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

Abstract

Substituted 3Ј-thiourea derivatives of ␤-thymidine (dThd) and 5Ј-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 3Ј-thiourea derivatives of ␤-dThd proved highly inhibitory to and selective for TK-2 (IC 50 value, 0.15-3.1 M). The 3Ј-C-branched p-methylphenyl (compound 1) and 3-CF 3 -4-Cl-phenyl (compound 7) thiourea derivatives of ␤-dThd showed competitive inhibition of TK-2 when dThd was used as the variable substrate (K i values, 0.40 and 0.05 M, respectively), but uncompetitive inhibition in the presence of variable concentrations of ATP (K i values, 15 and 2.0 M, 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.

3′-[4-Aryl-(1,2,3-triazol-1-yl)]-3′-deoxythymidine Analogues as Potent and Selective Inhibitors of Human Mitochondrial Thymidine Kinase

“…It is also interesting to note that 14a and 14b, both containing a BVDU moiety in place of the thymine, are only marginally more potent than 8c and 8f, respectively, an observation that is in line with the facts that (i) the Km value of BVDU when used as a substrate for Dm-DNK is only ~4-fold lower than that of AZT, 25 (ii) the nucleobase-binding site in Dm-DNK and human TK2 are highly comparable, and (iii) AZT and BVDU binding to Dm-DNK show similar characteristics, as inferred from comparison of PDB structures 2JJ8 and 2VQS. Finally, our finding that a 5'-O-trityl substituent renders the inhibitors in the present series inactive is also consistent with the proposed binding mode in so far as the 5'-hydroxyl is used, as described above and reported previously for thiourea-derived inhibitors, 11 to anchor the inhibitor in the substrate binding site through hydrogen bonding interactions with the catalytically important Arg134 and Glu81.…”

Recent Advances in Thymidine Kinase 2 (TK2) Inhibitors and New Perspectives for Potential Applications

“…Since our last report in 2008 [29], the research on TK2 inhibitors has been headed mainly by Van Calenbergh and collaborators who reported in 2009 a series of 3´-substituted thymidine derivatives as potent and selective inhibitors of human TK2 (Fig. 6) [40]. Originally designed and evaluated as inhibitors of Mycobacterium tuberculosis thymidylate kinase (TMPKmt) [41], the 3´-thiourea-dThd derivatives 24-31( Fig.…”

“…Thus it may be concluded that the acyclic nucleoside inhibitors occupy the thymidine binding site of TK2 only when the cosubstrate ATP is already bound to the enzyme. As regards the 3´-substituted nucleosides, the kinetic properties of compounds 30 [40] and 38 [42] have also been investigated against TK2. When tested against variable concentrations of dThd, these inhibitors inhibited TK2 in a purely competitive fashion with considerable low K i values of 0.054 and 0.012 μM, respectively.…”

Synthesis and Evaluation of α-Thymidine Analogues as Novel Antimalarials

“…21 Moreover, 5′-thiourea α-thymidine derivatives were completely inactive against human thymidine kinase 1 and only showed weak inhibition of human mitochondrial nucleoside kinase TK-2. 22,23 …”