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Based on the presumed binding mode of an earlier identified inhibitor, we herein report new 3'-modified nucleosides as potent and selective inhibitors of mitochondrial thymidine kinase (TK2). A series of thirteen 3'-amino-, 3'-guanidino- and 3'-tetrazole-containing nucleosides were synthesized and evaluated for their TK2 inhibitory activity. Within the tetrazole series, compounds with nanomolar inhibitory activity were identified. A homology model of TK2 allowed to elucidate the observed activities. Introduction of a 2-bromovinyl group on C-5 of the pyrimidine base of the most promising 3'-derivative further improved the inhibitory activity, and caused a significant increase in the selectivity for TK2 versus TK1. Interestingly, for the current series of analogues, a strong correlation was observed between TK2 and Drosophila melanogaster dNK inhibition, further substantiating the phylogenetic relationship between these two nucleoside kinases. ispartof: Organic & Biomolecular Chemistry vol:9 issue:3 pages:892-901 ispartof: location:England status: published

In an effort to increase the potency and selectivity of earlier identified substrate-based inhibitors of mitochondrial thymidine kinase 2 (TK-2), we now describe the synthesis of new thymidine analogues containing a 4- or 5-substituted 1,2,3-triazol-1-yl substituent at the 3'-position of the 2'-deoxyribofuranosyl ring. These analogues were prepared by Cu- and Ru-catalyzed cycloadditions of 3'-azido-3'-deoxythymidine and the appropriate alkynes, which produced the 1,4- and 1,5-triazoles, respectively. Selected analogues showed nanomolar inhibitory activity for TK-2, while virtually not affecting the TK-1 counterpart. Enzyme kinetics indicated a competitive and uncompetitive inhibition profile against thymidine and the cosubstrate ATP, respectively. This behavior is rationalized by suggesting that the inhibitors occupy the substrate-binding site in a TK-2-ATP complex that maintains the enzyme's active site in a closed conformation through the stabilization of a small lid domain. ispartof: Journal of Medicinal Chemistry vol:53 issue:7 pages:2902-12 ispartof: location:United States status: published

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

Deficient enzymatic activity of the mitochondrial deoxyribonucleoside kinases deoxyguanosine kinase (DGUOK) or thymidine kinase 2 (TK2) cause mitochondrial DNA (mtDNA)-depletion syndromes in humans. Here we report the generation of a Tk2-deficient mouse strain and show that the mice develop essentially normally for the first week but from then on exhibit growth retardation and die within 2-4 weeks of life. Several organs including skeletal muscle, heart, liver and spleen showed progressive loss of mtDNA without increased mtDNA mutations or structural alterations. There were no major histological changes in skeletal muscle, but heart muscle showed disorganized and damaged muscle fibers. Electron microscopy showed mitochondria with distorted cristae. The Tk2-deficient mice exhibited pronounced hypothermia and showed loss of hypodermal fat and abnormal brown adipose tissue. We conclude that Tk2 has a major role in supplying deoxyribonucleotides for mtDNA replication and that other pathways of deoxyribonucleotide synthesis cannot compensate for loss of this enzyme.

The multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) is studied as a candidate suicide gene for applications in combined gene/chemotherapy of cancer. We have created an engineered Dm-dNK nucleoside kinase that is targeted to the mitochondrial matrix. The enzyme was expressed in a thymidine kinase 1-deficient osteosarcoma cell line, and the sensitivity of the cells to cytotoxic nucleoside analogs was determined when the enzyme was targeted to either the nucleus or the mitochondrial matrix. Although the total deoxythymidine (dThd) phosphorylation activity was similar in cells expressing Dm-dNK in the nucleus or in the mitochondria, the cells expressing the enzyme in the mitochondria showed higher sensitivity to the antiproliferative activity of several pyrimidine nucleoside analogs, such as (E)-5-(2-bromovinyl)-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, and 5-fluoro-2'-deoxyuridine. Labeling studies using [3H]dThd showed that the cells expressing the mitochondrial enzyme had an increased incorporation of [3H]dThd into DNA, shown to be due to a higher [3H]dTTP specific activity of the total dTTP pool in the cells in which Dm-dNK was targeted to the mitochondria. The difference in the specific activity of the dTTP pool is a result of different contributions of the de novo and the salvage pathways for the dTTP synthesis in transduced cells. In summary, these findings suggest that mitochondrial targeting of Dm-dNK facilitates nucleoside and nucleoside analog phosphorylation and could be used as a strategy to enhance the efficacy of nucleoside analog phosphorylation and concomitantly their cytostatic potential.

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) is investigated for possible use as a suicide gene in combined gene/chemotherapy of cancer. The enzyme has broader substrate specificity and higher catalytic rate compared to herpes simplex type 1 thymidine kinase and other known dNKs. Although the enzyme has broad substrate specificity, it has a preference for pyrimidine nucleosides and nucleoside analogs. We have evaluated the substrate specificity and kinetic properties of Dm-dNK proteins containing M88R, V84A+M88R or V84A+M88R+A110D mutations in the amino-acid sequence. These engineered enzymes showed a relative increase in phosphorylation of purine nucleoside analogs such as ganciclovir, 9-beta-D-arabinofuranosylguanine and 2',2'-difluorodeoxyguanosine compared to the wild-type enzyme. The mutant enzymes were expressed in an osteosarcoma thymidine kinase-deficient cell line and the sensitivity of the cell line to nucleoside analogs was determined. The cells expressing the M88R mutant enzyme showed the highest increased sensitivity to purine nucleoside analogs with 8- to 80-fold decreased inhibition constant IC(50) compared to untransduced control cells or cells expressing the wild-type nucleoside kinase. In summary, our data show that enzyme engineering can be used to shift the substrate specificity of the Dm-dNK to selectively increase the sensitivity of cells expressing the enzyme to purine nucleoside analogs.

Few muscarinic antagonists differentiate between the M4 and M2 muscarinic receptors. In a structure activity study, aimed at discovering leads for the development of a M4 muscarinic receptor-selective antagonist, we have synthesized and tested at cloned muscarinic receptors the binding of a group of dioxolane- or oxadiazole-dialkyl amines, and compared them to our compound 1, which contains the furan nucleus. Although none of these agents were particularly potent at M4 receptors (Kd values were typically 30-70 nM), furan derivatives (-)1 and (+)1 were significantly more potent at M4 receptors than at M2 receptors (approximately 3- and 4-fold, respectively). The dioxolane derivatives 12b and 12c were more than 10-fold selective for the M4 versus the M2 receptors, while the dioxolane derivative 12e was 15-fold more potent at M4 receptors than for M2 receptors. However, these agents bound to M3 receptors with potencies like that for the M4 receptor, so they are not M4-selective. The M4/M2 relative selectivities of some of our compounds are similar to the better hexahydrosiladifenidol derivatives, and may provide some important structural clues for the development of potent and selective M4 antagonists.

Graphical abstract 5-Halo-2′,2′-difluorodeoxyuridines and their ProTides are prepared: the iodo and bromo nucleosides are specific low μM inhibitors of feline herpes virus., We report the synthesis of a series of novel 2′-deoxy-2′,2′-difluoro-5-halouridines and their corresponding phosphoramidate ProTides. All compounds were evaluated for antiviral activity and for cellular toxicity. Interestingly, 2′-deoxy-2′,2′-difluoro-5-iodo- and -5-bromo-uridines showed selective activity against feline herpes virus replication in cell culture due to a specific recognition (activation) by the virus-encoded thymidine kinase.

Mitochondria are important for normal blood-cell development, and several diseases linked to mitochondrial DNA (mtDNA) show hematological manifestations. We recently generated a mouse strain deficient in expression of the mitochondrial pyrimidine nucleoside kinase thymidine kinase 2 (Tk2), showing that these mice exhibit progressive mtDNA depletion in multiple organs. We used this mouse strain as a model for mtDNA depletion syndromes to investigate the effects of mtDNA depletion on hematopoiesis. MtDNA levels in spleen from the Tk2-deficient mice were decreased 50%, but in contrast to all other investigated organs, both thymus and peripheral blood leukocytes showed normal mtDNA levels. Analysis of peripheral blood and cell populations in spleen, thymus, and bone marrow showed normal findings in the Tk2-deficient mice. The total rates of thymidine phosphorylation—which also include phosphorylation catalyzed by cytosolic Tk 1—in both spleen and thymus from wild-type mice were >50-fold higher than in liver, brain, and muscle. In summary, our data show that blood cells are less dependent on mitochondrial Tk2 compared with several other tissues and that these cells can synthesize deoxyribonucleotides required for mtDNA replication by alternative pathways such as phosphorylation of thymidine by cytosolic Tk1.

Adenylate kinases are abundant nucleoside monophosphate kinases, which catalyze the phosphorylation of AMP by using ATP or GTP as phosphate donors. A previously cloned cDNA was named adenylate kinase 4 (AK4) based on its sequence similarity with known AKs but with no confirmed AK enzyme activity. In the present study the AK4 cDNA was expressed in Escherichia coli and the substrate specificity and kinetic properties of the recombinant protein were characterized. The enzyme catalyzed the phosphorylation of AMP, dAMP, CMP and dCMP with ATP or GTP as phosphate donors and AK4 also phosphorylated AMP with UTP as phosphate donor. The kinetic parameters of the enzyme were determined for AMP and dAMP with ATP as phosphate donor and for AMP with GTP as phosphate donor. AK4 showed its highest efficiency when phosphorylating AMP with GTP and a slightly lower efficiency for the phosphorylation of AMP with ATP. Among the three reactions for which kinetics were performed, dAMP was the poorest substrate. The AK4 mitochondrial localization was confirmed by expression of AK4 as a fusion protein with GFP in HeLa cells. The mitochondrial import sequence was shown to be located within the first N-terminal 11 amino acid residues, very close to the ATP-binding region of the enzyme. Import analysis suggested that the mitochondrial import sequence was not cleaved and thus the enzyme retained its activity upon entering the mitochondria. Site directed mutagenesis of amino acids Lys 4 and Arg 7 showed that these two residues were essential for mitochondrial import.

During a random screening of representative libraries of nucleoside analogues we discovered that the adenine derivatives FEVB28 and FEG118 were Flaviviridae inhibitors endowed with potency comparable, if not superior, to that of ribavirin. Those studies prompted us to design a new class of protected nucleoside analogs, reported herein, which displays interesting anti-bovine viral diarrhea virus (BVDV) activity and low cytotoxicity in cell-based assays (4, 23, 29 EC(50): 14, 11, 26 microM respectively, CC(50)100 microM) and appreciable activity in enzyme assays against the RNA dependent RNA polymerase (RdRp) of BVDV (4, 23, 29, RdRp inhibition activity 27, 16, 15 microM respectively). A molecular modeling study was also carried out to highlight the possible interactions between this compounds class and the corresponding hepatitis C virus (HCV) enzyme.

The thymidine kinases from feline herpesvirus (FHV TK) and canine herpesvirus (CHV TK) were cloned and characterized. The two proteins are closely sequence-related to each other and also to the herpes simplex virus type 1 thymidine kinase (HSV-1 TK). Although FHV TK and CHV TK have a level of identity of 31 and 35%, respectively, with HSV-1 TK, and a general amino acid similarity of approximately 54% with HSV-1 TK, they do not recognize the same broad range of substrates as HSV-1 TK does. Instead the substrate recognition is restricted to dThd and pyrimidine analogs such as 1-beta-d-arabinofuranosylthymine (araT), 3'-azido-2',3'-dideoxythymidine (AZT) and (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU). FHV TK and CHV TK differ in substrate recognition from mammalian cytosolic thymidine kinase 1 (TK1) in that TK1 does not phosphorylate BVDU and they also differ from mammalian mitochondrial thymidine kinase 2 (TK2), which, in addition to thymidine and thymidine analogs also phosphorylates dCyd. Although the nucleoside analog BVDU was a good substrate for FHV and CHV TK, the compound was poorly inhibitory to virus-induced cytopathic effect in FHV- and CHV-infected cells. The reason is likely the poor, if any, thymidylate kinase activity of FHV and CHV TK, which in HSV-1 TK-expressing cells convert BVDU-MP to its 5'-diphosphate derivative.

NDP kinase catalyzes the last step in the phosphorylation of nucleotides. It is also involved in the activation by cellular kinases of nucleoside analogs used in antiviral therapies. Adenosine phosphonoacetic acid, a close analog of ADP already proposed as an inhibitor of ribonucleotide reductase, was found to be a poor substrate for human NDP kinase, as well as a weak inhibitor with an equilibrium dissociation constant of 0.6 mM to be compared to 0.025 mM for ADP. The X-ray structure of a complex of adenosine phosphonoacetic acid and the NDP kinase from Dictyostelium was determined to 2.0 A resolution showing that the analog adopts a binding mode similar to ADP, but that no magnesium ion is present at the active site. As ACP may also interfere with other cellular kinases, its potential as a drug targeting NDP kinase or ribonucleotide reductase is likely to be limited due to strong side effects. The design of new molecules with a narrower specificity and a stronger affinity will benefit from the detailed knowledge of the complex ACP-NDP kinase.

The deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) has a broad substrate specificity and a higher catalytic rate than other known deoxyribonucleoside kinases. Therefore it is a natural candidate for possible use as a suicide gene in combined gene/chemotherapy of cancer. We have performed site directed mutagenesis and tested different truncated forms of the enzyme in order to increase the affinity for ganciclovir.

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) is sequence-related to three human deoxyribonucleoside kinases and to herpes simplex virus type-1 thymidine kinase. Dm-dNK phosphorylates both purine and pyrimidine deoxyribonucleosides and nucleoside analogues although it has a preference for pyrimidine nucleosides. We performed site-directed mutagenesis on residues that, based on structural data, are involved in substrate recognition. The aim was to increase the phosphorylation efficiency of purine nucleoside substrates to create an improved enzyme to be used in suicide gene therapy. A Q81N mutation showed a relative increase in deoxyguanosine phosphorylation compared with the wild-type enzyme although the efficiency of deoxythymidine phosphorylation was 10-fold lower for the mutant. In addition to residue Q81 the function of amino acids N28, I29 and F114 was investigated by different substitutions. All of the mutated enzymes showed decreased efficiency of thymidine phosphorylation in comparison with the wild-type enzyme supporting their importance for substrate binding and/or catalysis as proposed by the recently solved structure of Dm-dNK.

Continuing our investigations on inhibitors of ribonucleotide reductase (RNR), the crucial enzyme that catalyses the reduction of ribonu-cleotides to deoxyribonucleotides, we have now prepared and evaluated 5′-phosphonoacetic acid, amide and ester analogues of adenosine, uridine and cytidine with the aim to verify both substrate specificity and contribution to biological activity of diphosphate mimic moieties. A molecular modelling study has been conducted on the RNR R1 subunit, in order to verify the possible interaction of the proposed bioisosteric moieties. The study compounds were finally tested on the recombinant murine RNR showing a degree of inhibition that ranged from 350 μM for the UDP analogue 5′-deoxy-5′- N-(phosphon-acetyl)uridine sodium salt (amide) to 600 μM for the CDP analogue 5′- O-[(diethyl-phosphon)acetyl]cytidine (ester). None of the tested compounds displayed noteworthy cytostatic activity at 100–500 μM concentrations, whereas ADP analogue 5′- N-[(diethyl-phosphon) acetyl]adenosine (amide) and 5′-deoxy-5′- N-(phos-phon-acetyl)adenosine sodium salt (amide) showed a moderate inhibitory activity (EC50: 48 μM) against HSV-2 and a modest inhibitory activity (EC50: 110 μM) against HIV-1, respectively.