Inhibition Of Thymidine Kinase Research Articles

Role of deoxycytidine kinase (dCK) activity in gemcitabine's radioenhancement in mice and human cell lines in vitro

Background: Gemcitabine (dFdC, 2′,2′-difluorodeoxycytidine) is a deoxycytidine nucleoside analog which has a marked effect on several enzymes involved in DNA synthesis and repair. Gemcitabine has been tested as a radiosensitizer in various biological models, and radiation dose modification factors (DMF) have been reported in the range between 1.1 and 2.4. Gemcitabine is a prodrug that requires intracellular activation by phosphorylation into its active triphosphate dFdCTP form. Deoxycytidine kinase (dCK) is the enzyme involved in the first phosphorylation cascade, and several observations have suggested that dCK was a limiting factor for the cytotoxic activity of gemcitabine. Objective: In the present article, we investigated the relationship between dCK activity and gemcitabine's radiosensitization in four mice and two human cell lines. Materials and methods: Four mice and two human tumor cell lines were investigated. Radiosensitization was assessed on confluent cell incubated with 5 μM gemcitabine for 3 h prior to a single radiation dose. Enzymatic activity was assessed using deoxycytidine as substrate with (specific activity) or without (total activity) inhibition of thymidine kinase 2 activity. dCK protein level was assessed by immunoblotting using a rabbit anti-human dCK antibody. mRNA expression was assessed with Northern blot using β-actin as internal control. Results: Gemcitabine's radiosensitization was heterogeneous with DMF ranging from 0.8 to 1.5. A good correlation was observed between the specific dCK activity and the protein level or the mRNA expression indicating that in our cell systems no post-transcriptional or post-translational activation occurred. An excellent correlation ( r=0.99) was observed between the specific enzymatic activity and gemcitabine's radiosensitization. Cell lines that expressed a high enzymatic activity were the more radiosensitized by gemcitabine. This correlation holds when radiosensitization was plotted against the dCK mRNA expression and protein level. Conclusions: The present study has suggested the role of dCK activity in gemcitabine's radioenhancement in human and mice cell lines. The study suggests that determination of the enzymatic activity prior to a concurrent gemcitabine and radiotherapy treatment might represent a good predictive assay for tumor response. Such concept should deserve further testing in pre-clinical and clinical settings.

Database Searching for Thymidine and Thymidylate Kinase Inhibitors Using Three-dimensional Structure-based Methods

Structure-based drug design methods were used to search for novel inhibitors of herpes simplex virus type 1 (HSV-1) thymidine kinase and Mycobacterium tuberculosis thymidylate kinase. The method involved the use of crystal structure complexes to guide database searching for potential inhibitors. A number of weak inhibitors of HSV-2 were identified, one of which was found to inhibit HSV-1 TK and HSV-1 TK-deficient viral strains. Each compound tested against M. tuberculosis thymidylate kinase was found to have some activity. The best of these compounds was only 4.6-fold less potent than 3′-azido-3′-deoxythymidine-5′-monophosphate (AZTMP). This study demonstrates the utility of structure-based drug design methods in the search for novel enzyme inhibitors.

ChemInform Abstract: Nucleoside Analogues as Highly Potent and Selective Inhibitors of Herpes Simplex Virus Thymidine Kinase.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform Abstract: Design, Synthesis and Enzymatic Activity of Highly Selective Human Mitochondrial Thymidine Kinase Inhibitors.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Nucleoside Analogues as Highly Potent and Selective Inhibitors of Herpes Simplex Virus Thymidine Kinase

A series of carboxamide derivatives of 5′-amino-2′,5′-dideoxy-5-ethyluridine has been prepared as inhibitors of HSV-TK (herpes simplex virus thymidine kinase). The most potent compounds were derived from xanthene, thioxanthene and dihydroanthracene carboxylic acids. The lead compounds show subnanomolar IC 50 values against HSV TKs.

Design, Synthesis and Enzymatic Activity of Highly Selective Human Mitochondrial Thymidine Kinase Inhibitors

Highly selective arabinofuranosyl nucleosides, which inhibit the mitochondrial thymidine kinase (TK-2) without affecting the closely related herpes simplex virus type 1 thymidine kinase (HSV-1 TK), varicella-zoster virus thymidine kinase (VZV-TK), cytosolic thymidine kinase (TK-1) or the multifunctional Drosophila melanogaster deoxyribonucleoside kinase ( Dm-dNK), have been obtained. SAR studies indicate a close relation between the length of the substituent at the 2′ position of the arabinofuranosyl moiety and the inhibitory activity.

2′- O-Acyl/alkyl-substituted arabinosyl nucleosides as inhibitors of human mitochondrial thymidine kinase

Introduction of a bulky lipophilic acyl entity at the 2′-OH position of both 1-β- d-arabinofuranosylthymine (araT) and ( E)-5-(2-bromovinyl)-1-β- d-arabinofuranosyluracil (BVaraU), consistently resulted in a marked (∼10-fold) increase in the inhibitory activity of these new arabinosyl nucleoside analogues for the mitochondrial thymidine kinase (TK-2)-catalysed conversion of 2 μM [ methyl- 3H]dThd to [ methyl- 3H]dTMP. The most potent derivatives were inhibitory to [ methyl- 3H]dThd phosphorylation by TK-2 within the lower micromolar concentration range. Substitution of the arabinosyl nucleoside derivatives with the acyl groups also dramatically increased the selectivity of these compounds. The inhibitory activity of araT and BVaraU to dThd phosphorylation by other related nucleoside kinases, including herpes simplex virus type 1 TK, varicella–zoster virus TK, and cytosolic TK-1, was completely annihilated upon 2′- O-acyl substitution ( ic 50 ≥ 1000 μM). Kinetic analysis revealed purely competitive inhibition of 2′- O-acyl-BVaraU against TK-2-catalysed thymidine phosphorylation ( K i / K m : 2.3). However, 2′- O-acyl-BVaraU was extremely poorly converted to the corresponding arabinosyl nucleoside 5′-monophosphate by TK-2 as revealed by [γ- 32P]phosphate transfer studies from [γ- 32P]ATP. Thus, the 2′- O-acyl derivatives of BVaraU did not behave as substrates, but rather as potent and highly selective inhibitors of TK-2. This is the first report on such a highly selective arabinosyl nucleoside inhibitor of mitochondrial TK-2, and opens perspectives for the rational design of selective mitochondrial TK-2 inhibitors.

Anti-Epstein-Barr virus (EBV) activity of beta-L-5-iododioxolane uracil is dependent on EBV thymidine kinase.

beta-L-5-Iododioxolane uracil was shown to have potent anti-Epstein-Barr virus (EBV) activity (50% effective concentration = 0.03 microM) with low cytotoxicity (50% cytotoxic concentration = 1,000 microM). It exerts its antiviral activity by suppressing replicative EBV DNA and viral protein synthesis. This compound is phosphorylated in cells where the EBV is replicating but not in cells where the EBV is latent. EBV-specific thymidine kinase could phosphorylate beta-L-5-iododioxolane uracil to the monophosphate metabolite. The K(m) of beta-L-5-iododioxolane uracil with EBV thymidine kinase was estimated to be 5.5 microM, which is similar to that obtained with thymidine but about fivefold higher than that obtained with 2' fluoro-5-methyl-beta-L-arabinofuranosyl uracil, the first L-nucleoside analogue discovered to have anti-EBV activity. The relative V(max) is seven times higher than that of thymidine. The anti-EBV activity of beta-L-5-iododioxolane uracil and its intracellular phosphorylation could be inhibited by 5'-ethynylthymidine, a potent EBV thymidine kinase inhibitor. The present study suggests that beta-L-5-iododioxolane uracil exerts its action after phosphorylation; therefore, EBV thymidine kinase is critical for the antiviral action of this drug.

Novel ribofuranosylnucleoside lead compounds for potent and selective inhibitors of mitochondrial thymidine kinase-2.

The ribonucleoside analogues (E)-5-(2-bromovinyl)uridine (5-BV-Urd) and 3'-spiro-(4'-amino-1',2'-oxathiole-2',2'-dioxide)-5-methyluridine (3'-AOD-5-MeUrd) emerged as potent and selective competitive inhibitors of mitochondrial thymidine kinase (TK)-2 with respect to thymidine (K(i)/K(m) values of 9.0 and 1.2 respectively). Cytosolic TK-1 did not show measurable affinity for these compounds. [(32)P]Phosphate transfer studies from [gamma-(32)P]ATP to 5-BV-Urd and 3'-AOD-5-MeUrd revealed extremely poor substrate activity but potent inhibitory potential of the compounds. It was concluded that the ribonucleosides 5-BV-Urd and 3'-AOD-5-MeUrd represent two new lead compounds for potent and selective inhibitors of mitochondrial TK-2.

Novel ribofuranosylnucleoside lead compounds for potent and selective inhibitors of mitochondrial thymidine kinase-2

The ribonucleoside analogues (E)-5-(2-bromovinyl)uridine (5-BV-Urd) and 3´-spiro-(4´-amino-1´,2´-oxathiole-2´,2´-dioxide)-5-methyluridine (3´-AOD-5-MeUrd) emerged as potent and selective competitive inhibitors of mitochondrial thymidine kinase (TK)-2 with respect to thymidine (Ki/Km values of 9.0 and 1.2 respectively). Cytosolic TK-1 did not show measurable affinity for these compounds. [32P]Phosphate transfer studies from [γ-32P]ATP to 5-BV-Urd and 3´-AOD-5-MeUrd revealed extremely poor substrate activity but potent inhibitory potential of the compounds. It was concluded that the ribonucleosides 5-BV-Urd and 3´-AOD-5-MeUrd represent two new lead compounds for potent and selective inhibitors of mitochondrial TK-2.

Identification and proposed mechanism of action of thymidine kinase inhibition associated with cellular exposure to camptothecin analogs.

To investigate the effects of several camptothecin analogs including 9-aminocamptothecin (9-AC), SN38, topotecan, and irinotecan (CPT-11) on the enzymes involved in the pyrimidine salvage pathway including thymidylate synthase (TS). A COMPARE analysis using the NCI 60 cell line drug-screening panel suggested that there were similarities in the mechanisms of action of camptothecin analogs and TS inhibitors. TS enzymatic activity was measured by both an in situ tritium release assay using both the H630 colon cancer cell line and the CEM human leukemia cell line, and by a radiolabelled in vitro assay using partially purified human TS as the enzyme source. Thymidine kinase (TK) activity was measure by a radiolabelled in vitro assay using H630 colon cancer cell lysates as the enzyme source. In vitro studies indicated that none of the analogs directly inhibited TS enzymatic activity; however, utilization of a coupled TS/TK in situ assay with radiolabelled deoxyuridine as the precursor revealed marked inhibition by the camptothecin analogs. 9-AC, SN38, and topotecan yielded IC50 values of 1.3, 1.6, and 1.1 microM respectively. In contrast, there was no inhibition detected when deoxycytidine was used as the radiolabelled nucleoside precursor, suggesting that the drug effect was through inhibition of TK, rather than inhibition of TS. In vitro studies using cell lysates from H630 human colon cancer cells to measure TK activity showed no decrease in TK activity after 9-AC treatment. In addition, no changes were detected in the dATP and dTTP nucleotide pools. Permeabilizing the cell membranes with saponin did not abolish the inhibitory effect of the camptothecins indicating that altered cell transport was not responsible for the decreased activity in the in situ assay in intact cells. These studies suggest that there is inhibition of TK in intact cells associated with topoisomerase I inhibition by camptothecin analogs, and the inhibition of TK is the result of an indirect effect not related to feedback inhibition by changes in dTTP pools.

QSAR and molecular graphics analysis of N 2-phenylguanines as inhibitors of herpes simplex virus thymidine kinases

A quantitative structure-activity relationship study of N 2-(substituted)-phenylguanines (PHG) as inhibitors of herpes simplex virus thymidine kinase (HSV TK) was performed. The activity of a set of PHG derivatives were analyzed against the thymidine kinase of herpes simplex virus types 1 (HSV1 TK) and 2 (HSV2 TK). Classic and calculated physicochemical parameters were included in the analysis. The results showed that there is an important difference in the activity of the meta substituted PHG derivatives against HSV1 TK and HSV2 TK. The activity of the meta derivatives against HSV2 TK is influenced by a steric effect, which is not observed against HSV1 TK. The superposition of the three-dimensional structures of the active sites of HSV1 TK (crystal structure) and HSV2 TK (homology model) revealed that the amino acid Ile97 is located near the meta position in the HSV1 TK active site, whereas the amino acid Leu97 is located near the meta position in the HSV2 TK active site. This single difference in the active sites of both enzymes can explain the source of the steric effect and serves as an indication that our previously proposed binding mode for the PHG derivatives is plausible. However, another observed mutation in the active site region, Ala168 by Ser168, suggests that an alternative binding mode, similar to that of ganciclovir, could be possible.

Conformational analysis and the active conformation of N2-phenylguanine derivatives

A conformational analysis of four N2-phenyl-(substituted)-guanine (PHG) derivatives, which are herpes simplex virus thymidine kinase inhibitors (HSV TK), was performed mainly with the semiempirical AM1 method. Nonempirical (ab initio) HF and MP2 calculations were employed to refine some of the results obtained with the semiempirical method. The two dihedral angles that connect the phenyl and the guanine rings were varied for systematic conformational search. Four stable conformations were found: C1 (θ1=184°; θ2=58°); C2(θ1=174°; θ2=127°); C3(θ1=186°; θ2=233°) and C4(θ1=176°; θ2=303°), in which θ1=N1−C2−N11−C1′, and θ2=C2−N11−C1′−C2′. The conformation C3 was found to be equivalent to the bioactive conformation proposed in a previous paper.

Trichomonas vaginalis thymidine kinase: purification, characterization and search for inhibitors

We report that a thymidine kinase (TK) activity is present in Trichomonas vaginalis and can be separated from the deoxyribonucleoside phosphotransferase. T. vaginalis TK, purified 11200-fold to apparent homogeneity, has a molecular mass of 31500 Da. It phosphorylates not only thymidine (Km 0.18 microM) but also deoxycytidine (Km 0.88 microM) and deoxyuridine (Km 0.14 microM). In contrast with T. vaginalis deoxyribonucleoside phosphotransferase, the TK activity is strongly inhibited by novel deoxyuridine analogues such as 5-methyl-4'-thio-2'-deoxyuridine (MTdU) (Ki 20 nM) and 5-iodo-4'-thio-2'-deoxyuridine (ITdU) (Ki 24 nM). MTdU and ITdU are phosphorylated by T. vaginalis TK in vitro. In vivo they inhibit [3H]thymidine incorporation in T. vaginalis cultured cells and T. vaginalis growth (IC50 7.5 and 24 microM respectively; minimal lethal dose 100 microM). Thus the TK inhibitors described here demonstrate the key role of T. vaginalis TK for protozoal growth and viability and indicate TK as a new target for the design of antitrichomonal drugs.

Exploring the active site of herpes simplex virus type-1 thymidine kinase by X-ray crystallography of complexes with aciclovir and other ligands.

Antiherpes therapies are principally targeted at viral thymidine kinases and utilize nucleoside analogs, the triphosphates of which are inhibitors of viral DNA polymerase or result in toxic effects when incorporated into DNA. The most frequently used drug, aciclovir (Zovirax), is a relatively poor substrate for thymidine kinase and high-resolution structural information on drugs and other molecules binding to the target is therefore important for the design of novel and more potent chemotherapy, both in antiherpes treatment and in gene therapy systems where thymidine kinase is expressed. Here, we report for the first time the binary complexes of HSV-1 thymidine kinase (TK) with the drug molecules aciclovir and penciclovir, determined by X-ray crystallography at 2.37 A resolution. Moreover, from new data at 2.14 A resolution, the refined structure of the complex of TK with its substrate deoxythymidine (R = 0.209 for 96% of all data) now reveals much detail concerning substrate and solvent interactions with the enzyme. Structures of the complexes of TK with four halogen-containing substrate analogs have also been solved, to resolutions better than 2.4 A. The various TK inhibitors broadly fall into three groups which together probe the space of the enzyme active site in a manner that no one molecule does alone, so giving a composite picture of active site interactions that can be exploited in the design of novel compounds.

Overexpression of a Heterologous Thymidine Kinase Delays Apoptosis Induced by Factor Deprivation and Inhibitors of Deoxynucleotide Metabolism

Perturbing deoxyribonucleoside triphosphate (dNTP) metabolism with inhibitors of the de novo synthesis of dNTP causes apoptosis in the interleukin-3 (IL-3)-dependent pre-B cell line BAF3. Under these conditions apoptosis is prevented when deoxyribonucleosides for dNTP synthesis are supplied in the culture medium. On the other hand, removal of IL-3 from cultures of BAF3 cells resulted in down-regulation of thymidine kinase activity, rapid imbalance in dNTP levels, and apoptosis. In this study we show that overexpression of a heterologous thymidine kinase, herpes simplex virus thymidine kinase (TK), in BAF3 cells protects these cells from apoptosis induced by either inhibitors of dNTP synthesis or IL-3 deprivation. This protection against apoptosis is abrogated by 9-(4-hydroxybutyl)-N2-phenylguanine, a specific inhibitor of herpes simplex virus-1 TK. These results suggest that deoxyribonucleoside kinases, particularly TK, may be important in the regulation of apoptosis in hemopoietic cells.

Design and biochemical properties of orally active inhibitors of herpes simplex virus thymidine kinase

Design and biochemical properties of orally active inhibitors of herpes simplex virus thymidine kinase

Resolution and stereoselective synthesis of the herpes thymidine kinase inhibitor L-653180

Resolution of L-653180, (±)-9-{[(Z)-2-(hydroxymethyl)cyclohexyl]-methyl}guanine, a racemic, nonsubstrate inhibitor of HSV-TK, was achieved and the absolute stereochemistry of the enantiomers was determined. Enzyme inhibition was shown to reside in the enantiomer with [1 S,2 R] stereochemistry in the cyclohexyl sidechain. An enantioselective synthesis of the more active L-653180 stereoisomer was devised.

Potential Use of Interferon-α in Ophthalmological Disorders

The a interferons are a unique class of cytokines, apparently possessing ‘proquiescent’ properties which may become useful for the treatment of a range of ophthalmological diseases. Most of the recent clinical studies have employed interferon-α-2a or -2b. Although these agents demonstrate some efficacy against viral infections of the eye such as herpetic keratitis, particularly when given in combination with other antiviral treatments, they have been supplanted in this regard by the thymidine kinase inhibitors. Similarly, although it does appear to have a weak antiangiogenic effect, recent clinical trials have not supported the use of interferon-α for the treatment of choroidal neovascularisation in age-related macular degeneration. Interferon-α does, however, have an established role in the management of tumours occasionally seen by the ophthalmologist, such as basal cell carcinoma, haemangioma, Kaposi’s sarcoma, disseminated malignant melanoma and, possibly, corneal epithelial dysplasia.

Effect of 9-(4-hydroxybutyl)- N2-phenylguanine (HBPG), a thymidine kinase inhibitor, on clinical recurrences of ocular herpetic keratitis in squirrel monkeys

9-(4-Hydroxybutyl)- N 2-phenylguanine (HBPG) is a new viral thymidine kinase inhibitor that we tested for the ability to prevent recurrences of herpetic keratitis. Eighteen squirrel monkeys ( Saimiri scuireus) were infected in both corneas with the Rodanus strain of herpes simplex virus type 1 (HSV-1). All corneas showed typical dendritic keratitis 3 days after infection, followed by spontaneous healing. On day 21, the monkeys were randomized into two coded groups and ocular examinations were begun. One group received intraperitoneal (i.p.) injections of HBPG, 150 mg/kg, in a corn oil suspension every 8 h, and the other group received i.p. injections of the corn oil vehicle only. On day 22, recurrences were induced by reducing the temperature of the room in the late afternoon so that a low of 18°C was achieved during the night. After the morning treatment, room temperature was raised to the normal ambient temperature (24–27°C), and treatment was discontinued. Treatment was reinstituted on day 27, the room temperature was lowered again on day 28, and treatment was again discontinued as before. Third and fourth cycles of treatment and cold stress were begun on days 34 and 69. Ocular examinations were continued until day 73, at which point the code was broken. We found that the HBPG treatment significantly reduced the number of corneas with recurrences during the treatment periods, compared with recurrences in untreated, cold-stressed animals ( P=0.01).