Chapter 10 M o l e c u l a r Recognition of DNA by D a u n o r u b i c i n Jonathan B. Chaires
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Department of Biochemistry, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505
Solution studies of the interaction of the potent anticancer drug daunorubicin (daunomycin) with D N A have converged with structural and theoretical studies to provide a coherent picture of the preferential interaction of the antibiotic with the D N A sequences 5'(A/T)GC and 5 ' ( A / T ) C G , where the notation ( A / T ) indicates that either A or Τ may occupy the position. Daunorubicin is unique among monointercalating compounds both in its recognition of a 3 bp site and in its binding to D N A by a mixed mode. The anthraquinone ring system of daunorubicin intercalates, with concomitant unwinding and lengthening of the D N A helix, while the daunosamine moiety is bound within the minor groove. The sequence preference for daunorubicin binding arises from a combination of specific hydrogen bond formation between the drug and D N A base pairs and from a favorable stereochemical fit of the daunosamine in the minor groove in the vicinity of an A T base pair. This chapter summarizes the results of macroscopic studies used to characterize the physical chemistry of the binding of daunorubicin to D N A and discusses newer results obtained by DNAse I footprinting methods that have characterized the preferred D N A binding sites of daunorubicin, which in turn have begun to characterize the microscopic binding properties of the drug. The observed binding behavior is discussed in light of the extensive structural and theoretical results now available. The anthracycline antibiotics have been, and continue to be, important weapons in the chemical arsenal used against cancer. As recently as 1987, doxorubicin (adriamvcin) was the leading anticancer drug in terms of sales in the United States (1). Because of their proven utility in cancer chemotherapy, the anthracycline antibiotics have been intensively studied in attempts to understand the underlying chemical, biochemical, and pharmacological mechanisms that make them effective anticancer agents. Several excellent monographs have summarized much of the wealth of information obtained 0097-6156/95/0574-0156$08.00/0 © 1995 American Chemical Society
Priebe; Anthracycline Antibiotics ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
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10. CHAIRES
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about these important drugs over the last two decades of research (2,3). In spite of this intensive study, the fundamental mechanism(s) by which the anthracycline antibiotics act remain unclear. Further study is necessary to understand how these important drugs work at the molecular level. It is now clear that the mechanisms of action of the parental anthracycline antibiotics daunorubicin (daunomycin) and doxorubicin are pleiotropic (4). Convincing arguments may be made to implicate either D N A or membranes as targets for these drugs or to point to the ability of these compounds to generate free radicals as an integral part of their mode of action. Topoisomerase II has been identified as perhaps the key target for the anthracycline antibiotics (5—11), although the precise molecular mechanism by which inhibition of this key enzyme occurs remains to be defined. Historically, D N A has received considerable attention as an important target for doxorubicin and daunorubicin. Both compounds bind avidly to D N A by the process of intercalation (12—14). D N A binding results in inhibition of both D N A replication and R N A transcription, effects that may be observed both in vivo and in vitro (15—18). It has been argued that the magnitude of observed macroscopic DNA binding constants (» 10 M ) is too low to account for the physiological concentrations at which the anthracycline antibiotics exert their effects (19), and that something other than D N A alone must be the key target of the drugs. More will be said about this later on. Strong evidence implicating D N A as an important target for the anthracyclines has appeared. Valentini, et al, have found a positive correlation between D N A binding affinity and biological activity in studies utilizing 26 different anthracycline antibiotics (20). Microspectrofluorometry has shown that doxorubicin is quantitatively localized into the nuclei of living cells (21). Interestingly, it has been shown that doxorubicin selectively displaces a unique set of nuclear proteins from nuclei, a set distinct from those displaced by actinomycin, mitoxantrone, and amenatrone (22). Finally, recent studies have shown that D N A binding is necessary (but not sufficient) for the inhibition of topoisomerase II by anthracycline antibiotics (9,10). Whether or not D N A is the ultimate cellular target of the anthracyclines, their D N A binding properties are still of intense, fundamental interest to the physical biochemist and the medicinal chemist. One reason for this is that the anthracycline antibiotics have been (until just last year) the only monointercalators for which atomic level crystallographic structural information was available for their complexes with D N A oligonucleotides (23—27). Such structural information makes these compounds, at the least, important models for how intercalators bind to D N A . Knowledge of the structural details of their binding has provided information that may guide the rational design of new compounds with improved D N A binding properties. Kinetic and thermodynamic studies in solution are essential complements to these structural studies, along with synthetic efforts to produce new compounds with systematically altered binding properties. Such solution studies of anthracycline antibiotic binding to D N A have been the primary focus of this laboratory and will be summarized here. 6
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M A C R O S C O P I C BINDING STUDIES O F T H E D A U N O R U B I C I N - D N A INTERACTION Figure 1 shows a binding isotherm for the interaction of daunorubicin with calf thymus D N A . This figure is a composite, and was constructed by combining data from four different laboratories that had studied the binding reaction
Priebe; Anthracycline Antibiotics ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
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ANTHRACYCLINE ANTIBIOTICS
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under identical solution conditions (28—32; Satyanarayana, S., Chaires, J . B . , unpublished data). A variety of experimental approaches have been used to obtain these data, including fluorescence and absorption spectroscopy, equilibrium dialysis, and phase partition methods. The combined data, it is clear in Figure 1, are in excellent agreement, at least at binding ratios (r) above 0.05. Analysis of the data of Figure 1 (neglecting the data below r = 0.05) by the simple neighbor exclusion model of McGhee and von Hippel (33) yields a
r bound F I G U R E 1. Composite binding isotherm for the interaction of daunorubicin with calf thymus D N A at 20°C, pH 7.0, 0.2 M N a . Data were taken from reports from four different laboratories (28—32). Data above r=0.05 were fit to the neighbor exclusion model (33), yielding the best fit shown as the solid line. The filled symbols indicate the points omitted when fitting the data. The inset shows the residuals (experimental data — fit) plotted as a function of the binding ratio r. +
Priebe; Anthracycline Antibiotics ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
10. CHAIRES
binding constant (K) for the interaction of daunorubicin with an isolated D N A site of 6.6 (±0.2) χ 10 M and an exclusion parameter (n) of 3.3 ± 0 . 1 bp. The latter value is consistent with structural studies (23—27) that show that daunorubicin physically covers 3 bp when bound to D N A . It must be emphasized that the binding isotherm shown in Figure 1 is a macroscopic characterization of binding and that the Κ value obtained by the analysis using the neighbor exclusion model is obtained under the assumption of identical and noninteracting binding sites along the D N A lattice. Footprinting studies, to be summarized in a later section, reveal that this assumption is probably invalid. The macroscopic Κ value is, nonetheless, still a useful quantitative measure of binding, if it is kept in mind that it represents a complicated average binding constant that masks a distribution of binding constants for the interaction of daunorubicin at different sequences along the D N A lattice. By studying daunorubicin binding to D N A as a function of temperature, ionic strength, and base composition, it is possible to derive a complete macroscopic thermodynamic profile for the binding interaction. Table I shows such a profile. Several points emerge from Table I. Daunorubicin binding to calf thymus D N A is energetically favorable, with A G = -8.0 kcal mol . The favorable binding free energy results largely from a favorable binding enthalpy ( Δ Η = -12.0 kcal mol" ). The direct measurement, by calorimetry, of the binding enthalpy of daunorubicin for its interaction with a number of natural D N A samples and polynucleotides is an area of recent progress (34—35). The negative entropy that accompanies daunorubicin binding must arise from the complicated interplay of many contributions, including changes in both D N A and antibiotic hydration, ion release, the loss of translational and rotational freedom by the antibiotic upon binding, and D N A conformational changes upon intercalation. 5
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Molecular Recognition of DNA by Daunorubicin
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0
0
-1
1
T A B L E I. Thermodynamic Profile for the Interaction of Daunorubicin with Calf Thymus D N A
a
K= 6.6 (±0.2) χ 105 M - i (20°C) η = 3.3 (±0.1) bp A G O - —RTlnuf = -7.9 kcal m o H (20oC) -12.8 (±2.0) kcal m o H (van't Hoff)
Δ Η 0 =
-1
-11.4 (±0.7) kcal m o l (calorimetry) ASo= -14 cal m o H deg-i (20°C) +
(
