ENZYMATIC AND CHEMICAL FOOTPRINTING OF ANTHRACYCLINE ANTITUMOR ANTIBIOTICS AND RELATED SACCHARIDE SIDE-CHAINS

DNase I and three DNA chemical footprinting agents were used to compar e the DNA binding properties of the anthracycline antitumor antibiotic s daunomycin, aclacinomycin A, and ditrisarubicin B. These anthracycli nes contain a tetracyclic chromophore which intercalates into DNA and a monosaccharide, trisaccharide, and two trisaccharide side chains, re spectively. These side chains consist of between one and three 2,6-did eoxy, 1,4-diaxially linked sugars. Three chemical probes, fotemustine, dimethyl sulfate, 4-(2'-bromoethyl)phenol, and the enzymic probe DNas e I were used in the footprinting experiments. The chemical probes pro vided a clear picture of the binding pattern at 37 degrees C and more detailed information than that obtained using the standard DNase I foo tprinting assay. All three anthracyclines showed preferred binding to 5'-GT-3' sequences in both the chemical and enzymatic footprinting. DN ase I footprinting showed that the number of base pairs of DNA protect ed from cleavage increased with the number of saccharide groups presen t at particular sites and is consistent with DNA binding of the saccha ride side chains. Alkylation of runs of guanine by fotemustine was inh ibited by all three anthracyclines, while alkylation by dimethyl sulfa te was enhanced for most guanines. The probe 4-(2'-bromoethyl)phenol s howed that all three anthracyclines completely protected all of the ad enines in the minor groove from alkylation, and enhanced major groove guanine alkylation was observed with aclacinomycin A, daunomycin, and, to a much lesser extent, ditrisarubicin B. These results are consiste nt with intercalation of the aglycone ring and binding of the rigid, h ydrophobic saccharide side chains in the minor groove. Footprinting of four methyl glycosides related to the anthracyclines showed no eviden ce of DNA binding with any of the agents studied.