ELUCIDATION OF THE STRUCTURE OF THE CC-1065-DNA ADDUCT AND DETERMINATION OF THE DNA SEQUENCE SPECIFICITY OF CC-1065 AND THE PYRROLO(1,4)BENZODIAZEPINE ANTITUMOR ANTIBIOTICS (ANTHRAMYCIN, TOMAYMYCIN, SIBIROMYCIN)

CC-1065 and the pyrrolo(1,4)benzodiazepines are potent antitumor antibiotics whose antineoplastic effects are believed to be due to their ability to bind covalently to DNA (at N3 of adenine for CC-1065 and at N('2) of guanine for the pyrrolo(1,4)benzodiazepines). The reactions of these agents with DNA is studied with the goals of (1) elucidating the structure of the CC-1065-DNA adduct and (2) determining the DNA sequence specificity of these drugs. Thermal treatment of CC-1065-DNA adducts leads to DNA strand breakage. It is shown that the CC-1065 modification of DNA which leads to strand breakage is distinct from the primary alkylation event. Relative to the covalent binding site, DNA strand breakage occurs: (1) on the 3' side; (2) about one deoxyribose phosphate residue removed; and (3) on the same strand. No residual modification of DNA is detected on the opposite strand around the CC-1065 lesion. Using the early promoter element of SV40 DNA as a target, the DNA sequence specificity of CC-1065 has been examined. A consensus sequence analysis of CC-1065 binding sites on DNA reveals two distinct classes of sequences of which CC-1065 is highly specific, i.e., 5'PuNTTA and 5'AAAAA. The orientation of the DNA sequence specificity relative to the covalent binding site provides a basis to predict the polarity of drug binding in the minor groove. Stereo drawings of the CC-1065-DNA adduct are proposed which are predictive of features of the CC-1065-DNA adduct elucidated in this investigation. The sequence specificity of the pyrrolo(1,4)benzodiazepines are examined by two methods. In the first, sibiromycin and tomaymycin binding sites are detected by altered methylene blue-induced scission of DNA. In the second method, MPE "footprinting" reveals high affinity binding sites of anthramycin, tomaymycin and sibiromycin. The sequence specificities of these drugs are demonstrated to be 5'GG for anthramycin, 5'GG for tomaymycin and 5'GPu for sibiromycin. It is concluded that an ability selectively to target biologically important DNA sequences is probably not the basis for the potent antitumor effects of drugs in the pyrrolo(1,4)benzodiazepine class. Alternative hypotheses regarding the molecular basis for the potencies of these agents are discussed.