Biochemistry zyxwvut 1986, 25, zyxwvu 5933-5940 5933 Stopped-Flow Kinetic Analysis of the Interaction of Anthraquinone Anticancer Drugs with Calf Thymus DNA, Poly[ d(G-C)]ePoly[ d( G-C)], and P~ly[d(A-T>]*Poly[d(A-T)]~ C. R. Krishnamoorthy,t Shau-Fong Yen,* J. C. Smith,$ J. William Lawn,§ and zyxw W. David Wilson*** Department of Chemistry and Laboratory for Microbial and Biochemical Sciences, Georgia State University, Atlanta, Georgia 30303-3083, and Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada Received February 25, 1986; Revised Manuscript Received June 6 , 1986 ABSTRACT: The sodium dodecyl sulfate driven dissociation reactions of daunorubicin (l), mitoxantrone (2), ametantrone (3), and a related anthraquinone without hydroxyl groups on the ring or side chain (4) from calf thymus DNA, poly[d(G-C)],, and poly[d(A-T)], have been investigated by stopped-flow kinetic methods. All four compounds exhibit biphasic dissociation reactions from their DNA complexes. Daunorubicin and mitoxantrone have similar dissociation rate constants that are lower than those for ametantrone and 4. The effect of temperature and ionic strength on both rate constants for each compound is similar. An analysis of the effects of salt on the two rate constants for daunorubicin and mitoxantrone suggests that both of these compounds bind to DNA through a mechanism that involves formation of an initial outside complex followed by intercalation. The daunorubicin dissociation results from both poly[d(G-C)], and poly[d(A-T)], can be fitted with a single exponential function, and the rate constants are quite close. The ametantrone and 4 polymer dissociation results can also be fitted with single exponential curves, but with these compounds the dissociation rate constants for the poly[d(G-C)], complexes are approximately 10 times lower than for the poly [d(A-T)], complexes. Mitoxantrone also has a much slower dissociation rate from poly[d(G-C)], than from poly [d(A-T)] 2r but its dissociation from both polymers exhibits biphasic kinetics. Possible reasons for the biphasic behavior with the polymers, which is unique to mitoxantrone, are selective binding and dissociation from the alternating polymer intercalation sites and/or dual binding modes of the intercalator with both side chains in the same groove or with one side chain in each groove. A large number of DNA1-intercalator binding studies have been conducted, but there have been far fewer studies of the dynamics of the interactions. With their extensive analysis of the kinetics of the interaction of actinomycin analogues with DNA, Muller and Crothers (1968) clearly established the necessity of this type of investigation in developing an un- derstanding of DNA intercalation complexes. In addition, they established a potential link between the intercalation kinetics and the biological effects of the compound as an anticancer drug. The basic idea is that to be active a compound must have both strong binding to DNA and slow dissociation ki- netics. Either of these alone would be insufficient to block polymerase activities in target cells. Although the kinetics of dissociation of only a few inter- calator-DNA complexes have been analyzed in detail, the following general order of dissociation rate constants exists: acridines (Li zyxwvutsrqp & Crothers, 1969; Dourlent & Hogrel, 1976; Ramstein et al., 1976; Marcandalli et al., 1984) > phenan- thridines (Mandal et al., 1980; Ryan & Crothers, 1984; Wilson et al., 1985; Chandrasekaran et al., 1984; Macgregor et al., 1985) > anthracyclines (Wilson et al., 1976; Chaires et al., 1985; Fox et al., 1985) > actinomycin (Muller & Crothers, 1968; Shafer et al., 1980; Fox & Waring, 1984), nogalamycin (Fox et al., 1985). This is only a crude comparison since most of these compounds exhibit complex rate processes with DNA and can have significant base-pair-dependent interaction rates. Mitoxantrone type anthraquinone antitumor drugs (Mur- dock et al., 1979) are interesting from several standpoints: (1) they have shown excellent clinical anticancer activity (Roz- encweig et al., 1983); (2) they have a similar activity spectrum but less severe cardiotoxic effects than anthracyclines (Roz- encweig et al., 1983); (3) they cause large changes in chro- matin structure (Citarella et al., 1982); (4) as a consequence of the positioning of their two cationic substituent chains, they can have two distinct types of binding modes with both side chains in one groove or one side chain in each groove (Islam et al., 1985; Balaji et al., 1985; Lown et al., 1985; Lown & Hanstock, 1985). There is strong evidence that the interaction of mitoxantrone with cellular DNA contributes significantly to its specific cytotoxic action (Lom et al., 1984, 1985). The molecular relationship of structural changes in mitoxantrone derivatives to changes in DNA interactions and effects on activity are not known. We have recently investigated the mode of binding, binding strength, and binding specificity for mitoxantrone and a series of related derivatives (Lown et d., 1985). As might be expected from their structure, these compounds bind to DNA by intercalation. They show binding selectivity for G/C base pairs, but structural variations have significant effects on binding strength. Mitoxantrone, for example, with two hydroxyl groups on the substituted an- thraquinone ring system, has a binding constant similar to that This research was supported by grants from the NIH (GM 30267 to W.D.W.) and the National Cancer Institute of Canada (to J.W.L.) and by a contract between J.W.L. and the National Foundation of Cancer Research. ‘Georgia State University. 8 University of Alberta. 0006-2960/86/0425-5933$01.50/0 I Abbreviations: DNA, deoxyribonucleic acid; PIPES, piperazine- N,N’-bis(2-ethanesulfonic acid); poly[d(A-T)],, poly[d(A-T)].poly [d(A- T)] ; poly[d(G-C)],, poly[d(G-C)].poly[d(G-C)]; SDS, sodium dodecyl sulfate; EDTA, ethylenediaminetetraacetic acid; 2D, two dimensional; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; SSR, sum of the squared residuals. 0 1986 American Chemical Society