title: Human mitochondrial thymidine kinase is selectively inhibited by 3'-thiourea derivatives of beta-thymidine: identification of residues crucial for both inhibition and catalytic activity.
abstract: Substituted 3'-thiourea derivatives of beta-thymidine (dThd) and 5'-thiourea derivatives of alpha-dThd have been evaluated for their inhibitory activity against recombinant human cytosolic dThd kinase-1 (TK-1), human mitochondrial TK-2, herpes simplex virus type 1 (HSV-1) TK, and varicella-zoster virus TK. Several substituted 3'-thiourea derivatives of beta-dThd proved highly inhibitory to and selective for TK-2 (IC(50) value, 0.15-3.1 microM). The 3'-C-branched p-methylphenyl (compound 1) and 3-CF(3)-4-Cl-phenyl (compound 7) thiourea derivatives of beta-dThd showed competitive inhibition of TK-2 when dThd was used as the variable substrate (K(i) values, 0.40 and 0.05 microM, respectively), but uncompetitive inhibition in the presence of variable concentrations of ATP (K(i) values, 15 and 2.0 microM, respectively). These kinetic properties of compounds 1 and 7 against TK-2 could be accounted for by molecular modeling showing that two hydrogen bonds can be formed between the thiourea nitrogens of compound 7 and the oxygens of the gamma-phosphate of ATP. The importance of several active-site residues was assessed by site-directed mutagenesis experiments on TK-2 and the related HSV-1 TK. The low K(i)/K(m) ratios for compounds 1 and 7 (0.38 and 0.039 against dThd, and 0.75 and 0.12 against ATP, respectively) indicate that these compounds are among the most potent inhibitors of TK-2 described so far. In addition, a striking close correlation was found between the inhibitory activities of the test compounds against TK-2 and Mycobacterium tuberculosis thymidylate kinase that is strongly indicative of close structural and/or functional similarities between both enzymes in relation to their mode of interaction with these nucleoside analog inhibitors.
authors.affiliation: Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium. jan.balzarini@rega.kuleuven.be
authors.forename: Jan
authors.initials: J
authors.lastname: Balzarini
authors.forename: Ineke
authors.initials: I
authors.lastname: Van Daele
authors.forename: Ana
authors.initials: A
authors.lastname: Negri
authors.forename: Nicola
authors.initials: N
authors.lastname: Solaroli
authors.forename: Anna
authors.initials: A
authors.lastname: Karlsson
authors.forename: Sandra
authors.initials: S
authors.lastname: Liekens
authors.forename: Federico
authors.initials: F
authors.lastname: Gago
authors.forename: Serge
authors.initials: S
authors.lastname: Van Calenbergh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002478:Cells, Cultured; D003198:Computer Simulation; D004355:Drug Stability; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000378:metabolism; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D013937:Thymidine Kinase / Q000037:antagonists & inhibitors*
chemical_list: D004791:Enzyme Inhibitors; C105339:thymidine kinase 2; D013937:Thymidine Kinase; D013890:Thiourea