INHIBITION OF TOPOISOMERASE-I FUNCTION BY CORALYNE AND 5,6-DIHYDROCORALYNE

The antitumor agent coralyne and a number of structural analogues were found to be inhibitors of DNA topoisomerase I and were characterized biochemically. Several of these analogues stabilized the covalent bina ry complex formed between calf thymus topoisomerase I and pSP64 plasmi d DNA; coralyne and 5,6-dihydrocoralyne had the greatest potency as in hibitors in this assay. In common with camptothecin, the effects of co ralyne and 5,6-dihydrocoralyne were reversed in the presence of increa sing salt concentration or temperature, consistent with the interpreta tion that both functioned mechanistically in a fashion analogous to ca mptothecin. The sequence specificity of DNA cleavage by coralyne and 5 ,6-dihydrocoralyne was also studied in comparison with camptothecin us ing a 471-bp DNA duplex as a substrate for topoisomerase I. Seven site s of cleavage were apparent, four of which were shared in common by co ralyne, 5,6-dihydrocoralyne and camptothecin. Coralyne and 5,6-dihydro coralyne produced cleavage at one sequence, 5'-TCTC down arrow GTAA-3' , that was not apparent in the presence of camptothecin; corresponding ly, two cleavage bands appeared only when camptothecin was present. Co ralyne and 5,6-dihydrocoralyne also inhibited topoisomerase I-mediated relaxation of supercoiled plasmid DNA. Coralyne was the most potent i nhibitor of DNA relaxation; the effects of camptothecin and 5,6-dihydr ocoralyne were roughly equal. At high concentrations, coralyne complet ely suppressed the formation of the topoisomerase I-DNA covalent binar y complex.