was quantified by HPLC. Maximal inhibition obtained for ecto-apyrase and ecto-ATPase was 60% and 35%, respectively. Most PPADS analogs were better inhibitors of ecto-apyrase than of ecto-ATPase. Compound 8, a phosphate derivative, inhibited ecto-apyrase with no inhibition evident at ecto-ATPase. Comparison of pharmacological data of PPADS analogs at P2 receptors as previously determined showed that four PPADS analogs exhibited selectivity for P2X nucleotide receptors. None of these compounds inhibited ecto-ATPase, while two inhibited the ecto-apyrase. Compound 14, a bisphosphate derivative, inhibited ecto-ATPase without inhibition of ecto-apyrase. This compound only weakly antagonized P2X1receptors and was inactive at P2X2and P2Y1receptors, thus bearing some selectivity for ecto-ATPase. Compound 7, a 5-methylphosphonate derivative, a potent antagonist of P2X1receptors, was inactive at ecto-apyrase and only weakly inhibitory at ecto-ATPase. Thus, PPADS modifications that enhance selectivity among ecto-nucleotidases and P2 receptors have been identified.
authors.affiliation: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Carsten
authors.initials: C
authors.lastname: Hoffmann
authors.affiliation: AK Neurochemie, Biozentrum der J.W. Goethe-Universität, Frankfurt am Main, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Heine
authors.affiliation: AK Neurochemie, Biozentrum der J.W. Goethe-Universität, Frankfurt am Main, Germany.
authors.forename: Gabi
authors.initials: G
authors.lastname: Pradel
authors.affiliation: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Yong-Chul
authors.initials: YC
authors.lastname: Kim
authors.affiliation: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Jacobson
authors.affiliation: AK Neurochemie, Biozentrum der J.W. Goethe-Universität, Frankfurt am Main, Germany.
authors.forename: Herbert
authors.initials: H
authors.lastname: Zimmermann
keywords: CD39; P2 receptors; PPADS; ecto-ATPase; ecto-apyrase; enzyme-inhibition