Inhibition of ecto-apyrase and ecto-ATPase by pyridoxal phosphate-related compounds

Studies of nucleotide receptors (P2-receptors) in cells and tissues are com plicated by cleavage of phosphate groups from nucleotide agonist ligands by ecto-nucleotidases. Some P2 receptor antagonists may also inhibit ecto-nuc leotidases, making these studies even more complex. In order to systematica lly approach this problem, we investigated structure-activity relationships of pyridoxal-5'-phosphate-6-azophenyl-2,4-disulfonate (PPADS) and 14 deriv atives, many potent as antagonists at P2 receptors, as inhibitors of ecto-n ucleotidases. The compounds were tested for their ability to inhibit enzyma tic nucleotide breakdown by CHO cells stably transfected with plasmids cont aining the cDNA for rat ecto-apyrase (NTPDase1) and rat ecto-ATPase (NTPDas e2). All inhibitors were tested at a concentration of 100 muM and ATP hydro lysis was quantified by HPLC. Maximal inhibition obtained for ecto-apyrase and ecto-ATPase was 60% and 35%, respectively. Most PPADS analogs were bett er inhibitors of ecto-apyrase than of ecto-ATPase. Compound 8, a phosphate derivative, inhibited ecto-apyrase with no inhibition evident at ecto-ATPas e. Comparison of pharmacological data of PPADS analogs at P2 receptors as p reviously determined showed that four PPADS analogs exhibited selectivity f or P2X nucleotide receptors. None of these compounds inhibited ecto-ATPase, while two inhibited the ecto-apyrase. Compound 14, a bisphosphate derivati ve, inhibited ecto-ATPase without inhibition of ecto-apyrase. This compound only weakly antagonized P2X(1) receptors and was inactive at P2X(2) and P2 Y(1) receptors, thus bearing some selectivity for ecto-ATPase. Compound 7, a 5-methylphosphonate derivative, a potent antagonist of P2X(1) receptors, was inactive at ecto-apyrase and only weakly inhibitory at ecto-ATPase. Thu s, PPADS modifications that enhance selectivity among ecto-nucleotidases an d P2 receptors have been identified. Drug Dev. Res. 51:153-158, 2000. Publi shed 2001 Wiley-Liss, Inc.