Product analyses in DNA strand scission by antitumor antibiotic elsamicin A.

Elsamicin A is an antitumor antibiotic with fascinating chemical structure and a good candidate for pharmaceutical development. Molecular mechanism of DNA backbone cleavage mediated by Fe(II)-elsamicin A has been examined. Product analysis using DNA sequencing gels and HPLC reveals the production of damaged DNA fragments bearing 3'-/5'-phosphate and 3'-phosphoglycolate termini associated with formation of free base. In addition, hydrazine-trapping experiments indicate that C-4' hydroxylated abasic sites are formed concomitant with DNA degradation by Fe(II)-elsamicin A. The results lead to the conclusion that the hydroxyl radical formed in Fe(II)-elsamicin A plus dithiothreitol system oxidizes the deoxyribose moiety via hydrogen abstraction predominantly at the C-4' carbon of the deoxyribose backbone and ultimately produces strand breakage of DNA.     

Selective DNA cleavage by elsamicin A and switch function of its amino sugar group.

We report guanine-specific recognition and selective cleavage of DNA by the antitumor antibiotic elsamicin A equipped with an amino sugar and compare these results with cleavage by chartarin and chartreusin antibiotics. The preferential cutting sites of DNA strand scission with elsamicin A are on the bases adjacent to the 3'-side of guanine residues such as 5'-GN sites, in particular 5'-GG sites. The present results also indicate that (1) the aglycon portion binds intercalatively to the 3'-side of guanine in host DNA, (2) the guanine 2-amino group has an important effect on selective DNA binding of elsamicin A, and (3) the amino sugar residue of elsamicin A facilitates the drug binding into the minor groove of B-DNA. In addition, we found that an acetylation of the amino group on the elsamicin A sugar portion plays an interesting switch function for the activity of elsamicin A. The biological implication of this switch has also been discussed.     

Studies on binding of toromycin, an antitumor antibiotic, to DNA

Toromycin, an antitumor, bactericidal and antiviral compound, was found to bind to DNA in such a way as to interfere with the dissociation of double helix at an elevated temperature. The antibiotic did not introduce strand scission into DNA. Single-strand-specific nuclease S1-susceptibility of negatively supercoiled DNA was not influenced by its binding. The antibiotic was shown to bind to both of the alternating purine-pyrimidine copolymers, poly(dG-dC):poly(dG-dC) and poly(dA-dT):poly(dA-dT). The unique C-glycoside molecule of toromycin interacted with single-stranded DNA, but was found to have no affinity for RNA.     

Map of chartreusin and elsamicin binding sites on DNA

Three DNA restriction fragments designated tyrT, 102-mer and 70-mer, have been used as substrates for footprinting studies using DNase I in the presence of the structurally similar antibiotics chartreusin and elsamicin A. The sequence-selective binding sites of the antibiotics can be mapped in regions which are rich in guanine + cytosine. Chartreusin and elsamicin appear to recognize and bind preferentially to sequences containing a CpG step. Regions containing a TpG step also seem to be a good binding site. The binding of elsamicin to these sites appears to be more concentration-dependent. A comparative analysis is performed of the sizes and locations of the different binding sites, aimed to infer whether the different biological effects of chartreusin and elsamicin A can be correlated to differences in their sequence-selective binding to DNA.     

Specific binding of chartreusin, an antitumor antibiotic, to DNA

Chartreusin, an antitumor and antibacterial antibiotic, was found to inhibit negatively superhelical DNA-relaxation catalyzed by prokaryotic topoisomerase I and conversion of the superhelical DNA into unit length linear form catalyzed by single-strand-specific S1 nuclease. The inhibitory effect of the agent was due to the binding to DNA causing the alteration of tertiary structure. To characterize the binding specificity, we investigated the protection of DNA against cleavages by various restriction endonucleases. It was evidenced that the binding of the agent is not at random and correlates to the sequence 5'CGC 3' 3'GCG 5' on DNA stretch.     

Amino terminal amino acid sequence of macromomycin, a protein antitumor antibiotic.

Forty-five amino acids at the amino terminus of macromomycin, a protein antitumor antibiotic, have the following sequence: Ala-Pro-Gly-Val-Thr-Val-Thr-Pro-Ala-Thr-Gly-Leu-Ser- Asn-Gly-Glu-Thr-Val-Thr-Val-Ser-Ala-Thr-Gly-Leu-Thr- Pro-Gly-Thr-Val-Tyr-His-Gly-Glu-Ser-Ala-Val-Ala-Glu- Pro-Gly-Val-Ile-Gly-Pro- ..... Of the first 31 residues in this sequence 16 are homologous with the amino terminal sequence in neocarzinostatin, a protein antitumor antibiotic which also degrades DNA. Because of this conservation of structure, this region of the molecule may be involved in the mechanism of action of these proteins.     

Biosynthesis of fredericamycin A, a new antitumor antibiotic

Fredericamycin A (FM A), produced by a strain of Streptomyces griseus, represents a new structural class of antitumor antibiotics containing a spiro ring system. Studies on the producer organism showed that glucose in the fermentation medium is not utilized until late in the growth stage, just prior to synthesis of FM A. [14C]Glucose tracer experiments demonstrated that glucose is incorporated into FM A by catabolism to acetate. Biosynthetic enrichment of FM A with single- and double-labeled [13C]acetate showed that the entire carbon skeleton of the spiro ring system is derived from acetate. L-Methionine was shown to provide the only nonskeletal carbon in FM A, the methoxy carbon at position C-6. The direction of the polyketide chain and the position of the carbon lost during biosynthesis were established by using stable isotope experiments. A general model for FM A biosynthesis is proposed, and a possible scheme for the formation of the spiro carbon center is presented.     

Identification of DNA degradation products by antitumor antibiotic, esperamicin

Esperamicin A1 is a DNA-damaging agent characterized by a unique ten-membered ene-diyne core. We studied the detailed reaction mechanism by using synthetic DNA oligomers. The cleavage site and activity depend on the sequence of the oligomers. d(GGATCC) and d(GGTACC) were cleaved by the drug while d(CCATGG) and d(CCTAGG) were not cleaved under the present conditions. d(GGTACC) gave two major 5'-fragments. The result of partial nuclease digestion experiments suggests that these products are trimer and pentamer with a modified 3'-end.     

Structure of eremosamine--an amino sugar from the antibiotic eremomycin

A new amino sugar named eremosamine was isolated from hydrolysate of eremomycin, an antibiotic belonging to the group of polycyclic glycopeptides. Crystalline derivatives of the amino sugar i. e. methyleremosaminide and methyl-N,O-acetyleremosaminide (alpha- and beta-anomers) were prepared. The data on PMR study and optic properties of the compounds showed that eremosamine had the structure of 2,3,6-tridesoxy-3-amino-3-C-methyl-L-arabinohexose.     

Chartreusin,an antitumor glycoside antibiotic,induces DNA strand scission

The interaction of chartreusin with covalently closed circular PM2 phage DNA was studied. The antibiotic caused a single strand scission in the presence of reducing agents, such as dithiothreitol, ascorbic acid or NaBH₄. The degree of DNA breakage was dependent upon the drug concentration. The DNA-cleaving activity was enhanced by ferrous ion; but was completely blocked by catalase and partially by superoxide dismutase. The results suggest that reduction, chelate formation and auto-oxidation of the antibiotic, presumably the 5,12-dione moiety, produce free radicals, including $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and ^?OH, which are capable of inducing DNA strand scission.     

Naphthalimide intercalators with chiral amino side chains: effects of chirality on DNA binding, photodamage and antitumor cytotoxicity

Several novel heterocyclic-fused naphthalimides intercalators with chiral amino side chains were investigated. Their side chains' chiral configuration determines DNA binding activities in the order: S-enantiomers > R-enantiomers. And their DNA photodamaging activities were in good agreement with their DNA binding constants, the S-enantiomers could photocleave circular supercoiled pBR322 DNA more efficiently than their R-enantiomers. S-enantiomer B(3) could photodamage DNA at 0.2 microM and cleave supercoiled plasmid DNA from form I to form II completely at 50 microM. Almost all of these intercalators showed effective cytoxicities against human lung cancer cells and murine leukemia cells. S-enantiomers showed different antitumor cytotoxicity by comparison with R-enantiomers. This work may provide additional information for the role of amino side chains on intercalators as antitumor agents.     

Effect of the antitumor antibiotic bleomycin on DNA polymerase activity

Treatment with bleomycin activates considerably a repair synthesis of DNA in rat liver chromatin in vitro and can cause loosening of the nucleoprotein complex, which facilitates the accessibility or repair enzymes for lesions in chromatin DNA. The bleomycin action on DNA-template increases severalfold the rate of synthesis catalyzed by DNA polymerase beta inhibits the activity of DNA polymerase I from Escherichia coli and suppresses severalfold the activity of DNA polymerase alpha and DNA polymerase of bacteriophage T4. The effect of bleomycin consists in a prevailing increase of nicks and minimal gaps in DNA as compared to the rise of moderate gaps, thus suggesting that bleomycin is a gamma-mimetic.     

Molecular structure, conformation and interactions of antitumor antibiotic cyanonaphthridinomycin, a covalent binder of DNA

X-ray, NMR and molecular modeling studies on cyanonaphthridinomycin (C22H26N4O5), a DNA binding antibiotic, have been carried out to study the structure, conformation and interactions with DNA. The crystals belong to the space group P21 with the cell dimensions of a = 5.934(1)b = 20.684(4), c = 16.866(3)A, gamma = 90.9 degrees and Z = 4(two molecules/asymmetric unit). The structure was solved by direct methods and difference Fourier methods and refined to an R value of 0.087 for 4061 reflections. The conformation of the molecule is compared with that of naphthridinomycin. There are differences in the orientation of the methoxyl group and the saturated oxazole ring. 1 and 2D NMR studies have been carried out and the dihedral angles obtained from coupling constants have been compared with those obtained from the crystal structure. Molecular mechanics studies were carried out to obtain the energy minimized structure and its comparison with X-ray and NMR results. Molecular modelling studies were performed to propose models for drug-DNA interactions. Both partial intercalation and groove-binding models have been proposed.     

Experimental and modelling studies on the DNA cleavage by elsamicin A.

The ability of elsamicin A, an antitumour antibiotic, to cleave DNA in the presence of ferrous iron and reducing agents, has been analysed using experimental and theoretical approaches. Experimentally, the antibiotic causes DNA breakage in the presence of ferrous ions and a reducing agent. The DNA-cleaving activity appears to be partially blocked by the action of superoxide dismutase and catalase. These results indicate that the elsamicin aglycone moiety (chartarin) can be involved in the production of free radicals. We have performed a broad theoretical study based in the quantum-mechanical framework, which allow us to determine the redox properties of elsamicin that lead to the generation of radical species. Our results clearly show that elsamicin acts as a true catalyst in the production of superoxide radicals. Moreover, it is suggested that the oxidation/reduction mechanism of the aglycone moiety of elsamicin (a lactone), leading to DNA breakage, is different from the mechanism followed by other well-known anti-cancer drugs, whose chromophore is a quinone.     

Covalent modification and single-strand scission of DNA by a new antitumor antibiotic kapurimycin A3

Kapurimycin A3 is a new antitumor antibiotic isolated from a Streptomyces. It contains the anthrapyrone skeleton and a beta,gamma-unsaturated delta-keto carboxylic acid moiety in the structure. In vitro, kapurimycin causes single-strand cleavage of supercoiled pBR322 DNA. The diminished cytotoxicity and DNA cleaving activity for 13-decarboxykapurimycin A3 indicates that the beta, gamma-unsaturated delta-keto carboxylic acid moiety is important for the activity of kapurimycin. Kapurimycin A3 binds to calf thymus DNA at 4 degrees C, and the thermal treatment of this adduct results in release of a guanine covalently attached to C-16 of kapurimycin via one of its nitrogen atoms. Thus, the epoxide is the alkylating functional group of kapurimycin, and this is consistent with the lack of DNA cleaving and cytotoxic activities for 14,16-deoxy-14,16-dihydroxykapurimycin. These findings have revealed that DNA strand scission by kapurimycin is due to the alkylation of guanine by ring opening of the epoxide group of kapurimycin, depurination of modified guanine, and presumably subsequent hydrolysis of the phosphate ester backbone at the resultant apurinic sites.     

链霉菌AC-57及其产生的阿克拉霉素A的研究

During the course of screening for new antitumor antibiotics, a new anthracycline antibiotic--aclacinomycin A was separated from the broth and mycelium of Streptomyces AC-57. The strain AC-57 was isolated from the soil collected in the Shanghai suburbs. According to its culture and physiological characteristics the producer was identified as Str. galilaeus AC-57. The broth and mycelium were extracted and treated with solvents as usual way. The aclacinomycin A was separated by silica-gel column chromatography eluted with chrolo-form-methanol. Aclacinomycin A, its aglycone and sugar components were identified by comparison of their physico-chemical and spectral data (MS, UV, IR, 1H-NMR, and 13C-NMR) with authentic compound, purified from the market sample.     

Visible light induced DNA cleavage by the hybrid antitumor antibiotic dynemicin A

Dynemicin A, which is a hybrid antitumor antibiotic containing anthraquinone and enediyne cores, effectively breaks DNA strands upon irradiation with visible light of long wavelength. The preferential cutting sites of visible light induced DNA cleavage with dynemicin A are on the 3'-side of purine bases such as in 5'-AT and 5'-GT sequences. The observed nucleotide cutting specificity is remarkably similar to that of NADPH- (or thiol) induced DNA breakage with dynemicin A, suggesting the presence of the same DNA-cleaving intermediate. Indeed, the photoproduct of dynemicin A is chromatographically identical with the reaction product (dynemicin H) of the thiol-activated dynemicin A. On the basis of the present results, a reasonable mechanism for the visible light induced DNA cleavage of dynemicin A has been proposed.     

Biosynthetic studies on saframycin A, a quinone antitumor antibiotic produced by Streptomyces lavendulae

The biosynthesis of saframycin A, a heterocyclic quinone antitumor antibiotic isolated from Streptomyces lavendulae 314, was studied by feeding experiments with 14C and 13C precursors. Highly increased production of saframycin A and prolongation of the maximum production period of saframycin A were attained by constant pH control of the culture and by addition of chloramphenicol to the culture. The biosynthetic origin of the quinone skeleton common to the saframycin group was confirmed to be two tyrosine molecules which condense to generate the basic ring system of saframycin A. Feeding experiments with [1-13C]tyrosine showed specific labeling of C-11 and C-21 carbons of saframycin A, and the enrichment of the carbons was 40-fold over natural abundance. Two O- and two C-methyl and one N-methyl carbons arose directly from methionine, and alanine and glycine were the precursors for the pyruvoyl amide side chain of saframycin A.     

DNA strand scission by the novel antitumor antibiotic leinamycin

Leinamycin is a recently discovered antitumor antibiotic with an unusual 1,3-dioxo-1,2-dithiolane structure. It preferentially inhibits the incorporation of [3H]thymidine into the acid-insoluble fraction of Bacillus subtilis. In vitro, leinamycin causes single-strand cleavage of supercoiled double-helical pBR322 DNA in the presence of thiol cofactors. Scavengers of oxygen radical did not supress the DNA-cleaving activity. Thiol-activated leinamycin binds calf thymus DNA at 4 degrees C and thermal treatment of the leinamycin-DNA adduct released a chemically modified leinamycin from the complex. The lack of cytotoxicity and DNA-cleaving activity for S-deoxyleinamycin indicates that the 1,3-dioxo-1,2-dithiolane moiety is essential for the activity of leinamycin. Thus, the primary cellular target of leinamycin appears to be DNA. It binds DNA and causes single-strand break at low concentrations, which may account for the potent antitumor activity.