The Role of the Aminosugar and Helix Binding in the Thiol-Induced Activation of Calicheamicin for DNA Cleavage
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- 作者：Moneesh Chatterjee ; Paul J. Smith ; and Craig A. Townsend
- 刊名：Journal of the American Chemical Society
- 出版年：1996
- 出版时间：February 28, 1996
- 年：1996
- 卷：118
- 期：8
- 页码：1938 - 1948
- 全文大小：436K
- 年卷期：v.118,no.8(February 28, 1996)
- ISSN：1520-5126
文摘
The diynene antitumor antibiotic calicheamicin
1I (CLM1I)cleaves DNA in the presence of thiols andmolecular oxygen. The proposal that interaction ofCLM1I with DNA enhances the rate of thiscleavage processhas been addressed. The kinetics of CLM activation for DNA cuttingby aminoethanethiol and glutathione (GSH)have been investigated for the drug free in solution and primarilybound to DNA. The second-order rate constantsfor the disappearance of the trisulfideCLM1I and for the slower reaction of theprincipal disulfide intermediate inthe activation process have been determined and reveal that both ofthese reactions are slower in the presence ofDNA. In earlier solution studies the second-order reaction rate ofCLM1I was compared to the rates measuredforN-acetylCLM and CLM
3, derivatives lacking aninternal free amine. Little difference was observed amongtheserates, a finding inconsistent with the contention that the ethylaminosugar serves as a general base in the activationprocess. The absence of intramolecular amine participation inthiol activation concluded from these rate comparisonshas been reinforced by reactions of these CLM derivatives in thepresence of DNA. Again no comparative rateadvantage was seen for CLM1I. Thevalidity of the continuous UV assay used in these experiments tomonitor thereaction of the intermediate GSH-CLM disulfide was confirmed by directkinetic measurements of the mixed disulfideitself and by independent PAGE cleavage assays. Recent claims thatthe calicheamicins are not soluble under theconditions used in these experiments are refuted by four independentexperimental means including light scattering,UV spectral comparisons, centrifugation experiments and adherence toBeer's Law. The present studies permit amuch simpler picture to be drawn of the reductive activation processand the roles played by the aminosugar andDNA interaction than previously proposed.
1I (CLM1I)cleaves DNA in the presence of thiols andmolecular oxygen. The proposal that interaction ofCLM1I with DNA enhances the rate of thiscleavage processhas been addressed. The kinetics of CLM activation for DNA cuttingby aminoethanethiol and glutathione (GSH)have been investigated for the drug free in solution and primarilybound to DNA. The second-order rate constantsfor the disappearance of the trisulfideCLM1I and for the slower reaction of theprincipal disulfide intermediate inthe activation process have been determined and reveal that both ofthese reactions are slower in the presence ofDNA. In earlier solution studies the second-order reaction rate ofCLM1I was compared to the rates measuredforN-acetylCLM and CLM3, derivatives lacking aninternal free amine. Little difference was observed amongtheserates, a finding inconsistent with the contention that the ethylaminosugar serves as a general base in the activationprocess. The absence of intramolecular amine participation inthiol activation concluded from these rate comparisonshas been reinforced by reactions of these CLM derivatives in thepresence of DNA. Again no comparative rateadvantage was seen for CLM1I. Thevalidity of the continuous UV assay used in these experiments tomonitor thereaction of the intermediate GSH-CLM disulfide was confirmed by directkinetic measurements of the mixed disulfideitself and by independent PAGE cleavage assays. Recent claims thatthe calicheamicins are not soluble under theconditions used in these experiments are refuted by four independentexperimental means including light scattering,UV spectral comparisons, centrifugation experiments and adherence toBeer's Law. The present studies permit amuch simpler picture to be drawn of the reductive activation processand the roles played by the aminosugar andDNA interaction than previously proposed.