Search

Resveratrol, a natural compound found in many dietary plants and in red wine, plays an important role in the prevention of many human pathological processes, including inflammation, atherosclerosis and carcinogenesis. We have shown that the antiproliferative activity of resveratrol correlated with its ability to inhibit the replicative pols (DNA polymerases) α and δ in vitro [Stivala, Savio, Carafoli, Perucca, Bianchi, Maga, Forti, Pagnoni, Albini, Prosperi and Vannini (2001) J. Biol. Chem. 276, 22586–22594]. In this paper, we present the first detailed biochemical investigation on the mechanism of action of resveratrol towards mammalian pols. Our results suggest that specific structural determinants of the resveratrol molecule are responsible for selective inhibition of different mammalian pols, such as the family B pol α and the family X pol λ. Moreover, the resveratrol derivative trans-3,5-dimethoxy-4-hydroxystilbene, which is endowed with a strong antiproliferative activity (Stivala et al., 2001), can inhibit pols α and λ and also suppress the in vitro SV40 DNA replication. The potency of inhibition is similar to that of aphidicolin, an inhibitor of the three replicative pols α, δ and ε. Our findings establish the necessary background for the synthesis of resveratrol derivatives having more selective and potent antiproliferative activity.

The HIV-1 accessory protein Vif was found to modulate the RNA- and DNA-dependent DNA synthesis activity of the viral RT (reverse transcriptase) in two ways: (i) it stimulated the binding of the viral RT to the primer by increasing the association rate kcat/Km and by decreasing the thermodynamic barrier ΔH[ES] for complex fomation, and (ii) it increased the polymerization rate of HIV-1 RT. A Vif mutant lacking the final 56 amino acids at the C-terminus failed to stimulate the viral RT. On the other hand, another Vif mutant lacking the first 43 amino acids at the N-terminus, which are involved in RNA binding and interaction with the viral protease, was able to stimulate RT activity. In addition, Vif was found to promote the bypass of an abasic site by HIV-1 RT.

The antiviral activity of several nucleoside analogues is often limited by their rapid degradation by pyrimidine nucleoside phosphorylases. In an attempt to avoid this degradation, several modified nucleosides have been synthesized. A series of 4´-thio-2´-deoxyuridines exhibits an anti-[herpes simplex virus (HSV)] activity significantly higher (20–600 times) than that shown by the corresponding 4´-oxy counterpart. We investigated the mode of action of these compounds and we found that: (i) several 4´-thio-2´-deoxyuridines are phosphorylated to the mono- and di-phosphates by HSV-1 thymidine kinase (TK) more efficiently than their corresponding 4´-oxy counterpart; (ii) both are inhibitors of cellular thymidylate synthase; (iii) 4´-thio-2´-deoxyuridines are resistant to phosphorolysis by human thymidine phosphorylase; (iv) both 4´-oxy- and 4´-thio-2´-deoxyuridines are phosphorylated to deoxyribonucleotide triphosphate in HSV-1-infected cells and are incorporated into viral DNA; (v) 4´-thio-2´-deoxyuridines are better inhibitors than their 4´-oxy counterparts of [3H]thymidine incorporation in HSV-1-infected cells; (vi) 4´-thio-2´-deoxyuridines are not recognized by HSV-1 and human uracil-DNA glycosylases. Our data suggest that 4´-thio-2´-deoxyuridines, resistant to pyrimidine phosphorylase, can be preferentially or selectively phosphorylated by viral TK in HSV-infected cells, where they are further converted into triphosphate by cellular nucleotide kinases. Once incorporated into viral DNA, they are better inhibitors of viral DNA synthesis than their corresponding 4´-oxy counterpart, either because they are not recognized, and thus not removed, by viral uracil-DNA glycosylase, or because they preferentially interfere with viral DNA polymerase.

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 µM) but also deoxycytidine (Km 0.88 µM) and deoxyuridine (Km 0.14 µM). 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. vaginaliscultured cells and T. vaginalis growth (IC50 7.5 and 24 µM respectively; minimal lethal dose 100 µM). 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.

Our discovery that Herpes virus thymidine kinase (TK) and cellular deoxycytidine kinase lack enantioselectivity, being able to phosphorylate both D- and L-enantiomers of the substrate, suggested the use of unnatural L-nucleoside analogues as antiviral drugs (Herpes, hepatitis and immunodeficiency viruses). Several L-nucleoside analogues have displayed a short-term cytotoxicity much lower than their corresponding D-counterpart. Since the delayed cytotoxicity of a drug often depends on its effects on mitochondrial metabolism, we have investigated the degree of enantioselectivity of human mitochondrial thymidine kinase (mt-TK). We demonstrate that mt-TK does not show an absolute enantioselectivity, being able to recognize, although with lower efficiency, the L-enantiomers of thymidine, deoxycytidine and modified deoxyuridines, such as (E)-5-(2-bromovinyl)-2ʹ-deoxyuridine and 5-iodo-2ʹ-deoxyuridine. Interestingly, the reported negative co-operativity of mt-TK phosphorylating β-D-2ʹ-deoxythymidine (D-Thd), disappears when the deoxyribose moiety has the inverted configuration, resulting in the preferential phosphorylation of D-Thd even in the presence of high concentrations of the L-enantiomer. This, coupled with the higher Km for β-l-2ʹ-deoxythymidine (L-Thd), makes mt-TK resistant to high concentrations of L-Thd and L-Thd analogues, minimizing the mitochondria-dependent delayed cytotoxicity that might be caused by the administration of l-nucleoside analogues as antivirals.

From oocysts of the protozoan parasite Eimeria tenella, responsible for avian coccidiosis, we have partially purified and characterized a novel enzymic activity which specifically phosphorylates guanosine to GMP. The enzyme is able to use several phosphate donors, in the order: acetyl phosphate (Ac-P) > ATP > UTP > CTP > phosphoribosyl pyrophosphate (PRPP) > dUTP > or = dATP. The low specificity of this enzyme for the phosphate donor suggested that it be named guanosine phosphotransferase (GPTase). This enzyme is biochemically distinct from the previously described adenosine kinase (AK) and hypoxanthine/xanthine/guanine phosphoribosyltransferase (HXGPRTase), and may enable the parasite to synthesize guanine nucleotides under conditions of imbalance between adenine and guanine nucleotides. Because of its possible role in the purine salvage pathways, we have studied the effect of several guanine and guanosine analogues, recently synthesized in our laboratory, on the activity of GPTase in vitro. GPTase is specifically inhibited in the micromolar range by several substituted N2-phenylguanine bases. These results indicate that, as previously found for AK and HXGPRTase, GPTase could be a potential target for antiparasitic chemotherapy.

Bacteriophage-T4 and human type I DNA ligases were found capable of self-adenylating upon exposure to both ribo- and deoxyribo-[alpha-35S]thio-ATP. However, the joining reaction does not take place in the presence of the deoxyribotriphosphates. Enzyme adenylation is reversed in all cases by an excess of PPi, but the rate of reversion is lower with thio derivatives. Therefore thio derivatives can be used to study the adenylation of DNA ligases and to search for specific inhibitors of the first step of the ligation reaction. In addition we show that thio derivatives can be used to detect DNA ligase adenylation activity covalently bound to a solid matrix.

We demonstrate that l-ATP is recognized by some enzymes that are involved in the synthesis of nucleotides and nucleic acids. l-ATP, as well as its natural d-enantiomer, acts as a phosphate donor in the reaction catalysed by human deoxycytidine kinase, whereas it is not recognized by either enantioselective human thymidine kinase or non-enantioselective herpes virus thymidine kinase. l-ATP strongly inhibits (Ki 80 µM) the synthesis of RNA primers catalysed by DNA primase associated with human DNA polymerase α, whereas RNA synthesis catalysed by Escherichia coli RNA polymerase is completely unaffected. Moreover, l-ATP competitively inhibits ATP-dependent T4 DNA ligase (Ki 25 µM), suggesting that it interacts with the ATP-binding site of the enzyme. Kinetic studies demonstrated that l-ATP cannot be used as a cofactor in the ligase-catalysed joining reaction. On the other hand, l-AMP is used by T4 DNA ligase to catalyse the reverse reaction, even though a high level of intermediate circular nicked DNA molecules accumulates. Our results suggest that a lack of enantioselectivity of enzymes is more common than was believed a few years ago, and, given the absence of selective constraints against l-nucleosides in Nature, this may depend on chance more than on evolutionary strategy.

It is known that the Herpes simplex virus type 1 (HSV-1)-encoded thymidine kinase (TK) co-purifies with an associated thymidylate kinase (TMPK) activity and that thymidylate (TMP) inhibits the phosphorylation of thymidine by the HSV-1 TK. Here we demonstrate that: (i) TMP phosphorylation catalysed by the viral TMPK is competitively inhibited by thymidine (TdR) with a Ki equal to its Km as substrate for the viral TK; (ii) L-thymidine (L-TdR), the enantiomer of the naturally occurring D-TdR and a substrate for the HSV-1 TK [Spadari, Maga, Focher, Ciarrocchi, Manservigi, Arcamone, Capobianco, Caruso, Colonna, Iotti and Garbesi (1992) J. Med. Chem. 35, 4214-4220], is a powerful inhibitor of the HSV-1 TMPK activity with a Ki value identical with its Km as a substrate for the viral TK; (iii) both viral TK and TMPK activities are inhibited, in a competitive way and with identical Ki values, by novel, non-substrate inhibitors of HSV-1 TK, N2-phenylguanines; (iv) L-TdR is phosphorylated to L-TMP by the viral TK, but L-TMP is not phosphorylated to L-TDP by the viral TMPK activity; and (v) L-TMP inhibits competitively and with identical potencies the phosphorylation of TdR and TMP catalysed respectively by the HSV-1 TK and TMPK activities. In conclusion, our data demonstrate that both TK and TMPK activities encoded by HSV-1 share a common active site which is very tolerant in accepting modified nucleosides, but cannot readily accommodate modified nucleoside monophosphates.

We have partially purified suid pseudorabies virus (PRV) thymidine kinase from infected thymidine kinase- mouse cells, and cytosolic swine thymidine kinase from lymphatic glands, and we have found that PRV thymidine kinase, unlike the host enzyme, shows no stereospecificity for D- and L-beta-nucleosides. In vitro, unnatural L-enantiomers, except L-deoxycytidine, function as specific inhibitors for the viral enzyme in the order: L-thymidine >> L-deoxyguanosine > L-deoxyuridine > L-deoxyadenosine. Contrary to human and swine thymidine kinases and like herpes simplex virus-1 and -2 thymidine kinases, PRV thymidine kinase phosphorylates both the natural (D-) and the unnatural (L-) thymidine enantiomers to their corresponding monophosphates with comparable efficiency. The kinetic parameters Vmax/Km for D- and L-thymidine are 3.7 and 2.3 respectively. Our results demonstrate that the lack of stereospecificity might be a common feature of the thymidine kinases that are encoded by human and animal herpes viruses. These observations could lead to the development of a novel class of antiviral drugs.

We have purified Herpes simplex type 1 (HSV1) uracil-DNA glycosylase from the nuclei of HSV1-infected HeLa cells harvested 8 h post-infection, at which time the induction of the enzyme is a maximum. The enzyme has been shown to be distinct from the host enzyme, isolated from HeLa cells, by its lack of sensitivity to a monoclonal antibody to human uracil-DNA glycosylase. Furthermore, several uracil analogues were synthesized and screened for their capacity to discriminate between the viral and human uracil-DNA glycosylases. Both enzymes were inhibited by 6-(p-alkylanilino)uracils, but the viral enzyme was significantly more sensitive than the HeLa enzyme to most analogues. Substituents providing the best inhibitors of HSV1 uracil-DNA glycosylase were found to be in the order: p-n-butyl < p-n-pentl = p-n-hexyl < p-n-heptyl < p-n-octyl. The most potent HSV1 enzyme inhibitor, 6-(p-n-octylanilino)uracil (OctAU), with an IC50 of 8 microM, was highly selective for the viral enzyme. Short-term [3H]thymidine incorporation into the DNA of HeLa cells in culture was partially inhibited by OctAU, whereas it was unchanged when 6-(p-n-hexylanilino)uracil was present at concentrations that completely inhibited HSV1 uracil-DNA glycosylase activity. These compounds represent the first class of inhibitors that inhibit HSV1 uracil-DNA glycosylase at concentrations in the micromolar range. The results suggest their possible use to evaluate the functional role of HSV1 uracil-DNA glycosylase in viral infections and re-activation in nerve cells.

We have investigated the substrate specificity of human, viral and bacterial uracil-DNA glycosylases employing as substrate double-stranded oligonucleotides containing in the same position of the 5′-32P-labelled strand an uracil residue facing, on the complementary strand, guanine (mimicking cytosine deamination) or adenine (mimicking dUTP misincorporation). The enzyme removal of uracil was monitored and quantified by the generation of alkali-sensitive apyrimidinic sites. All three uracil-DNA glycosylases excise uracil from mispaired oligonucleotides (U/G) more efficiently than from paired oligonucleotides (U/A). The enzymes also remove uracil from single-stranded oligonucleotide with an efficiency similar to that observed with U/A paired oligonucleotide. The efficient recognition of U/G mispair by uracil-DNA glycosylase is important in minimizing miscoding transcripts and C/G-->T/A transitions in proliferating cells.

A total of 19 anthracycline derivatives were tested for their ability to interfere in vitro with the action of the human replicative DNA ligase. Only those with the sugar devoid of unmodified amino groups or with large configurational modifications were found to be inactive. Maximal inhibition of DNA-joining activity was found to require a 4′-deoxy-3′-amino sugar. Self-adenylation of DNA ligase was largely insensitive to these drugs. An important general finding is that slight modifications of the anthracycline structure have pronounced effects on DNA-ligase-inhibitory activity and might be related to the specificity of anthracycline anti-tumour activity.

A number of DNA intercalating and externally binding drugs have been found to inhibit nick sealing, cohesive and blunt end ligation, AMP-dependent DNA topoisomerization and EDTA-induced DNA nicking mediated by bacteriophage T4 DNA ligase. The inhibition seems to arise from drug-substrate interaction so that formation of active DNA-Mg2(+)-AMP-enzyme complex is impaired while assembled and active complexes are not disturbed by drug binding to the substrate.

A radioautographic examination of nuclear DNA synthesis in unirradiated and u.v.-irradiated HeLa cells, in the presence and in the absence of aphidicolin, showed that aphidicolin inhibits nuclear DNA replication and has no detectable effect on DNA repair synthesis. Although the results establish that in u.v.-irradiated HeLa cells most of the DNA repair synthesis is not due to DNA polymerase alpha, they do not preclude a significant role for this enzyme in DNA repair processes.