Antitumor Active Drugs as lntercalators of Deoxyribonucleic Acid Molecular Models of Intercalation Complexes Ulf ~indur,'Manfred Haber, and Kristin Sattler Department of Chemistry and Pharmacy, University of Mainz, Saarstr. 21, D-6500 Mamz 1, Federal Republic of Germany One of the most important cytostatic mechanisms of action of wplanar annelated polycyclic compounds is their intercalation with human bDNA. In this paper, stmturedependent models of this intercalation with DNA as well as that with base-paired oligonucleotides are presented at the molecular level for some cytostatic agents of the anthraquinone and carbazole series as well as for actinomycin D, triostin A, the bleomycins, and amsacrine. The results are based mainly on molecular spectroswpic data in combination with the computer-graphics-aidedmethods of molecular modeling. The application of these procedures that is particularly interesting to teachers and their students is that they also provide a rational foundation for the development of new intercalating drugs. Intercalation Model with Deoxyribonucleic Acid (DNA) Figure 1. Scheme of the intercalation Drocess of Watson-Crick t v ~ The molecular mechanisms of action of clinically used tubDNA. mor-inhibiting active principles are now relatively well understood in their broad outlines (Id).In the present article, details of the molecul a r interactions between intercalating antibiotics, carbazole alkaloids, and other heterocyclic compounds with DNA will be described and exemplified (6). The proposed mechanisms are based principally on molecular spectroscopic results and theoretical considerations. Fmm a rational point of view, the exact description of the active compound/receptor interaction model constitutes an extremely important foundation for the development of new cytostatic agents. In the cases of the numerous anticancer drugs to be discussed here, a relationship between the affinity to DNA(DNA as an endogenous receptor) and the in vitro as well as in vivo antitumor activity has been established unequivocally (5). The experimentally determined association constants Figure 2. Molecular structures of human Watson-Crick bDNA derived by computer graphics: a) CPK bali'Author to whom CorrespOn- and-socket model; b) skeletal structure showing the helical wurse (SYBYL molecular modeling program, dence should be addressed. BIOPOLYMER module (11)).in both cases viewed parallel to the axis of the helix (sideviews).
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of DNA-com lexing drugs are in =' M-'. the range 10i-10" Intercalation of active principles with human DNA (e.g., bDNA) is understood a s the insertion (Latin, intercalare = to insert) of a chromophoric (planar) part of a molecule between two stacked base pairs (Fig. 1). The DNA primary and secondary structures remain intact in this process. The DNA tertiary structure (helix) is partially lengthened and thus somewhat unwound in comparison to the original structure (7, 8). Upon intercalation, the average separation between two stacked base pairs increases from 3.4 to approximately 7-8 As a result of the changed DNA topology, a blockade of the matrix functions occurs a t the biochemical level. Thus, for example, inhibition of RNA polymerase andlor topoisomerase I1 takes place (2). The first experimental evidence for an intercalation with human bDNA was obtained for acridines by means of X-ray fiber diffractometry and hydrodynamic studies (8, 9).
A
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Daunorubicin
A.
In Figure 2, the computer-graphics-supported representations of Watson-Crick bDNA (bDNA constitutes the more strongly hydrated basic form in comparison 4 to the less hydrated, paracrystallAclarubiiin ine aDNA) are shown to illustrate the molecular shapes. The geometries were derived from fiber and single crystal X-ray diffraction structural analyses (8, 10).Figure 2a shows the ball-and-socket model; Figure 2b illustrates the typical right spiralling directionof the Watson-Crick double helix. Single crystal X-ray structural analyses of about 30 intercalation complexes with base-paired oligonucleotides as DNA equivalents (37) as well as molecular calculations have shown that the major contribution to intercalative bind&Y ing in DNA arises from electroOC"3 static, van der Waals, and in vitro, most importantly, hydrophobic 5 forces of interaction. Numerous Nogalamycin physical studies have now provided reliable information about For an optimal intercalation, the planar part of the molthe structural prerequisites for intercalation of a molecule ecule (the chromophore) must possess a minimum surface into DNA(8). In addition to pure a f f i t y analyses such as area of 28 (optimum at three to four riws). The molecviscosimetry (increase), uv measurements (bathochromic ular building blocks of nucleic acids are the nucleotides. In effects), sedimentation rate (lowering of sedimentation the case of an intercalation in base-paired nucleic acid douconstants), and electrophoresis (changesin eledmphoretic ble strands (e,g,, DNA),the torsional angles of the sugar the modern methods of spedmscopy phosphate skeleton change significantly. Together with (X-ray structural analyses, ID-, 2D-, and 3D-NMR techthis so-called sugar-puckering (conformational changes of niques) are of decisive relevance for investigations on the the 2'-deoxyfuranoses, among others by pseudorotation), geometric properties (e.g., localization, configuration, the torsions of the glycosidic angle, X , and the phosphoric add ester angle, P, which increase to more than 50' upon conformation)of the complexes.
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intercalation, are of particular importance (8). In addition, the so-called "unwinding" angle of the helix a, which is generally defined as the difference to the mrresponding basic angle of bDNA [a = 36, (a)],also plays a part. Molecular Modeling Methods for DNA Intercalation Complexes The use of theoretical methods for intercalation analysis in the past few years has provided increasingly detailed insights into molecular properties such as topologies, bind-
Bisanthracycline,
n=1,2; m s - 8
Ellipticine
6K-~lido[4.3-b]carbazole
8
9
7
c'3w
D
9
Dnercalinium 11
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Figure 3. Single crystal X-ray structure of the daunorubicind(CpGpTpApCpG)zcomplex (modifiedfrom (14)). a) View parallel to the base pair plane, b) view practically parallel to the helix axis. In b) only half of the complex is drawn because the molecule complex represents a dyad. ing sites, base selectivities, and energy parameters of the mmplexes. In comparison to single crystal Xray structural analysis which provides only a single (usually an approximation to a local minimum) comolex aeometrv on t h e inElliptinium tercalation energyhypersurface, 10 computer-graphics-supported theoretical methods of "molecular modeling" make it possible to represent complete energy maps, to follow molecular changes visually, and to calculate defined interaction me t i in combination energies. These model with investigamolecu-
lar spectroscopic (experimental) data also smooth the way to "drawing board construction" of new intercalating cytostatic agents. Empirical processes for force fields (molecular mechanics, mo2-a-L-ArabinosyC9-hydroxyelliplicinium bromae lecular dynamics) and semiempi+ 12 cal (or in individual cases, ab initio) chemical methods provide the theoretical background for the methods (12,13). With the currently available highly refined computer graphics that are based on quantum chemical charge calculations, the molecu l a r charge distribution, the molecular electrostatic potential (MEP) (e.g., presented on the van Volume 70 Number 4 April 1993
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Figure 4. (a, above) Intercalation of mitoxantrone (6)in a tetradeoxynucleotide duplex; stereoscopic representation, side view parallel to the axis of the helix (19). (b,at right) Energy-minimized intercalation complex of mitoxantrone (6)and a 2-deoxycytidylyl-3',5'-guanosine duplex [MAXIMIN 2 molecular mechanics calculations with the SYBYL program (II)].The molecular electrostatic potential is visualized on the van der Waals surface ('dotted surface" representation). Color code: blue = negative; green = neutral; yellow, orange, red = positive. Charge calculations were performed according to GasteigerHOckel method. The atomic coordinates of the basepaired dinucleotide were taken from an X-ray analysis (8).
der Waals surface), or the isopotential lines can be visualized clearly on the computer monitor. In this way, further-reaching conclusions can be drawn concerning the intercalative interactions as well as, for e&mple, the dynamic entry process and the orientation of the intercalator in the large or small grooves of DNA. More recently, b i n d i i d i t i e s and sequence selectivities to intercalation have been calculated with the help of special algorithms based on the intramolecular energy [SIBFA/AGNAS procedures (211, the conformational changes, and the (intermolecular) ligand-polynwleotide interaction energies; these results were then compared with the geometries obtained from X-ray analyses. In principle, a maximum of 16 intercalation sites are feasible in bDNA for intercalation with a small foreign molecule. Anthracycline Antibiotics and mitoxantrone
In the series of anthracycline antibiotics, daunorubicin (daunomycin; I), doxorubicin (2), t h e 4'-epimeric epirubicin (3)(less cardiotoxic than doxombicin),the novel aclarubicin (4). and noealamvcin (5) are freouentlv used cytostatic agents. ~ i t k a n t r o n e(6) represents a-novel, comoletelv svnthetic substance with a basic functionalized antdraqu&dne structure. A prerequisite for activity in the anthracycline series of antibiotics is the couplineof a coplanar hydrophobic region (anthraquinone) with a
