Camptothecins inhibit the utilization of hydrogen peroxide in the ligation step of topoisomerase I catalysis.
The antitumor compounds camptothecin and its derivatives topotecan and irinotecan stabilize topoisomerase I cleavage complexes by inhibiting the religation reaction of the enzyme. Previous studies, using radiolabeled camptothecin or affinity labeling reagents structurally related to camptothecin, suggest that the agent binds at the topoisomerase I-DNA interface of the cleavage complexes, interacting with both the covalently bound enzyme and with the +1 base. In this study, we have investigated the molecular mechanism of camptothecin action further by taking advantage of the ability of topoisomerase I to couple non-DNA nucleophiles to the cleaved strand of the covalent enzyme-DNA complexes. This reaction of topoisomerase I was originally observed at moderate basic pH where active cleavage complexes mediate hydrolysis or alcoholysis by accepting water or polyhydric alcohol compounds as substitutes for a 5'-OH DNA end in the ligation step. Here, we report that a H2O2-derived nucleophile, presumably, the peroxide anion, facilitates the release of topoisomerase I from the cleavage complexes at neutral pH, and we present evidence showing that this reaction is mechanistically analogous to DNA ligation. We find that camptothecin, topotecan, and SN-38 (the active metabolite of irinotecan) inhibit H2O2 ligation mediated by cleavage complexes not containing DNA downstream of the cleavage site, indicating that drug interaction with DNA 3' to the covalently bound enzyme is not strictly required for the inhibition, although the presence of double-stranded DNA in this region enhances the drug effect. The results suggest that camptothecins prevent ligation by blocking the active site of the covalently bound enzyme.
Department of Molecular and Structural Biology, University of Aarhus, Denmark.
M
M
Lisby
B O
BO
Krogh
F
F
Boege
O
O
Westergaard
B R
BR
Knudsen
D002166:Camptothecin / Q000494:pharmacology*; D002384:Catalysis / Q000187:drug effects; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D008956:Models, Chemical; D010636:Phenols / Q000378:metabolism; D013379:Substrate Specificity; D013481:Superoxides / Q000378:metabolism; D059004:Topoisomerase I Inhibitors
D010636:Phenols; D059004:Topoisomerase I Inhibitors; D013481:Superoxides; D004247:DNA; D006861:Hydrogen Peroxide; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin