THE ROLE OF THE AMINOSUGAR AND HELIX BINDING IN THE THIOL-INDUCED ACTIVATION OF CALICHEAMICIN FOR DNA CLEAVAGE

摘要

The diynene antitumor antibiotic calicheamicin gamma(1)(I) (CLM gamma(1)(I)) cleaves DNA in the presence of thiols and molecular oxygen. The proposal that interaction of CLM gamma(1)(I) with DNA enhances the rate of this cleavage process has been addressed. The kinetics of CLM activation for DNA cutting by aminoethanethiol and glutathione (GSH) have been investigated for the drug free in solution and primarily bound to DNA. The second-order rate constants for the disappearance of the trisulfide CLM gamma(1)(I) and for the slower reaction of the principal disulfide intermediate in the activation process have been determined and reveal that both of these reactions are slower in the presence of DNA. In earlier solution studies the second-order reaction rate of CLM gamma(1)(I) was compared to the rates measured for N-acetylCLM and CLM alpha(3), derivatives lacking an internal free amine. Little difference was observed among these rates, a finding inconsistent with the contention that the ethylamino sugar serves as a general base in the activation process. The absence of intramolecular amine participation in thiol activation concluded from these rate comparisons has been reinforced by reactions of these CLM derivatives in the presence of DNA. Again no comparative rate advantage was seen for CLM gamma(1)(I). The validity of the continuous UV assay used in these experiments to monitor the reaction of the intermediate GSH-CLM disulfide was confirmed by direct kinetic measurements of the mixed disulfide itself and by independent PAGE cleavage assays. Recent claims that the calicheamicins are not soluble under the conditions used in these experiments are refuted by four independent experimental means including light scattering, UV spectral comparisons, centrifugation experiments and adherence to Beer's Law. The present studies permit a much simpler picture to be drawn of the reductive activation process and the roles played by the aminosugar and DNA interaction than previously proposed.