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Proc. Int. Symp. Biomol. Struct. Interactions, Suppl. J. Biosci., Vol. 8, Nos 3 & 4, August 1985, pp. 689–697 © Printed in India. Conformational aspects of drug-DNA interactions: Studies on anthracycline antibiotics and psoralen derivatives M. PALUMBO, L. CAPASSO, G. PALÙ* and S. MARCIANIMAGNO † Biopolymer Research Centre, CNR, Institute of Organic Chemistry, Via Marzolo 1–35100 Padova, Italy * Institute of Microbiology, University of Padova, Medical School, Via Gabelli 63–35100 Padova, Italy † Centro di Studi sulla Chimica del Farmaco e dei Prodotti Biologicamente Attivi, CNR, Department of Pharmaceutical Sciences, Via Marzolo 5–35100 Padova, Italy Abstract. The interaction of anticancer agents, analogues of adriamycin and of photochemotherapeutic compounds of the psoralen structural type with DNA was investigated using spectroscopic, hydrodynamic and chiroptical techniques. The nucleic acid may undergo conformational changes from the Β form to more compact structures as a result of the binding process to charged compounds. Different complex geometries adopted byvarious drugs were observed and discussed in terms of intercalation into the polynucleotide double helix and orientation of the ligand in the base-pair pocket. The binding of chemotherapeutic agents to functionally organized DNA was also studied. Lower binding affinities and modified spectral responsesareindicativeofdifferent drug-DNA complexation patternsinthiscase. The results of these studies allow a better understanding of drug-nucleic acid interactions at a molecular level. Keywords. Chemotherapy; photochemotherapy; DNA, interaction; conformation. Introduction The biological activity of a number of drugs, causing inhibition of cellular DNAdependent replication and transcription processes, may often be explained in terms of a physical interaction with DNA, so as to distort its structure and function (Waring, 1981). In many instances a direct relationship has been found between complex formation ability and antibacterial or antitumor properties of the drugs, thus supporting the above point of view (Arlandini et al., 1977). Important chemotherapeutic agents such as anthracycline antibiotics interact with DNA by intercalative processes. In fact their flat polycyclic aromatic ring system may slip between the stacked base-pairs of the DNA double helix, leading to the observed biological effects (Waring, 1970). For the photochemotherapeutic drugs of the psoralen structural type, the same binding mechanism leads to the formation of a reversible adduct. Subsequent irradiation with UV-Α light causes a covalent conjugation with pyrimidine bases (in particular thymine) of DNA (Song and Tapley, 1979). The photoreaction consists of a C4 cyclo addition between the pyrimidine 5,6 double bond and the 3,4 and/or 4',5' double bonds of the drug. Further contributions to complex stability both in chemotherapeutic and photochemotherapeutic compounds may arise from positively charged groups bound to the drug, as to allow electrostatic and/or hydrogen bonding interactions with the polynucleotide backbone. We recently synthesized a number of 689 690 Palumbo et al. anthracenedione and psoralen derivatives, sharing the key features of a planar polycyclic structure and a linear aliphatic side chain with positively charged terminal groups. These compounds are reported in figure 1. In the present paper we will focus on the conformational aspects of drug–DNA interactions, in particular considering the intercalation process into the polynucleotide double helix and ligand orientation in the base pair pocket. The binding of the above chemotherapeutic agents to functionally organized DNA will be finally presented as a convenient approach to the investigation of in vivo interactions. Figure 1. Chemical structures of examined compounds. Conformational aspects of drug-DNA interactions 691 Materials and methods Materials The synthesis and characterization of compounds I and II will be reported elsewhere. Compounds III–V were obtained as described (Antonello et al., 1979). Adriamycin hydrochloride was kindly provided by Farmitalia (Italy). Calf thymus DNA was purchased from Sigma Chemical Co. (USA) and purified as described (Palumbo and Marciani, 1983). Sonicated calf thymus DNA, with =4·3·105, was obtained according to literature procedures (Doty et al., 1958). Soluble chromatin was isolated from calf thymus nuclei (Tatchell and Van Holde, 1977). H1-depleted nucleosomes were prepared from frozen calf thymus glands (Chaires et al., 1983). Both preparations were dialysed extensively against 0·01 Μ Tris and 10 – 3 Μ EDTA, pH 7·0. All physicochemical measurements were performed in this buffer at 25°C. Methods Spectrophotometric measurements were performed in a Perkin Elmer Lambda 5 instrument, equipped with a Haake F3C thermostate. Fluorimetric results were obtained on a Perkin Elmer MPF2A instrument. Experiments were usually performed by addition of known amounts of DNA to solutions containing a given concentration of the ligand. Alternatively, known amounts of drug were added at constant DNA concentration. In the equilibrium dialysis measurements DNA solutions were dialyzed against equal volumes of solvent containing the examined compounds, using Thomas dialysis tubing (12,000 D cutoff). Equilibrium was usually reached within 18 h. The concentration of free ligand in the dialysate was measured directly from absorbance readings at the maximum wavelength. The amount of total drug in the retentate was determined after dissociation of the drug-DNA complex by the addition of equal volumes of 0·1 M lithium chloride in methanol. Experimental data were analysed using the neighbor exclusion model (McGhee and Von Hippel, 1974). Viscometric titrations were performed with a suspended level multigradient Ubbelohde dilution viscometer. A constant temperature bath, controlled by a Haake F3C thermostate, was used to maintain a temperature of 25°C. Appropriate control experiments showed that the presence of the drug had negligible effects on solvent viscosity, at least within the concentration range used. Known (small) volumes of the compounds were sequentially added to a solution of sonicated DNA, to obtain the desired drug/DNA ratio. Circular dichroism studies were carried out in a Jasco J 500 A spectropolarimeter equipped with a Jasco model DP-501 data processor. Four to sixteen scans were accumulated for each sample. Thermal denaturation experiments were performed in a Perkin-Elmer model 554 spectrophotometer. Samples were continuously heated at a rate of approximately 0·5°C min –1. 692 Palumbo et al. Results and discussion DNA- binding mechanism Due to their chemical structure, an intercalative binding mechanism might be reasonably expected for all compounds considered in this work (figure 1), except perhaps for compound V, which exhibits a planar moiety of only two condensed rings and is therefore expected to overlap less efficiently with the DNA base-pairs. Incidentally, it shows very poor, if any, biological activity (Antonello et al., 1979). Enhancement in specific viscosity of sonicated rodlike nucleic acid, increase in melting temperature of the macromolecule, bathochromicity and hypochromicity in the ligand absorption bands and dramatic changes in drug fluorescence quantum yields were used altogether as reasonably safe probes for an intercalative binding mechanism, even though none of these lines of evidence constitutes by itself a firm diagnostic tool (Waring, 1981). All compounds satisfy the above requirements but derivative II, for which the mentioned techniques give responses totally dissimilar from the expected ones. We are confident in concluding that derivatives I, III, IV and V intercalate into DNA, while compound II, notwithstanding its chemical structure, represents an example of non-intercalated, outside-bound drug. In addition to this unexpected finding, our results indicate that the coumarin derivative V binds the same way as the related compounds III and IV. It is worth mentioning that intercalative binding has been proposed for a few other systems containing two condensed rings (Lybrandt et al., 1981; Ishida et al., 1983). The dramatic drop in biological activity of compound V should accordingly be ascribed to a remarkable loss of intercalation energy, as indicated by the rather low intrinsic binding constant value (~ 2·103 M–1), obtained from fluorimetric and equilibrium dialysis measurements, rather than by a modified interaction with DNA. Conformational studies in the DNA absorption region Insertion of a planar molecule into B-DNA causes extension and local unwinding of the polynucleotide double helix (Lerman, 1961). Thus, in the complex region, the relative positions of the bases are substantially changed. The modified structure containing the planar ligand, besides affecting the hydrodynamic properties of DNA, dramatically alters the chiroptical response of the polynucleotide. Studies on intercalating drugs including aminoacridines, proflavin, β-carbolines and 9-AMSA (Dalgleish et al., 1971; Duportail, 1981; Hudecz et al., 1981) show that the circular dichroism (CD) spectrum of DNA of 275 nm considerably increases in intensity as a consequence of drug insertion into base-pairs. These effects might be ascribed either to alteration of the DNA structure or to shielding effects of the intercalated dye molecules. Moreover, ligand–base and ligand-ligand dipole interactions should be considered. The results obtained with our intercalating compounds are totally consistent with the above findings. The maximum increase in CD response of the positive DNA absorption is reported in table 1 for the examined compounds. Anthraquinone, furocoumarin and coumarin derivatives behave quite similarly, conforming the fact that intercalation causes well comparable spectral changes, irrespectively on the ligand chemical and spectral features. The conclusion follows that in intercalated systems of the above Conformational aspects of drug-DNA interactions 693 Table 1. Maximum increase in ellipticity of the 275 nm positive band of DNA upon addition of different intercalating drugs. a Data from Duportail (1981). structural type ligand–base and ligand-ligand interactions do not appear to contribute substantially to the complex CD in the ultraviolet region, as proposed for 9aminoacridine and proflavine (Dalgleish et al., 1971), whereas in particular shielding effects should be predominant. In the case of adriamycin, however, a remarkably lower spectral modification is observed. Considering that the drug is in itself optically active, it might be concluded that the CD spectrum reflects opposite contributions from the polynucleotide and the drug. However positive CD is exhibited also by adriamycin in the 280 nm region, with a cross-over point at 270 nm. The limited change in optical activity at this wavelength might therefore reflect different structural perturbations caused by adriamycin with reference to the other derivatives. Interestingly, the apparent DNA-unwinding angle by this drug is among the lowest found for intercalating compounds (Waring, 1975). These findings suggest a possible relationship between intercalation characteristics and CD response. In this connection it is interesting to note that the calculated magnitude of the DNA CD band at 275 nm is a linear function of the helix winding angle (Johnson et al., 1981). In any event, it appears that chiroptical properties allow monitoring the onset of intercalative binding for a wide range of compounds and could thus be used as an additional useful tool in investigating the modes of drug–DNA interactions. Let us now consider the CD results obtained with the non-intercalating compound II, which are presented in figure 2. The CD spectrum characteristic of the Β form is greatly affected by addition of compound II especially as far as the positive absorption is concerned. A new maximum appears, located around 260 nm, which increases in intensity up to ligand/DNA ratios of the order of 0·2. The original band remains present as a shoulder. At higher binding ratios the new absorption decreases in intensity until phase separation occurs. The appearance of a new band at shorter wavelength is a totally unusual feature when dealing with intercalating drugs. Thus CD measurements support the hypothesis based on hydrodynamic and fluorescence results that compound II is a non-intercalating one. Even though a detailed examination of the macromolecule conformational state does not appear to be straightforward, the position and intensity of the new band in the complex points toward the presence of regions of the nucleic acid in Α-like form. Under this hypothesis compound II causes a conformational transition of the polynucleotide from the Β to the A form, as a 694 Palumbo et al. Figure 2. Ultraviolet CD spectra for the system II-DNA at 25°C and 0·017 Μ ionic strength, pH 7·0. (1) R (drug/nucleotide ratio) = 0; (2) R = 0·050; (3) R = 0·098; (4) R = 0·200; (5) R = 0·405. C DNA = 1·26·10 – 4 MR. consequence of specific interactions which stabilize the new structure. While in the Β conformation the phosphate oxygens from the two polynucleotide chains are directed outward, in A-DNA they are directed toward each other. This arrangement might allow the "bidentate" ligand to bind the oxygens from different strands through its two amino groups, possibly favouring further contacts between the anthraquinone moiety and the polynucleotide. As to confirm the non-intercalating nature of the binding of compound II to DNA, it is known that intercalating agents inhibit transitions of the Β → A type (Sobell, 1980). It is finally interesting to note that compound II resembles to a certain degree polyamines such as spermine or spermidine, which also show larger interaction energy with DNA in the A form (Zhurkin et al., 1980). The dramatic decrease in intensity at high ligand concentrations might finally reflect the tendency of complexed DNA toward a more compact form, which causes phase separation (Lerman, 1971). Conformational aspects of drug-DNA interactions 695 These findings are further supported by a viscosity drop of the complex solution before a precipitate is observed. Chiroptical studies in the ligand absorption region Not only does CD give informations on the DNA structural modifications upon binding, but also on the complex geometry in the case of intercalated ligands. A model has been recently proposed, which predicts that the degree of alignment between basepairs and ligand may be directly probed through the sign of the circular dichroism induced in an adduct transition of known polarization (Schipper et al., 1980). In fact the non degenerate contribution to the CD of intercalated dyes simplifies, when averaged over random base-pairs combinations into a factor f, depending on DNA, and a factor related on the orientation of the transition moment of the dye. According to the theoretical calculations, when the transition moments of the adducts lie essentially parallel to the longest dimension of the base-pair pocket a negative induced CD is to be expected; when they are perpendicular a positive rotational strength should appear. For the intercalating anthraquinone I and the coumarin and furocoumarins investigated by us a negative band is observed (table 2), consistent with an intercalation geometry in which the aromatic system is oriented in a parallel fashion with reference to the basepairs. Interestingly the above arrangement reflects the optimum orientation recently proposed for anthraquinone compounds in a theoretical work on computer design of chromophoric intercalating agents (Miller and Newlin, 1982). The same structural features are required for a psoralen/DNA complex to obtain photoreaction (Rodighiero et al., 1984). For adriamycin a complication arises from the drug's intrinsic optical activity. Nonetheless an additional positive contribution is observed in the presence of DNA (table 2). The same behaviour is observed for daunomycin (Gabbay et al., 1976). According to the theoretical approach the intercalation geometry coresponds to the case in which the long-axis-polarized transition moment lies more or less perpendicular to the base-pair longest dimension. In total agreement, the crystal structure of daunomycin bound to a self-complementary DNA-fragment exhibits the aglycone chromophore oriented at right angles to the long axis of the DNA base-pair pocket (Quigley et al., 1980). Table 2. Induced CD in the ligand transition region upon intercalation of different drugs into double helical DNA. a b Data from Gabbay et al. (1976). Data from Hudecz et al. (1981). 696 Palumbo et al. These results, when combined with the data from other spectroscopic and hydrodynamic techniques, appear to be encouraging in defining more precisely the intercalation stereochemistry and in giving deeper insight into structure–activity relationships. Different geometries exhibited by different compounds could explain how some compounds, able to strongly intercalate into DNA, show only a limited biological activity. Besides tight binding, appropriate side groups should be properly located in the major or minor groove of the double helix to ensure the desired drug action. It has to be emphasized that binding kinetics may represent another important discriminating factor (Feigon et al., 1984). Drug binding to structurally organized DNA Drug-binding studies are usually performed with free nucleic acid, not organized as in the native functional state. As a result, comparison of the physico-chemical studies with biological results might be poorly significant and prevent from a sound evaluation of structure–function relationships. In fact, on one hand a number of binding sites of the "naked" macromolecule might be eliminated in the presence of protein–DNA and histone-DNA interactions, on the other hand new or modified sites might possibly result from the superstructural arrangement of the polynucleotide. As an example ethydium bromide exhibits a greater intrinsic affinity for nucleosomes than for DNA (Wu et al., 1980), whereas the reverse is found for daunomycin (Chaires et al., 1983). Compound I and II, structurally related to the latter anthracycline, show a similar trend, binding quite poorly to the organized polynucleotide as indicated by spectrophotometric and fluorimetric experiments. Also furocoumarins III and IV appear to behave the same way. Their photobinding response is in fact largely reduced when using nucleosomes or chromatin as the nucleic acid. CD results in the far UV region point toward an increased tendency to compaction and phase separation of the polynucleotide bound to positively charged drugs. Thus condensation phenomena might Figure 3. Visible CD spectrum for the system I–Chromatin at 25°C and 0·017 Μ ionic strength, pH 7·0. The drug/nucleotide ratio was 0·14. Conformational aspects of drug-DNA interactions 697 additionally contribute to DNA-synthesis inhibition, in keeping with recent results obtained on the interaction of adriamycin with chromatin (Waldes and Center, 1981) As a final observation splitting patterns observed for the ligand transitions (figure 3), indicate preferential drug accumulation in specific regions of the organized polynucleotide. Although preliminar, these results confirm the importance of DNA structure in directing complex formation and allow a better understanding of drug-nucleic acid interactions at a molecular level. 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