Structural basis for the DNA affinity of bleomycins

Abstract

The role of the bithiazole moiety of bleomycin in the interaction of the antibiotic with DNA has been studied by the use of synthetic bithiazole derivatives. The DNA affinity of individual C-terminal (bithiazole) analogues of bleomycin was measured in terms of the ability of these species to block the binding of bleomycin to DNA, as judged by diminution of the DNA degradation that attends bleomycin binding. DNA degradation was monitored both by release of [3H]thymine from radiolabeled PM-2 DNA and by alteration of bleomycin-treated DNA oligomers of defined sequence derived from Escherichia coli plasmid pLJ3. It was found that the affinity of the bithiazole derivatives for DNA depended on the presence of the bithiazole moiety itself but more importantly on the number and spacing of positively charged groups; 2'-(2-aminoethyl)-2,4'-bithiazole-4-[3-[(4-aminobutyl) amino]propyl]carboxamide (14), having three positively charged groups at neutral pH, was a reasonably effective inhibitor of DNA degradation by bleomycin. Consistent with the importance of the spacing of the positively charged groups, tetrapeptide S (12) was found to be significantly less inhibitory toward DNA degradation by bleomycin than tripeptide S, in spite of their equal number of positively charged groups and the greater structural similarity of the former to bleomycin A2. Bleomycin is known to cleave DNA perferentially at certain sequences. It was shown that the inhibitors employed in this study diminished DNA cleavage proportionately at each cleavage site; no alteration was observed in the specificity of cleavage. A number of the bithiazole analogues employed as inhibitors of bleomycin-mediated DNA degradation were also utilized in fluorescence quenching experiments with calf thymus DNA. Consistent with the belief that these species inhibit bleomycin degradation by competitive binding to the DNA substrate, the best inhibitors exhibited the greatest fluorescence quenching upon admixture of DNA.

Molecular basis for potentiation of bleomycin‐mediated degradation of DNA by polyamines. Experimental and molecular mechanical studies

“…Accordingly, many studies have been devoted to the understanding of the mode of binding of this small molecular fragment of bleomycin. Spectroscopic investigations (Lin and Grollman, 1981;Kross et al, 1982b;Sakai et al, 1982) and computer modelling studies (Miller et al, 1985;Dickerson, 1986) are consistent with the model of binding in which this bleomycin fragment interacts with the minor groove of B form DNA. This interaction may involve intercalation of one thiazole ring only, or partial intercalation of the two rings of the bithiazole system with DNA base pairs (Henichart et al, 1985;Houssin et al, 1986).…”

“…Due to a large difference in the DNA binding constants for 1 and 4, the inhibitory concentrations of 4 are three orders of magnitude smaller than those for 1 under otherwise identical conditions. Additional examples are the bithiazole fragment of bleomycin, closely related analogues (Kross et al, 1982b;Fisher et al, 1985), and distamycin A (Sugiura and Suzuki, 1982), which exhibit the same minor groove specificity as bleomycin. As expected, these compounds inhibit bleomycin degradation by competitive binding to DNA substrate.…”

Degradation of structurally modified DNAs by bleomycin group antibiotics

“…Most of the experiments directed toward understanding the nature of bleomycin-DNA interactions support a partial intercalation model, with most or all of the drug oriented in the minor groove (Kross et al, 1982a,b;Booth et al, 1983;Fisher et al, 1985;Henichart et al, 1985). Because the intercalation by bleomycin must almost certainly involve the bithiazole moiety (Chien et al, 1977;Povirk et al, 1979;Kross et al, 1982a;Kilkuskie et al, 1985), and the ability of the metal chelated N-terminus of BLM to effect transformations at C-4' of deoxyribose (Burger et al, 1980;Giloni et al, 1981;Wu et al, 1983Wu et al, , 1985aMurugesan et al, 1985;Sugiyama et al, 1985aSugiyama et al, ,b, 1986 argues for its localization in the minor groove, the only portion of bleomycin that appears to have the possibility of direct interaction in the major groove is the C substituent attached to the bithiazole. Therefore, to test whether methylation-induced diminution of bleomycin strand scission was caused by direct steric interaction of BLM with the methyl group of 5-methylcytidine in the major groove, the effect of modification of the C substituent was investigated.…”

DNA Strand Scission by Bleomycin Group Antibiotics

“…clinically used mixture of bleomycins, consists of several species that differ only at the C-terminus (10,11), i.e., within the putative DNA binding domain (12)(13)(14). The major constituents of this mixture, bleomycin A2 and bleomycin B2, have been analyzed with respect to their sequence specificity of DNA strand scission; interestingly, no significant differences were observed for these two congeners (15), although a few different lines of evidence suggest that individual C-substituents can influence the nature of bleomycin-DNA interaction (16).…”