N-Substituted Thymine Derivatives as Mitochondrial Thymidine Kinase (TK-2) Inhibitors

Abstract

Novel N1-substituted thymine derivatives related to 1-[(Z)-4-(triphenylmethoxy)-2-butenyl]thymine have been synthesized and evaluated against thymidine kinase-2 (TK-2) and related nucleoside kinases [i.e., Drosophila melanogaster deoxynucleoside kinase (Dm-dNK) and herpes simplex virus type 1 thymidine kinase (HSV-1 TK)]. The thymine base has been tethered to a distal triphenylmethoxy moiety through a polymethylene chain (n = 3-8) or through a (2-ethoxy)ethyl spacer. Moreover, substitutions at position 4 of one of the phenyl rings of the triphenylmethoxy moiety have been performed. Compounds with a hexamethylene spacer (18, 26b, 31) displayed the highest inhibitory values against TK-2 (IC50 = 0.3-0.5 microM). Compound 26b competitively inhibited TK-2 with respect to thymidine and uncompetitively with respect to ATP. A rationale for the biological data was provided by docking some representative inhibitors into a homology-based model of human TK-2. Moreover, two of the most potent TK-2 inhibitors (18 and 26b) that also inhibit HSV-1 TK were able to reverse the cytostatic activity of 1-(beta-D-arabinofuranosyl)thymine (Ara-T) and ganciclovir in HSV-1 TK-expressing OST-TK-/HSV-1 TK+ cell cultures.

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

“…This finding could account for the observed unusual kinetic behavior of this type of compounds against the enzyme. It is noteworthy that the substituted thiourea dThd analogs kinetically behave like the acyclic 5Ј-trityl nucleoside analogs earlier described as TK-2 inhibitors Hernandez et al, 2006). Indeed, TK-2 inhibition by such inhibitors [i.e., (E)-5-(2-bromovinyl)-1-[(Z)-4-triphenylmethoxy-2-butenyl]uracil and 1-[(Z)-6-pyridyldiphenylmethoxy)hexyl]thymine] is also competitive with respect to dThd but uncompetitive with respect to ATP.…”

“…Indeed, TK-2 inhibition by such inhibitors [i.e., (E)-5-(2-bromovinyl)-1-[(Z)-4-triphenylmethoxy-2-butenyl]uracil and 1-[(Z)-6-pyridyldiphenylmethoxy)hexyl]thymine] is also competitive with respect to dThd but uncompetitive with respect to ATP. A rationale for these kinetic data was provided by docking some of the representative inhibitors into a homology-based model of TK-2 (Hernandez et al, 2006). Our current molecular model can also account for the finding that the 3Ј-thiourea ␤-dThd analogs were consistently found to be markedly more inhibitory to TK-2 than the 5Ј-thiourea ␣-dThd congeners.…”

“…The threedimensional structure of human TK-2 has not been solved experimentally but a reliable molecular model has been built using a profile-based -fold recognition method (Hernandez et al, 2006). A Mg 2ϩ -bound ATP molecule was added to this TK-2 structure, and the complex was further refined by remodeling the loop that closes over the reaction substrates using as a template the X-ray crystal structure of human thymidylate kinase cocrystallized with the inhibitor P 1 -(5Ј-adenosyl)P 5 -(5Ј-(3Ј-azido-3Ј-deoxythymidyl))pentaphosphate (PDB code 1E9A) (Ostermann et al, 2000).…”

“…Further synthetic efforts subsequently led to the identification of and detailed studies on tritylated acyclic pyrimidine nucleoside analogs as TK-2 inhibitors . These compounds act as non-nucleosidic inhibitors and are endowed with a pronounced inhibitory activity against the enzyme (50% inhibitory concentrations as low as 0.3 M) (Herná ndez et al, 2002Balzarini et al, 2003;Priego et al, 2004;Hernandez et al, 2006;Pérez-Pérez et al, 2008). In this study, we report on the discovery of a series of substituted 3Ј-or 5Ј-thiourea derivatives of ␤-and ␣-dThd, respectively, as TK-2 inhibitors and the identification of several derivatives that were highly potent in their anti-TK-2 action.…”

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

“…10 11 Several substituted 3'-thiourea derivatives of β-dThd proved highly inhibitory to and selective for human mitochondrial TK-2 compared to the other enzymes. Compound 6, which emerged as the most potent analogue of this series, inhibited TK-2 at concentrations 2,100-fold lower than those required to inhibit cytosolic TK-1 (IC 50 : TK-1: 316 µM; TK-2: 0.15 µM).…”