Abstract
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.
Footnotes
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This work was supported by the Bijzonder Onderzoeksfonds (Universiteit Gent); the Fonds voor Wetenschappelijk Onderzoek (Vlaanderen); the Geconcerteerde Onderzoeksacties Vlaanderen [Grant GOA-05/19]; the Spanish Comisión Interministerial de Ciencia y Tecnologica [Grant SAF2006-12713-C02-0]; Comunidad de Madrid [Grants BIPEDD-CM, S-BIO/0214/2006]; and the Swedish Medical Research Council.
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ABBREVIATIONS: TK, thymidine kinase; DMSO, dimethyl sulfoxide; HSV, herpes simplex virus; VZV, varicella zoster virus; HPLC, high-performance liquid chromatography; TMPKmt, Mycobacterium tuberculosis-encoded thymidylate kinase; dThd, derivative of β-thymidine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; PBS, phosphate-buffered saline; PDB, Protein Data Bank; MD, molecular dynamics.
- Received November 28, 2008.
- Accepted February 20, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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