Topoisomerase Poisoning Activity of Novel Disaccharide Anthracyclines FULVIO GUANO, PHILIPPE POURQUIER, STELLA TINELLI, MONICA BINASCHI, MARIO BIGIONI, FABIO ANIMATI, STEFANO MANZINI, FRANCO ZUNINO, GLENDA KOHLHAGEN, YVES POMMIER, and GIOVANNI CAPRANICO Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy (F.G., S.T., Mo.Bi., F.Z., G.C.); Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (P.P., G.K., Y.P.); and Menarini Ricerche Sud, Pomezia, Italy (Ma.Bi., F.A., S.M.) Received September 25, 1998; accepted April 14, 1999 This paper is available online at http://www.molpharm.org ABSTRACT Doxorubicin and idarubicin are very effective anticancer drugs in the treatment of human hematological malignancies and solid tumors. These agents are well known topoisomerase II poisons; however, some anthracycline analogs recently have been shown to poison topoisomerase I. In the present work, we assayed novel disaccharide analogs and the parent drug, ida- rubicin, for their poisoning effects of human topoisomerase I and topoisomerases II and II. Drugs were evaluated with a DNA cleavage assay in vitro and with a yeast system to test whether the agents were able to poison the enzymes in vivo. We have found that the test agents are potent poisons of both topoisomerases II and II. The axial orientation of the second sugar relative to the first one of the novel disaccharide analogs was shown to be required for poisoning activity and cytotoxic- ity. Interestingly, idarubicin and the new analogs stimulated topoisomerase I-mediated DNA cleavage at low levels in vitro. As expected, the cytotoxic level of the drug was highly affected by the content of topoisomerase II; nevertheless, the test agents had a yeast cell-killing activity that also was weakly dependent on cellular topoisomerase I content. The results are relevant for the full understanding of the molecular mechanism of topoisomerase poisoning by anticancer drugs, and they define structural determinants of anthracyclines that may help in the rational design of new compounds directed against to- poisomerase I. Human DNA topoisomerases I and II are the targets of several compounds with antitumor activity (Gupta et al., 1995; Capranico et al., 1997). These agents interfere with enzyme functions by stabilizing a reaction intermediate, in which DNA strands are cut and covalently linked to tyrosine residues of the protein. This action poisons the enzyme, transforming it into a DNA-damaging agent (Froelich-Am- mon and Osheroff, 1995). The phenomenon of chemical poi- soning of DNA topoisomerases has been conserved during evolution, probably because it is a very efficient mechanism of cell killing. Doxorubicin, an effective agent in the treat- ments of human cancers (Arcamone, 1981), is a potent poison of type II DNA topoisomerases (Binaschi et al., 1998). The molecular action of topoisomerase poisons is peculiar. It is likely that these agents bind to a protein-DNA interface at the enzyme active site, thus hindering strand religation by topoisomerases (Capranico et al., 1997). This view is based on several lines of investigation, including sequence specificity of poison action (Capranico et al., 1997), photolabeling of DNA by cross-reactive poison analogs (Freudenreich and Kreuzer, 1994; Pommier et al., 1995), drug binding data (Shen et al., 1989; Hertzberg et al., 1989, 1990), and struc- tural determinants of drug site selectivity (Capranico et al., 1994a, 1995, 1998). In the case of camptothecin, a topoisom- erase I poison, drug receptor models have been suggested based on the crystal structures of enzyme-DNA complexes (Redinbo et al., 1998) and molecular modeling (Fan et al., 1998). In these models, camptothecin contacts specific amino acid residues as well as the GC base pair (bp) at the +1 position of the cleavage site that was shown to be required for camptothecin activity (Jaxel et al., 1991). It is interesting that poisons such as actinomycin D, into- plicine, saintopin, and others (Trask and Muller, 1988; Was- sermann et al., 1990; Poddevin et al., 1993; Leteurtre et al., 1994; Nabiev et al., 1994; Makhey et al., 1996) can act against both topoisomerases I and II, suggesting that some structural features of the drug receptor or the molecular mechanisms are shared by the two enzymes. Among anthra- cycline-related molecules, 3'-morpholinyl-doxorubicin, noga- lamycin, and aclacinomycin A (Fig. 1) have been shown to be poisons of topoisomerase I but not of topoisomerase II (Was- sermann et al., 1990; Nitiss et al., 1997; Sim et al., 1997). These findings point to the possibility of modifying the an- This work was supported in part by a research grant to G.C. from Associa- zione Italiana per la Ricerca sul Cancro, Milan, Italy. ABBREVIATIONS: SV40, simian virus 40; bp, base pair. 0026-895X/99/010077-08 Copyright © 1999 by U.S. Government All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 56:77–84 (1999). 77 at ASPET Journals on February 6, 2016 molpharm.aspetjournals.org Downloaded from