DNA biosynthesis

Reddy et al29 studied the molecule N6,N6-dimethyl-2,6-diaminobenz[cd]-indole, which is shown in Figure 6. The aim was to assess its suitability as an inhibitor of thymidylate synthase, an enzyme which is important in DNA biosynthesis. Three structures were investigated N1H, which is the amine form depicted, and N2H, the imine form, in anti- and syn-conformations, i.e. with the proton on Ni away from or towards the N2 proton, respectively. In the gas phase it was found that the order of stability is N1H > anti-N2H > syn-N2H, in a ratio of 73.1 20.3 6.6. Solvation then further favours the N1H form, and the ratios in solution are calculated to be 98.5 0.5 1.0. Unfortunately, the syn-N2H form is likely to be the conformation that binds most strongly to the enzyme, and so the calculations indicate that this molecule is unlikely to be a suitable inhibitor. 

As already mentioned, RNR is the metalloenzyme in which the first definitively characterized stable amino acid radical (1), later identified as a tyrosyl radical, was found in 1972. The RNR enzymes catalyse the reduction of ribonucleotides to their corresponding deoxyribonucleotides utilized in DNA biosynthesis. There are three unique classes of this enzyme, differing in composition and cofactor requirements all of them, however, make use of metal ions and free radical chemistry. Excellent reviews on RNRs are available (60, 61, 70, 89-97). 

Novobiocin (Fig. 8) is a glycosidic antibiotic isolated from Streptomyces species.Its mode of action was identified by Smith as DNA biosynthesis inhibitor and it was found to target the B subunit of DNA Gyrase, a DNA replication enzyme (Fig. 1). 

Another important target for cancer therapy is the inhibition of the RDPR. This homodimeric enzyme catalyses a crucial step of the DNA biosynthesis, the... 

Fludarabine (Fludara ) is a 2-fluorocytarabine (Fig. 34) which inhibits the DNA biosynthesis via the inhibition of DNA polymerase a and of RDPR. It is clinically used for the treatment of leukaemia (chronic lymphomytic leukaemia, CLL). 

The development of drugs with actions on intermediary metabolism of proliferating cells has been important both conceptually and clinically. While biochemical properties unique to all cancer cells have yet to be discovered, there are a number of quantitative differences in metabolism between cancer cells and normal cells that render cancer cells more sensitive to the antimetabolites. Many of these agents have been rationally designed and synthesized based on knowledge of critical cellular processes involved in DNA biosynthesis. 

A key step in DNA biosynthesis, that of conversion of deoxyuridylate (dUMP) to deoxythymidylate (dTMP), is catalyzed by thymidylate synthetase which uses (25) as cofactor. This reaction involves both the transfer of a one carbon unit at the formaldehyde level and hydride transfer (from C-6 of (25)) to produce 7,8-dihydrofolate (27) and dTMP... 

Most of the genetic information of bacteria is contained in a single structure of fixed DNA content, a giant circular DNA molecule that replicates semi-conservatively. The enzymatic reactions involved in the biologically fundamental processes of DNA biosynthesis and genetic recombination are being elucidated in studies with bacterial systems. 

Table 10. The effect of Hchloromethyl)silatrane on the DNA biosynthesis in regenerating liver of rats...

FIGURE 33-2 Folic acid metabolism in bacterial cells. Certain antibacterial drugs [e.g., sulfonamides and trimethoprim] inhibit the dihydrofolate synthetase and reductase enzymes, thus interfering with DNA biosynthesis. 

Folate in the human organism is converted to tetrahydrofolate (THF or FH4) by a reductive process. The reduction reaction is a stepwise one folate to dihydrofolate, then to THF. A single enzyme, dihydrofolate reductase, catalyzes both steps. The reaction is inhibited by folate analogues and the antitumor agents methotrexate and aminopterin (Figure 6.3). Because THF is required for DNA biosynthesis (Chapter 10) and tumors have a very high level of DNA biosynthetic activity, even modest decreases in THF availability will inhibit tumor growth. 

The control of ribonucleotide reductase activity is affected in the classic feedback fashion by cellular nucleotide concentrations. dATP inhibits the reduction of all four ribonucleoside diphosphates. dTTP inhibits the reduction of only CDP and UDP. ATP is the positive effector for the reduction of these two nucleotides, and both dTTP and dGTP stimulate the reduction of GDP and ADP. Hydroxyurea, an antitumor agent, inhibits ribonucleotide reductase, and this depletes the deoxyribonucleotide supply required for tumor DNA biosynthesis. 

Ribonucleotide reductase controls DNA biosynthesis via various feedback effects involving ribo- and deoxyribonucleotides. 

The literature is replete with reports of minor inhibitions of some biochemical or physiological process by chemotherapeutic drugs. In my own laboratory it was found that quinacrine caused a slight inhibition of protein biosynthesis in susceptible bacteria which subsequently was verified in a cell-free poly U system, polymerizing phenylalanine 18 Since it was also established, however, that the drug is a strong inhibitor of DNA biosynthesis 17 and that this effect accounts for the bactericidal action of quinacrine, the slight effect on protein biosynthesis was not mistaken as the mode of action. 

Brought to logical conclusion, it was shown that the probit transformations of bacterial growth inhibition and inhibition of DNA biosynthesis by Nitroakridin 3582 were superposable while the same functions for inhibition of RNA and protein biosyntheses were superposable upon each other but indicated a lesser susceptibility of the test organism. This led to the conclusion that the mode of antibacterial action of the nitroacridine was its inhibition of DNA biosynthesis27. ... 

One classical and early example of selective inhibition of DNA biosynthesis is shown in Fig. 3 for the anitbiotic, mitomycin C29 A concentration of 0.1 pg/ml (3 x 10 7 M) completely inhibited DNA synthesis in E. coli B, while RNA synthesis, protein synthesis and growth meaning turbidity, i.e. cell mass increase, continued. However, after the experimental period of only 90 min, the number of viable bacteria had decreased by 85 per cent. By that time, bacterial filaments were visible under the microscope. 

With the introduction of radiochemical methods, DNA biosynthesis and its inhibition is usually followed either by measuring the incorporation of radioactive thymine into thymine auxotrophs of bacteria or the incorporation of radioactive thymidine into prototrophic organisms. In the latter instance, it is practical to include in the experimental medium a large excess of non-radioactive deoxyadenosine in order... 

A large number of antibiotic and synthetic inhibitors of DNA biosynthesis form complexes with DNA and act as template poisons for the replication of chromosomal DNA6,7. These substances are preferential inhibitors of eucaryotic cells such as protozoa or neoplastic cells. The only antibacterial chemotherapeutic drug which is a selective inhibitor of DNA biosynthesis is nalidixic acid. This synthetic compound does not form complexes with DNA but acts in an unknown manner which appears to involve a perturbation of the interaction of DNA with a bacterial membrane system which is instrumental in DNA replication30. ... 

For example, in cultures of Bacillus subtilis, protein synthesis had ceased entirely after one fourth of a doubling time when RNA synthesis was specifically and completely inhibited by actionomycin D33. Since the initiation of a new round of DNA biosynthesis requires the ad hoc synthesis of initiator protein(s), DNA biosynthesis comes to a standstill after a lag as the result of the failure of initiator protein synthesis. This interesting sequence of events was first described by Kirk34 for the action of actinomycin D in Staphylococcus aureus. 

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