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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
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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.
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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
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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).
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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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