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Human Mitochondrial Thymidine Kinase Is Selectively Inhibited by 3'-Thiourea Derivatives of β-Thymidine: Identification of Residues Crucial for Both Inhibition and Catalytic Activity

Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium (J.B., S.L.); University of Ghent, Ghent, Belgium (I.V.D., S.V.C.); Departamento de Farmacologia, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain (A.N., F.G.); and the Karolinska Institute, Huddinge University Hospital, Huddinge, Sweden (N.S., A.K.)

Substituted 3'-thiourea derivatives of β-thymidine (dThd) and 5'-thiourea derivatives of -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 β-dThd proved highly inhibitory to and selective for TK-2 (IC₅₀ value, 0.15-3.1 µM). The 3'-C-branched p-methylphenyl (compound 1) and 3-CF₃-4-Cl-phenyl (compound 7) thiourea derivatives of β-dThd showed competitive inhibition of TK-2 when dThd was used as the variable substrate (K_i values, 0.40 and 0.05 µM, respectively), but uncompetitive inhibition in the presence of variable concentrations of ATP (K_i values, 15 and 2.0 µM, 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 -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.

Received for publication November 28, 2008.

Accepted for publication February 20, 2009.

Address correspondence to: Dr. Jan Balzarini, Rega Institute for Medical Research, K. U. Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail: jan.balzarini{at}rega.kuleuven.be