TY - JOUR T1 - Human Mitochondrial Thymidine Kinase Is Selectively Inhibited by 3′-Thiourea Derivatives of β-Thymidine: Identification of Residues Crucial for Both Inhibition and Catalytic Activity JF - Molecular Pharmacology JO - Mol Pharmacol SP - 1127 LP - 1136 DO - 10.1124/mol.108.053785 VL - 75 IS - 5 AU - Jan Balzarini AU - Ineke Van Daele AU - Ana Negri AU - Nicola Solaroli AU - Anna Karlsson AU - Sandra Liekens AU - Federico Gago AU - Serge Van Calenbergh Y1 - 2009/05/01 UR - http://molpharm.aspetjournals.org/content/75/5/1127.abstract N2 - Substituted 3′-thiourea derivatives of β-thymidine (dThd) and 5′-thiourea derivatives of α-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 (Ki values, 0.40 and 0.05 μM, respectively), but uncompetitive inhibition in the presence of variable concentrations of ATP (Ki 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 γ-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 Ki/Km 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. The American Society for Pharmacology and Experimental Therapeutics ER -