beta-Carbolinedione derivatives as topoisomerase I inhibitors.

Pyrrolo[3,4-c]-beta-carbolinedione dimers 5-14 were synthesized from furo[3,4-c]-beta-carbolinediones and diamines by solvent-free TaCl5/silica catalyzed reaction under microwave irradiation. The inhibitory property of these target compounds, the starting materials 2, 31, 32, and the N-alkylated pyrrolo[3,4-c]-beta-carbolinediones 16, 17, 20-30 was tested against the relaxation of supercoiled pRB322 DNA by calf thymus topoisomerases I and II. Some of these compounds, especially 7 and 23 proved to be selective inhibitors of topoisomerase I.

Binding of 2-azaanthraquinone derivatives to DNA and their interference with the activity of DNA topoisomerases in vitro.

We have investigated the binding ability to DNA of compounds belonging to the 2-azaanthraquinone-type structure and have examined the effect on the activity of DNA gyrase as well as on mammalian topoisomerases in vitro. Using different biophysical techniques it was found that one of these ligands, 9-((2-dimethylamino)ethyl)amino)-6-hydroxy-7-methoxy-5, 10-dihydroxybenzo[g]isoquinoline-5,10-dione (TPL-I), is an intercalating DNA binding agent, whereas the parent compound tolypocladin (TPL) and a derivative (TPL-II) showed almost no similar affinity to DNA. CD measurements demonstrated a significant and selective binding tendency of TPL-I to alternating purine/pyrimidine sequences with some preference for poly(dA-dT). poly(dA-dT). Tm values were increased of the ligand complex with the alternating AT-containing duplex polymer. The binding to various DNAs was characterized by CD and visible absorption spectral changes. From the latter, different binding constants of 6.2 x 10(5) and 1.5 x 10(5) M-1 were obtained for poly(dA-dT).poly(dA-dT) and poly(dA). poly(dT), respectively. Sedimentation measurements with supercoiled pBR322 plasmid DNA clearly indicated an intercalative binding mechanism associated with an unwinding angle of about 18 degrees. These results suggest that the intercalative binding of TPL-I is promoted by the 2-(dimethylamino)ethylamino group substituted on carbon 9 of the anthraquinone system. The cytotoxic compound TPL-I, but not TPL or TPL-II, effectively inhibited the DNA supercoiling reaction of DNA gyrase and the activity of mammalian topoisomerases I and II as measured by the relaxation assay. TPL-I affects the cleavage reaction of topoisomerases on a single site located in alternating purine-pyrimidine sequence regions. The inhibitory potency of TPL-I can be ascribed to a blocking of cleavage sites on the DNA substrate, which correlates with the sequence preference of the ligand.

DNA binding studies and influence on the activity of DNA topoisomerases of bis-netropsins: different effects of analogs containing cis and trans ethylene linkers.

Binding to DNA and synthetic duplex polymers of two bifunctional netropsins and effects on supercoiled plasmid DNA as well as their inhibitory potency on DNA topoisomerases have been investigated. Characteristic differences were found in the DNA binding properties of the two bis-netropsins containing a cis and trans tether as reflected by CD, thermal melting and sedimentation measurements. CD results indicate, that the bis-netropsins interact with DNA by a two-step binding mode depending on the ligand concentration. The trans bis-netropsin may form stable complexes with different DNA's at high salt concentration, whereas for cis bis-netropsin DNA complexes the second binding step is completely abolished. The variations in the DNA binding ability of trans and cis bis-netropsin show a close relationship to the differences observed in their inhibitory effects on DNA topoisomerases. It appeared that trans bis-netropsin more strongly blocks topoisomerase activity than the cis isomer and represents the most potent inhibitor of DNA gyrase. Differences in the DNA. binding ability of the bis-netropsins and their inhibitory potency on topoisomerase activity are explained in terms of bidentate and monodentate binding mode of the trans and cis isomer, respectively.

DNA topoisomerases from Streptomyces noursei: influence of coumarins and quinolones on the enzymic activity.

DNA topoisomerases have been purified from Streptomyces noursei. DNA gyrase and topoisomerase I show a differential sensitivity against quinolones and coumarins compared to the E. coli enzymes. Streptomyces gyrase is resistant to much higher levels of various drugs than is the E. coli enzyme. The observed differences between the gyrases from streptomycetes and E. coli are discussed in the light of present literature data.

Studies on the ability of minor groove binders to induce supercoiling in DNA.

The effect of various non-intercalating minor groove binders on closed circular DNA in the presence of topoisomerase I has been studied by means of agarose gel electrophoresis. Analogues of the netropsin series (lexitropsins) and SN-6999 effectively produce positive supercoils, as indicated by analysis of the topoisomers in the presence of chloroquine and the evaluated linking number changes. Analogues of the distamycin series are less effective, and bisquaternary heterocycles, as well as DAPI and pentamidine, were found to be ineffective ligands. The large differences observed in the ability of minor groove binders to induce positive supercoils are discussed.

Binding to DNA of selected lexitropsins and effects on prokaryotic topoisomerase activity.

The binding behaviour toward DNA of some minor groove binders related to distamycin was studied by means of circular dichroism. In addition their influence on the activity of topoisomerases isolated from Streptomyces noursei has been investigated. The monocationic imidazole containing ligands (lexitropsins) show a decreased affinity to AT pairs but an increased affinity to GC pairs which contrasts the AT-preferred binding of Dst-2 and Dst-3. For the monocationic triimidazole containing lexitropsin the affinity for GC over AT pairs was most pronounced. It was also found that the imidazole containing lexitropsins are inhibitors of topoisomerases. These minor groove binders interfere more strongly with the DNA gyrase activity than with the prokaryotic topoisomerase I. Our results indicate that Dst-3 most effectively inhibits gyrase and topoisomerase I activity. However, the inhibitory effect is neither related to the base pair specificity nor to the binding strength of different ligands. The mechanism of interference of minor groove binders with topoisomerase activity is more complex. It is considered that different factors, such as the nature of the ligand together with their DNA binding parameters and the target sequences of the enzymes play a role in the inhibitory effects of minor groove binders.

Minor-groove binders are inhibitors of the catalytic activity of DNA gyrases.

Non-intercalating DNA minor-groove binders may effectively inhibit the supercoiling activity of gyrases by influencing the enzyme recognition and cleavage site on DNA. For gyrase from Streptomyces noursei a wide range of inhibitory potency for different classes of ligands is observed. This can be explained by a number of structural and binding factors of the ligands competing with the gyrase on the target site of DNA, the mechanism of which is different from the classical gyrase inhibitors.

Microbial DNA topoisomerases and their inhibition by antibiotics.

Supercoiling of bacterial DNA is regulated by topoisomerases and influences most of the metabolic processes involving DNA. The present review is devoted to a brief outline of the supercoiled state of DNA in bacteria and to all microbial topoisomerases hitherto described. Recent studies on topoisomerases of archaebacteria led to the discovery of a so-called reverse gyrase, the properties of which are also discussed. Special emphasis is given to a selective treatment of the effects of those antibiotics which act as gyrase inhibitors.

DNA topoisomerase I from Diplococcus pneumoniae.

A type I topoisomerase has been purified from Diplococcus pneumoniae using phosphocellulose and hydroxylapatite chromatography. The purified enzyme catalyses the relaxation of negatively supercoiled DNA. The relaxation requires Mg2+ and is favoured by 0.2 M monovalent cations. The enzyme does not exhibit catenating or supercoiling activities. Using circular pBR322 DNA from dam+- and dam- -hosts as substrates for the enzyme, the relaxation reaction proceeds with somewhat higher efficiency with plasmids containing methylated adenine in GATC sequences. Plasmids from dcm+- and dcm- -hosts show no difference in reactivity.

[Effect of violamycin BI on the process of excision repair in Escherichia coli K 12 following UV irradiation]. / Einfluxss von Violamycin BI auf den Ablauf der Excisionsreparatur in Escherichia coli K12 nach UV-Bestrahlung.

The influence of violamycin B I on the process of excision repair of DNA-damages after UV-irradiation has been studied by observing the capacity to rejoin single-strand breaks introduced in the DNA at the beginning of the repair process. The number of single-strand breaks remaining unrepaired in the DNA was higher in presence of violamycin B I. Sedimentation analysis of the DNA of unirradiated cells showed in presence of violamycin B I only a small change in the molecular weight. As a possible reason for the lower capacity of the cells to accomplish repair steps following incision in presence of violamycin B I an inhibition of the function of repair enzymes by interaction of the antibiotic with the DNA-template is discussed.

Characterization of the antibiotic resistance plasmid ERL1 from Streptococcus pyogenes.

The streptococcal plasmid ERL1 determining inducible resistance to erythromycin, lincomycin, and staphylomycin S was isolated by dye-buoyant density centrifugation and shown to have a molecular weight of about 17.5 Mdal, as revealed by sedimentation through neutral sucrose gradients. In SM60 cells entering the stationary phase its covalently closed circular form was present to the extent of 5 copies per chromosomal genome equivalent, ERL1 was subject to the DNA restriction and modification mechanism discovered in strain 56188. It did not appear to exercise restriction of phage DNA but mediated a partial release of the restricted growth of A25.