title: Nucleotide and nucleoside analogues as inhibitors of cytosolic 5'-nucleotidase I from heart.
abstract: Substrate and product specificity studies were used to develop inhibitors of the cytosolic 5'-nucleotidase I (c-N-I) from myocardium. As measured by Vmax/Km, c-N-I preferred pyrimidine 2'-deoxyribonucleotides as substrates with thymidine monophosphate (TMP) being the most efficient. In product inhibition studies, thymidine inhibited noncompetitively and inorganic phosphate inhibited competitively, consistent with an ordered release of nucleoside prior to phosphate. Mirroring nucleotide substrate specificities, pyrimidine nucleosides were more potent product inhibitors than purine nucleosides. Thus, pyrimidine nucleotide and nucleoside analogues were developed as inhibitors. Phosphonate analogues of TMP were synthesized by a novel method. The most potent was the 5'-phosphonate of 3'-deoxythymidine (ddT) (apparent Ki value of 63 nM). In addition, pyrimidine nucleoside analogues were inhibitors with 5-ethynyl-2',3'-dideoxyuridine being the most potent (apparent Ki value of 3.7 microM). The most potent nucleotide and nucleoside inhibitor were both greater than 1000-fold more potent inhibiting c-N-I than the cytosolic 5'-nucleotidase II. The nucleoside analogue was also greater than 1000-fold more potent against c-N-I than the membrane ecto-5'-nucleotidase (e-N). Because the phosphonate analogues measurably inhibited e-N (apparent Ki values of 6-12 microM), the selectivity of the phosphonates for c-N-I versus e-N was less (40-200-fold). Because of the high selectivity for c-N-I versus both of the other 5'-nucleotidases, the nucleoside inhibitors of c-N-I may be useful biochemical tools in discerning the role that c-N-I plays in generating adenosine within myocardium.
authors.affiliation: Division of Biochemistry, Glaxo Wellcome, Research Triangle Park, North Carolina 27709, USA. epg34374@glaxo.com
authors.forename: E P
authors.initials: EP
authors.lastname: Garvey
authors.forename: G T
authors.initials: GT
authors.lastname: Lowen
authors.forename: M R
authors.initials: MR
authors.lastname: Almond
mesh_terms: D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors*; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D003600:Cytosol / Q000201:enzymology*; D003853:Deoxyribonucleosides / Q000494:pharmacology*; D003854:Deoxyribonucleotides / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D009206:Myocardium / Q000201:enzymology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011817:Rabbits; D013379:Substrate Specificity; D013936:Thymidine / Q000494:pharmacology
chemical_list: D003853:Deoxyribonucleosides; D003854:Deoxyribonucleotides; D004791:Enzyme Inhibitors; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D015720:5'-Nucleotidase; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D013936:Thymidine