Promising New Inhibitors of Tyrosyl-DNA Phosphodiesterase I (Tdp 1) Combining 4-Arylcoumarin and Monoterpenoid Moieties as Components of Complex Antitumor Therapy.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an important DNA repair enzyme in humans, and a current and promising inhibition target for the development of new chemosensitizing agents due to its ability to remove DNA damage caused by topoisomerase 1 (Top1) poisons such as topotecan and irinotecan. Herein, we report our work on the synthesis and characterization of new Tdp1 inhibitors that combine the arylcoumarin (neoflavonoid) and monoterpenoid moieties. Our results showed that they are potent Tdp1 inhibitors with IC50 values in the submicromolar range. In vivo experiments with mice revealed that compound 3ba (IC50 0.62 µM) induced a significant increase in the antitumor effect of topotecan on the Krebs-2 ascites tumor model. Our results further strengthen the argument that Tdp1 is a druggable target with the potential to be developed into a clinically-potent adjunct therapy in conjunction with Top1 poisons.

Halogenated benzimidazoles and benzotriazoles as selective inhibitors of protein kinases CK I and CK II from Saccharomyces cerevisiae and other sources.

Several halogeno benzimidazole riboside inhibitors of animal and plant protein kinases CK I and CK II (also known as casein kinases I and II), were found to be effective inhibitors of Saccharomyces cerevisiae CK II, but not of the 27-kDa CK.I or the 45-kDa CK I. The previously reported 5,6-dichloro-2-azabenzimidazole, which preferentially inhibits plant CK II relative to CK I, discriminates even more effectively between the yeast CK I and CK II enzymes. Two new analogues, tetrahalogeno-2-azabenzimidazoles, are even more potent inhibitors of CK II and much less so of CK I from yeast and animal sources. All inhibitors are competitive with respect to ATP (and GTP with CK II), the two latter with Ki values in the range 0.2-0.6 microM for CK II from yeast and mammalian sources.

Vector methylation inhibits transcription from the SV40 early promoter.

Methylation of a plasmid containing the SV40 promoter linked to the chloramphenicol acetyl transferase (CAT) gene, with either murine DNA methylase or methylase SssI results in inhibition of the expression of the reporter gene after transfection into cultured cells. Methylation of the plasmid with the methylases HhaI and HpaII has no effect on the expression of this gene. Protein-DNA interactions in the SV40 promoter are not affected by the presence of methylcytosine suggesting that inactivation results from the formation of an inactive chromatin structure that is dependent on the high CG content of the plasmid.

Subcellular localization of casein kinase I.

An anti-yeast CKI antiserum was shown to cross-react with CKI isolated from Krebs II mouse ascites tumour cells. The mammalian CKI showed virtually the same molecular mass (app. 45 kDa) as the yeast enzyme. By immunofluorescence it could be shown that CKI is preferably located in the nucleolus.

An enzyme which specifically splits a covalent bond between picornaviral RNA and VPg.

An enzyme which specifically splits a covalent bond between VPg and picornaviral RNAs (tentatively designated Y-pUpN PDE) has been partially purified from mouse ascites Krebs II cells. Using substrates labelled in vitro with 125I-Bolton-Hunter reagent and a new assay based on Kieselgel thin-layer chromatography, several biochemical characteristics of the enzyme have been determined, depending on the pH and on Mg2+, K+, spermidine and PEG concentrations, etc. We found that the enzyme does not 'unlink' VPg from comoviral RNA. We suggest that Y-pUpN PDE represents a new class of enzymes.

Enhanced casein kinase II activity during mouse embryogenesis. Identification of a 110-kDa phosphoprotein as the major phosphorylation product in mouse embryos and Krebs II mouse ascites tumor cells.

Mouse embryos at various stages of development were used to study the relationship of protein kinase activities with normal embryogenesis. Casein kinase II (CKII) activity in developing mouse embryos shows a 3-4-fold activity increase at day 12 of gestation. Together with the CKII activity, increased phosphorylation of a 110-kDa protein is observed. Treatment of the embryo extracts with heparin, a highly specific inhibitor of CKII activity, results in a drastic reduction of the 110-kDa protein phosphorylation indicating that the protein might be a CKII-specific substrate. Rapidly proliferating mouse tumour cells also show an enhanced CKII activity. Here too, a 110-kDa phosphoprotein was the major phosphoryl acceptor. Partial proteolytic digestion shows that both proteins are identical. Other protein kinases tested (cAMP- and cGMP-dependent protein kinases) only show a basal level of enzyme activity with minor alterations throughout the different stages of embryogenesis investigated.

[Characteristics of an enzyme hydrolyzing the covalent bond between RNA and protein VPg of the encephalomyocarditis virus]. / Kharakteristika fermenta, gidrolizuiushchego prirodnuiu kovalentnuiu sviaz' mezhdu RNK i belkom VPg virusa entsefalomiokardita.

The enzyme termed by us as uridilylpolynucleotide-(5'P----O)-tyrosine phosphodiesterase (Y-pUpN PDE) was isolated from mouse ascites Krebs II cells by ion-exchange and affinity chromatography. The enzyme was found to specifically split the natural covalent bond between VPg and EMC or polio viral RNAs. The enzyme is completely inactivated at 55 degrees C and partially by EDTA. The enzyme preparation isolated by the above-mentioned procedure is not homogeneous and contains inhibiting admixture(s). Possible role of the enzyme in living cells is discussed.

Nuclear matrix-associated DNA methylase.

Previous procedures for the extraction of DNA methylase (EC 2.1.1.37) from nuclei of mouse ascites cells have involved the use of buffers containing 0.2M NaCl. Whilst such 'soluble' methylase accounts for the bulk (70-80%) of DNA methylase activity a further portion of activity is detectable in a 'bound' form firmly associated with 2 M NaCl-resistant nuclear matrix-like structures. This association, which in part requires continuing DNA replication and protein synthesis, can, however, be disrupted in vitro with high concentrations of ammonium sulphate, and the enzymic properties of the 'bound' form of DNA methylase are similar to those described for the 'soluble' form.

Stimulation of de novo methylation following limited proteolysis of mouse ascites DNA methylase.

The ability of mouse Krebs II ascites cell DNA methylase to add methyl groups to native, unmethylated DNA (de novo activity) is stimulated by limited proteolysis. The affinity of the enzyme for DNA is not altered by this treatment but the rate of reaction is increased so that 40% or more of methylatable sites are methylated within 4.5 h. The activation is associated with a decrease in size of the enzyme to 6.2 S.

The ribosomal proteins phosphorylated in vitro by protein kinase activities from Krebs II ascites cells.

Studies were performed to identify in cytoplasmic extracts of Krebs II ascites cells protein kinase activities that might be responsible for the phosphorylation of the ribosomal proteins previously identified as phosphoproteins in these cells in vivo. Column chromatography resolved a casein kinase activity that could use ATP or GTP as a phosphoryl donor to phosphorylate, in ribosomes, exclusively the acidic 60S phosphoprotein(s) phosphorylated in vivo. A second casein kinase fraction could use ATP, only, in a similar reaction, but also contained protein kinase activity with respect to other ribosomal proteins, including the basic ribosomal protein phosphorylated in vivo, ribosomal protein S6. This latter was also among several proteins phosphorylated by an activity in the cyclic AMP-independent histone kinase fraction.

An RNA-dependent nucleoside triphosphate hydrolase from Krebs-II ascites tumor cells. Detection and preliminary characterization.

A novel enzymatic activity, RNA-dependent, NTPase, was isolated from Krebs-II ascites tumor cells. This activity is associated with ribosomes and can be detached from them by washing in KCl solutions of a higher than 0.3 M concentration. The enzyme hydrolyzes all the four nucleoside triphosphates to the corresponding nucleoside diphosphates and orthophosphate. The rate of NTP hydrolysis increases about 10-fold in the presence of natural RNAs and synthetic polyribonucleotides [except poly(G)]. Natural DNAs, both double and single-stranded, are poor cofactors, although pol(dA) and poly(dT) stimulate, to a certain extent, the rate of ATP hydrolysis. Possible involvement of RNA-dependent NTPase in protein biosynthesis is discussed.

Affinity labelling and characterization of the ppp(A2'p)nA-dependent endoribonuclease from different mammalian sources.

The ppp(A2'p)nA-dependent endoribonucleases from a number of different mammalian sources have been investigated. The enzyme from reticulocyte lysates shows optimal activity of 50-150 mM KCl and requires the presence of Mg2+. Whilst the enzyme is inactivated after passage of reticulocyte lysates through Sephadex columns in the absence of ATP, it retains full activity provided ATP is included in the column buffer. The activity of the partially purified nuclease was unaffected by the addition of reticulocyte RNase inhibitor, which, in contrast, effectively inhibited other endogenous endonucleases. The ppp(A2'p)nA-dependent Rnase co-purified with a ppp(A2'p)nA-binding protein and with a protein which could be specifically covalently labelled with an oxidised radioactive analogue of ppp(A2'p)nA. This covalent labelling could be carried out either with the partially purified RNase or in crude extracts from rabbit reticulocytes, mouse Krebs and Ehrlich ascites tumour cells and human lymphoblastoid (Daudi) or HeLa cells. In each case the affinity labelled protein migrated to a position corresponding to a apparent molecular weight of about 85 000 on electrophoresis on dodecylsulphate/polyacrylamide gels. In all cases labelling could be prevented by the addition of an excess of unlabelled ppp(A2'p)nA but not, for example, by a similar excess of the biologically inactive dimer ppp(A2'p)'A. It is concluded that the RNase and ppp(A2'p)nA binding activities are likely to reside in the same molecule.

Type I DNA topoisomerases from mammalian cell nuclei interlock strains and promote renaturation of denatured closed circular PM2 DNA.

Type I DNA topoisomerases from mouse ascites cell nuclei and from rat liver cell nuclei act on denatured viral closed circular PM2 DNA to produce molecules with a highly contracted structure as well as fully duplex non-supercoiled covalently closed circular molecules. Highly contracted DNA molecules contain a novel type of topological linkage in which a strand in one region of the double-stranded molecule passes between the strands in another region of the circular molecule one or more times. Since it is also found that the action of the topoisomerase promotes renaturation of complementary strands in denatured closed circular DNA, it is suggested that formation of contracted DNA structures proceeds through renatured, duplex intermediates with highly negative superhelix densities that contain small single-stranded regions.

Isolation and characterization of a ribonuclease activity specific for double-stranded RNA (RNase D) from Krebs II ascites cells.

We have extensively purified from Krebs II ascites cells, although not until homogeneity, a ribonuclease which preferentially cleaves natural or synthetic double-stranded RNA substrates (RNase D); this specificity is also supported by its sensitivity to inhibition by 10(-5) M ethidium bromide. It does not degrade RNA-DNA hybrids and is, therefore, clearly distinct from previously characterized RNases H (Cathala, G., Rech, J., Huet, J., and Jeanteur, Ph. (1979) J. Biol. Chem. 254, 7354-7361). It shows no requirement for a divalent cation and is inhibited by all kinds of nucleic acids regardless of their secondary structure. It acts exclusively as an endonuclease, as shown by the analysis of degradation products, and yields 5'-phosphate termini. This enzyme is able to introduce discrete nicks into purified HeLa 45 S preribosomal RNA as well as into HeLa heterogenous nuclear RNA packaged within naturally occurring nuclear ribonucleoprotein particles. It is, therefore, an interesting candidate for an RNA-processing enzyme.

Mouse DNA methylase: methylation of native DNA.

An improved method of purification of DNA methylase from Krebs II ascites cells is reported. The enzyme sediments at 8.3 S on glycerol-gradients and a major band on SDS polyacrylamide gel electrophoresis has a molecular weight of 184 000. Aggregation occurs at low salt and this may interfere with enzymic activity. The preferred double stranded DNA substrate is that rendered partially unmethylated by an in vitro repair mechanism or by isolation from methionine starved cells. Methylation of native partially methylated DNA is favoured under conditions of low salt and high temperature; conditions which encourage 'breathing' of the DNA. Methylation of native, unmethylated DNA also involves breathing but results in formation of a salt resistant tight binding complex between the enzyme and the DNA.