Poisoning of topoisomerase I by an antitumor indolocarbazole drug: Stabilization of topoisomerase I-DNA covalent complexes and specific inhibition ofthe protein kinase activity

We have investigated the mechanism of topoisomerase I inhibition by an indo locarbazole derivative, R-3, The compound is cytotoxic to P388 leukemia cel ls, but not to P388CPT5 camptothecin-resistant cells having a deficient top oisomerase I. R-3 can behave both as a specific topoisomerase I inhibitor t rapping the cleavable complexes and as a nonspecific inhibitor of a DNA-pro cessing enzyme acting via DNA binding. In addition, the drug is a potent in hibitor of the kinase activity of topoisomerase I, Unlike camptothecin, R-3 completely inhibits the phosphorylation of SF2/ASF, a member of the SR pro tein family, in the absence of DNA, The inhibitory effect is also observed using mutant enzyme Y723F that lacks DNA cleavage/religation activity but d oes not affect phosphotransferase activity, indicating, therefore, that R-3 acts independently at both DNA cleavage and protein kinase sites. R-3 is t he only compound known thus far that interferes specifically with the kinas e activity of topoisomerase I and not with other kinases, such as protein k inase C and the cdc2 kinase, The study reinforces the view that topoisomera se I is a dual enzyme with a DNA cleavage site juxtaposed to a functionally independent kinase site and shows for the first time that indolocarbazole drugs can inhibit both the DNA cleavage/religation and kinase activities of the enzyme.