Mapping Drug Interactions at the Covalent Topoisomerase II-DNA
Complex by Bisantrene/Amsacrine Congeners*
Giovanni
Capranico
,
Fulvio
Guano,
Stefano
Moro§,
Giuseppe
Zagotto§,
Claudia
Sissi§,
Barbara
Gatto§,
Franco
Zunino,
Ernesto
Menta¶, and
Manlio
Palumbo§
From the Division of Experimental Oncology B, Istituto Nazionale
per lo Studio e la Cura dei Tumori, via Venezian 1, 20133 Milan,
the § Department of Pharmaceutical Sciences, University of
Padua, Via Marzolo 5, 35131 Padua, and the ¶ Boehringer
Mannheim Italia, Research Center, 20052 Monza, Italy
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ABSTRACT |
To identify structural determinants for the
sequence-specific recognition of covalent topoisomerase II-DNA
complexes by anti-cancer drugs, we investigated a number of bisantrene
congeners, including a 10-azabioisoster, bearing one or two
4,5-dihydro-1H-imidazol-2-yl hydrazone side chains at
positions 1, 4, or 9 of the anthracene ring system. The studied
bisantrene/amsacrine (m-AMSA) hybrid and bisantrene isomers
were able to poison DNA topoisomerase II with an intermediate activity
between those of bisantrene and m-AMSA. Moving the side
chain from the central to a lateral ring (from C-9 to C-1/C-4) only
slightly modified the drug DNA affinity, whereas it dramatically
affected local base preferences of poison-stimulated DNA cleavage. In
contrast, switching the planar aromatic systems of bisantrene and
m-AMSA did not substantially alter the sequence specificity
of drug action. A computer-assisted steric and electrostatic alignment
analysis of the test compounds was in agreement with the experimental
data, since a common pharmacophore was shared by bisantrene,
m-AMSA, and 9-substituted analogs, whereas the 1-substituted isomer showed a radically changed pharmacophoric structure. Thus, the relative space occupancy and electron distribution of putative DNA binding (aromatic rings) and enzyme binding (side chains) moieties are fundamental in directing the specific action of
topoisomerase II poisons and in determining the poison
pharmacophore.
 |
INTRODUCTION |
The elucidation of structural determinants of the
sequence-specific recognition of DNA by small molecules is fundamental
for a rational design of gene-specific DNA binders that are effective in the therapy of human diseases. Several DNA-interactive compounds are
known that may bind to the double helix in a site-selective manner;
however, the degree and mechanisms of the specificity are very
different among them (1-4). A wide variety of antitumor drugs, with
and without the ability to bind to naked DNA, have been shown to poison
DNA topoisomerases with a high sequence selectivity (2, 5-7). DNA
topoisomerases are ubiquitous enzymes deputed to resolve topological
problems that arise during various nuclear processes including
transcription, recombination, and chromosome partitioning at cell
division (8-10). Type II enzymes make transient double-stranded breaks
into one segment of DNA and pass an intact duplex through the broken
DNA, before resealing the break (2, 8-10). Anticancer agents able to
poison the mammalian enzymes stabilize a key intermediate of the
catalytic reaction wherein DNA strands are broken and covalently linked
to the protein. Thus, the poisoning action results in increased DNA
cleavage levels in living cells that eventually trigger a cell death
process.
Classical topoisomerase poisons stimulate DNA cleavage in a
sequence-selective manner, yielding drug-specific cleavage intensity patterns in agarose as well as sequencing gels (2, 5, 6, 11, 12). Each
pattern reflects the recognition of specific features of the enzyme-DNA
covalent complex that are likely dictated by the nucleotide sequence at
the site of cleavage. In fact, while preferred bases distant from the
cleaved bond are similar regardless of the class of the poison used
(enzyme-specific preferences), preferred nucleotides close to the 5'-
or 3'-termini (positions +1 or
1, respectively) are poison-specific,
examples being an adenine at +1 for
m-AMSA1 and
bisantrene, a cytosine at
1 for teniposide (VM-26) and mitoxantrone, an adenine at
1 for doxorubicin, and a thymine at +2 for
streptonigrin (2). Poison localization in the ternary complex has been
directly shown using a photoactivable m-AMSA analog (13).
Upon activation, the compound was found to be covalently linked to DNA
bases at the +1 and
1 positions, only when T4 topoisomerase II was
present in the reaction mixture. Analogous results have been obtained in the case of camptothecin and topoisomerase I (14, 15). Thus,
several independent results demonstrate that poison receptors are
localized at the protein/DNA interface at the site of DNA cleavage.
Early studies suggested that topoisomerase II poisons may fit into a
"loose" pharmacophore, constituted by a planar ring system with DNA
intercalation or intercalation-like properties, and one or two
protruding side chains, possibly interfering with the protein side of
the covalent enzyme-DNA complex (16). The lack of structural restrictions on this pharmacophore can be ascribed mainly to the receptor heterogeneity that is determined by different nucleic acid
sequences at the enzyme-active site. An approach to the
characterization of the drug receptor site has been based on molecular
modeling analyses of topoisomerase II poisons along with the
determination of their sequence-specific DNA cleaving activity (17).
The relative positions of planar ring systems and side chains of many
clinically useful antitumor topoisomerase II poisons have been
suggested to determine the site of enzyme-mediated DNA cleavage (17). As a matter of fact, only compounds sharing defined steric and electronic features can trap the enzyme at the same DNA sites. In
particular, bisantrene and m-AMSA have the same base
preference for DNA cleavage stimulation, and despite the different
chemical structures, they were shown to be characterized by very
similar electronic and steric properties, suggesting a common fit into the receptor site (17).
Therefore, in order to challenge the idea that the drug shape and
electron density determine the sequence specificity of the poison
action, bisantrene analogs and an m-AMSA-bisantrene hybrid (Fig. 1) were synthesized and characterized for their DNA binding properties, topoisomerase II-mediated DNA cleavage, base sequence preferences, and conformational and electronic features. The studied compounds were formally derived from the exchange of putative pharmacophoric domains of m-AMSA and bisantrene or were
structural isomers of bisantrene (Fig. 1). In full agreement with the
original hypothesis, our combined experimental and theoretical work
demonstrates that alterations of the shape and electron density of the
drug molecule markedly affect the sequence-specific interaction of the
poison with topoisomerase II-DNA complexes. This might be useful in
providing a physicochemical map of poison receptors.
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EXPERIMENTAL PROCEDURES |
Materials--
m-AMSA and bisantrene were
obtained by the Drug Synthesis and Chemistry Branch, NCI, National
Institutes of Health, Bethesda, and by Lederle, Copenhagen, Denmark,
respectively. The IHA compounds were synthesized as described and
characterized previously (18). All the compounds used were stored at
20 °C in Me2SO or deionized water and diluted in
deionized water prior to use. DNA from calf thymus (
= 6600 M
1 cm
1), was purchased from
Sigma and used with no further purification. DNA topoisomerase II was
purified from nuclei of murine P388 leukemia cells and stored as
described (19). Human topoisomerase II
was purified from yeast cells
carrying a plasmid-borne human top2
cDNA, as described already
(20). Simian virus 40, restriction endonucleases, other enzymes,
agarose, and acrylamide were from Life Technologies, Inc., or from New
England Biolabs. [
-32P]ATP was from Amersham Pharmacia
Biotech (Milan, Italy).
DNA Binding Studies--
Measurements were carried out in 10 mM Tris-HCl, pH 7.0, 1 mM EDTA, 0.15 M NaCl at 25 °C. Binding was followed
spectrophotometrically or fluorometrically in the ligand absorption or
emission region upon adding scalar amounts of DNA to a freshly prepared
drug solution. To avoid large systematic inaccuracies due to
experimental errors in extinction coefficients or fluorescence quantum
yield, the range of bound drug fractions was 0.15-0.85. Data were
evaluated according to Equation 1 of McGhee and Von Hippel for
non-cooperative ligand-lattice interactions (21),
|
(Eq. 1)
|
where r is the molar ratio of bound ligand to DNA;
m is the free ligand concentration; Ki is
the intrinsic binding constant; and n is the exclusion
parameter. Spectrophotometric measurements were performed with a
Perkin-Elmer Lambda 5 apparatus and fluorometric studies on a MPF66
fluorometer, both equipped with a Haake F3-C thermostat.
Topoisomerase II-mediated DNA Cleavage--
SV40 DNA was
linearized with a restriction enzyme, treated with calf intestinal
phosphatase, and uniquely 5'-end-labeled using T4 polynucleotide kinase
and [
-32P]ATP prior to digestion with a second
endonuclease (11). Labeled DNA was incubated for 20 min at 37 °C
with topoisomerase II (10-30 units) with or without drugs in 40 mM Tris-HCl, pH 7.5, 0.5 mM dithiothreitol, 100 mM NaCl, 10 mM MgCl2, 1 mM ATP, 15 µg/ml bovine serum albumin, and 1% Triton
X-100. The presence of the surfactant reduces the extent of DNA-protein
aggregation that would otherwise affect drug-stimulated DNA cleavage
(22). Reactions were stopped by incubation with 1% SDS and 0.1 mg/ml
proteinase K for 45 min at 42 °C. Samples were then electrophoresed
in a 1% agarose gel in 89 mM Tris, 89 mM boric
acid, 2 mM EDTA, pH 8, and 0.1% SDS. For sequencing gels,
after proteinase K treatment, DNA was ethanol-precipitated, resuspended
in 80% formamide, 10 mM NaOH, 1 mM EDTA, 0.1%
dyes, heated for 2 min at 90 °C, chilled on ice, and loaded onto a
8% polyacrylamide denaturing gel. Gels were run for 2 h at 70 watts, dried, and autoradiographed to Amersham Hyperfilms (Amersham
Pharmacia Biotech). The reduction of the full-length labeled DNA band
was used to measure drug stimulation or suppression of enzyme-mediated DNA cleavage. The level of radioactivity of the uncut DNA band in each
lane was quantified by volume integration using the ImageQuant program
on a Molecular Dynamics PhosphorImager 425 model.
Statistical Analysis of Cleavage Site
Specificity--
Poison-stimulated DNA cleavage sites were mapped by
comparison with Maxam-Gilbert purine markers (11). The collected site sequences were gathered into drug-specific groups, and base frequencies at each position around the cleaved bonds were analyzed by using statistical tests as described in detail elsewhere (11, 17, 23).
Computational Chemistry--
Ab initio calculations
were performed at Hartree-Fock level with Gaussian basis set 3-21G(*)
(24). All geometries were fully optimized without geometry constraints.
Vibrational frequency calculations were used to characterize the minima
stationary points (zero imaginary frequencies). The software package
Spartan 3.1 (Wavefunction Inc., Irvine, CA) was used for all quantum
mechanical calculations. The optimized geometry and the atomic charges
allowed us to perform a steric and electrostatic analysis (SEA) (25). For this purpose, the Superimposition/Similarity facility to Spartan 4.0 (Wavefunction Inc., Irvine, CA) was employed for the alignment of
molecular structures. Optimized alignments were achieved by maximizing
the electrostatic potential field and steric overlap of
three-dimensional structures. This method performs the alignment many
times for a single pair of molecules and keeps only the best results,
which are finally sorted according to a functional form of the
similarity measure. The functional forms were generated as
three-dimensional grids surrounding each molecule to analyzed. In this
work a correlation coefficient (r), as shown in Equation 2,
was used as a similarity index,
|
(Eq. 2)
|
where n is the grid number and
xi,yi the corresponding
coordinates (26). All molecular modeling calculations were performed on
an IBM RISC System 6000 model 250 Unix workstation.
NMR Experiments--
Nuclear Overhauser (NOE) experiments
were performed in deuterated chloroform using a Varian Gemini 300 MHz
spectrometer. To freeze drug conformations, the temperature was reduced
to
50 °C in a number of runs. Since, as expected, the relevant
structural information could be obtained from the data concerning the
imine proton, this has been especially considered in the NOE
spectra.
 |
RESULTS |
Drug Stimulation of DNA Cleavage--
Levels of topoisomerase
II-dependent DNA cleavage in the presence of the studied
compounds (Fig. 1) were determined in
5'-end 32P-labeled SV40 DNA fragments by PhosphorImager
analysis of agarose gels. With the exception of 1,4-IHA, the new
compounds stimulated DNA cleavage mediated by murine native enzyme and
recombinant human topoisomerase II
(Fig.
2). The parent drugs m-AMSA
and bisantrene were the most and the least active compounds,
respectively. 9-IHA stimulated intermediate cleavage levels; 1-IHA
stimulated a low level of cleavage only at 10 µM, and
aza-9-IHA was as active as bisantrene (Fig. 2). Potency was also
reduced for all the new analogs, since cleavage was detectable at 1-10
µM, whereas bisantrene and m-AMSA stimulated
cleavage at 0.2-1 µM (Figs.
3 and 4).
No cleavage suppression was detected up to 10 µM of the
analogs and m-AMSA, in contrast to bisantrene that at the
same concentration suppressed even background cleavage. 1,4-IHA was
completely ineffective and abolished enzyme-mediated DNA cleavage even
at 0.1 µM (Figs. 2 and 3). This is likely due to the high
DNA binding affinity constant and precipitation of drug-nucleic acid
aggregates (see below).

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Fig. 2.
Levels of topoisomerase II DNA cleavage by
m-AMSA/bisantrene congeners. An SV40 DNA fragment,
32P-labeled at the 5'-end of the BamHI site, was
incubated with topoisomerase II and poisons at 37 °C for 20 min.
Reactions were stopped with SDS (1%) and proteinase K (0.1 mg/ml). DNA
cleavage was then examined by 1% agarose gel electrophoresis, and
cleavage levels were determined with PhosphorImager analyses. Numbers
above columns indicate the stimulation (above 1)
or suppression (below 1) factor, calculated as the ratio of
the cleaved DNA fraction with the drug over that without drugs.
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Fig. 3.
DNA cleavage intensity patterns by murine
native topoisomerase II. SV40 DNA fragments were reacted with
murine topoisomerase II with or without drugs for 20 min at 37 °C,
stopped with SDS and proteinase K, and then analyzed on a 8%
polyacrylamide sequencing gel. Lanes are as follows: C,
control DNA, and T, topoisomerase II without drugs.
Drug-treated samples are as indicated above each lane.
A, numbers indicate selected cleavage sites; an
asterisk indicates a weak cleavage site common to 1-IHA and
m-AMSA. B, arrowheads indicate sites
of cleavage stimulated by the studied compounds.
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Fig. 4.
Cleavage patterns by human topoisomerase
II at the replication origin region of simian virus 40. An SV40
DNA fragment was labeled at the BanI site, and cleavage
reactions were performed as described in the legend to Fig. 3. Drugs
were used at 10 µM. Numbers on the
right indicate cleavage sites. Symbols are as follows:
upward arrows, 21-base pair repeats; RY,
alternating purine/pyrimidine sequences; downward arrows,
72-base pair repeats. Numbers correspond to SV40 genomic
positions. Lanes are as follows: C, control DNA;
T, enzyme alone; m, purine markers; A,
m-AMSA; 9, 9-IHA; and 1, 1-IHA. The
purine marker lanes are from a 5-fold less exposed
autoradiography.
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Cleavage Intensity Patterns with Murine Native Topoisomerase II and
Human Topoisomerase II
Form--
To compare drug-stimulated
cleavage patterns, topoisomerase II-dependent DNA cleavage
was further investigated with sequencing gels. Intensity cleavage
patterns of 9-IHA, aza-9-IHA, and m-AMSA were somewhat
different even though many cleavage sites were common (Figs. 3 and 4).
This is shown by cleavage stimulation with murine native topoisomerase
II at the genomic positions 2401, 2430, 2438, 2441, and 2465 (Fig.
3A; see also sites indicated by arrowheads in
Fig. 3B). In contrast, 1-IHA stimulated cleavage at a
different subset of sites (genomic positions 2408, 2445, 2447, and the
asterisk in Fig. 3). Some sites stimulated by 1-IHA were
also stimulated by 9-IHA but not by m-AMSA and aza-9-IHA
(Fig. 3, sites 2447 and 2408). However, these
sites were a minor fraction of all the sites stimulated by 9-IHA.
Drug-stimulated DNA cleavage was also investigated with human
recombinant topoisomerase II
in the region of the SV40 replication origin (Fig. 4). IHA congeners stimulated main cleavage sites in the
purine/pyrimidine repeats within the 72-base pair repeats of SV40 DNA.
Again sites stimulated by the congeners (see sites 263, 204, 200, and 142, in Fig. 4) were also sites of
mAMSA-stimulated DNA cleavage. However, other main amsacrine
sites (see sites 252, 241, 223 and others in Fig. 4) were
not stimulated by the analogs, which are responsible for somewhat
different intensity patterns among the studied drugs.
Sequence Specificity of Poison Interactions with the Cleavable
Complex--
Statistical analyses of drug-specific cleavage site
sequences were performed for m-AMSA, 9-IHA, and 1-IHA. Even
though the new analogs were less active than m-AMSA, we
could collect enough cleavage sites for 9-IHA and 1-IHA to evaluate
base sequences with statistical tests (Table
I and Fig.
5). In the same DNA fragments, more than
150 sites stimulated by m-AMSA were also collected, and the
results confirmed a strong preference for adenine at +1 positions (2,
17). In the case of 9-IHA, we found that the highest base preference
was present at position +1 for adenine, and in addition a preference
for cytosines at position
1. Previously, a secondary preference for
thymines was observed at position
1 in the case of m-AMSA
(2). In the case of 1-IHA, no statistically relevant preference has
been observed at positions
1 and +1, and an exclusion of guanines was
noticed at position +6 only (Fig. 5 and Table I).
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Table I
Base frequencies (%) at the sites of topoisomerase II DNA cleavage
stimulated by the indicated poison
Site sequences were aligned with respect to the observed cleaved
phosphodiester bond, which is between nucleotides 1 and +1. The
numbers of cleavage sites were 66 and 37 for 9-IHA and 1-IHA,
respectively; for m-AMSA, the statistical data of the
analysis of 48 strong sites are reported. Base frequencies in SV40 DNA
are 29.6% of A and T, and 20.4% of G and C. In bold, statistically
significant high frequencies (11, 23).
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Fig. 5.
Base preferences at the site of
poison-stimulated DNA cleavage. Nucleotide sequences of sites
stimulated by m-AMSA (left), 9-IHA
(middle), and 1-IHA (right) were collected and
analyzed by statistical tests (11, 23). Log(p) values
indicate the chance of observing that deviation or more as either
excess or deficiency (above or below the zero line,
respectively) relative to the expected frequency of each base (G = C = 20.6%; A = T 29.4%).
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These findings demonstrated that the main base preference was identical
for 9-IHA and m-AMSA (A at +1), even though the former but
not the latter preferred a cytosine at
1 position. Moreover, since
1-IHA completely lost the base preference found for m-AMSA and bisantrene, our data indicated a substantial modification in
recognition of topoisomerase II-DNA covalent complexes by 1-IHA as
compared with the other congeners.
Thermodynamics of DNA Binding--
All test compounds (Fig. 1)
were able to interact effectively with naked DNA as shown by
spectrophotometric and fluorometric determinations of binding
parameters at physiological conditions (Table
II). The Ki values for
1,4-IHA could not be determined since the drug-DNA complex tended to
precipitate even at low binding ratios (not shown), demonstrating an
outstandingly high binding constant. In agreement with published
reports (3, 27, 28), m-AMSA and bisantrene showed a modest
and very large affinity for DNA, respectively (Table II). The new
bisantrene analogs, bearing a side chain only, had intermediate values
of DNA binding affinity. A comparison of aza-9-IHA with
m-AMSA, having the same planar portion, indicates that the
dihydroimidazolyl hydrazone side chain generated remarkably stronger
interactions with the nucleic acid than the methane sulfone
m-anisidide group (Table II). The location of the side chain
on the planar ring system plays a role in directing complex formation,
since 1-IHA had a 3-fold lower constant than the 9-substituted isomers.
The exclusion parameter n was close to 2 base pairs for all
9-substituted compounds, in agreement with an intercalative process of
binding. On the other hand a considerably lower n value was
found for 1-IHA (Table II), suggesting an important non-intercalative
component to the binding process.
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Table II
Thermodynamic properties of binding to calf thymus DNA by the test
compounds
DNA binding affinity constants were determined in 10 mM
Tris-HCl, pH 7.0, 1 mM EDTA, 0.15 M NaCl at
25 °C.
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Molecular Drug Conformations: Computer Simulation and NMR
Studies--
Structural information about the orientation of the side
chain groups relative to the planar ring system was experimentally obtained by NOE spectra of 9- and 1-IHA relative to irradiation of the
iminic protons (Fig. 6A). Both
compounds gave intense signals in the NOE difference spectra. The data
for 9-IHA gave 26.9% enhancement for protons H-1 and H-8 (exhibiting
the same chemical shift) of the anthracene ring, whereas enhancements
observed in the presence of 1-IHA were 11.6% for H-9 and 14.9% for
H-2 referred to the same ring system. These data are consistent with a
relatively free rotation of the side chain around the
Caromatic-Ciminic bond.

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Fig. 6.
Conformational analyses of 1-IHA and 9-IHA.
A, NMR examination of the side chain orientation relative to
the aromatic ring system of drugs in solution. 1H NMR
(bottom) and NOE difference spectra (top) of the
aromatic protons of 9-IHA (left) and 1-IHA
(right). B, atomic charges and vector composition
of electric dipole moment of the studied IHA agents. The values were
obtained by using an ab initio RHF/3-21G(*) methodology for
the model of 1-IHA and 9-IHA structures. Atoms are shown as
circles: dark gray, nitrogen; light
gray, carbon; and white, hydrogen.
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In addition to bisantrene and m-AMSA (17), we have
investigated low energy conformations of the IHA drugs by
computer-aided modeling techniques to identify the structural
determinants of the drug sequence-specific action. Each structure was
first energy-minimized to find the most stable conformations, as
obtained from a systematic conformational analysis. For each compound,
structures within 3 kcal/mol of the minimum energy were considered,
since most biologically relevant conformations are normally expected to
be included in such an energy range. As previously reported (17), and
consistent with the nuclear Overhauser experiments (Fig.
6A), the dihydroimidazolyl hydrazone side chains of
bisantrene were characterized by a high degree of conformational
freedom, and low energy dynamic structures of the drug displayed two
symmetric sets of chain space occupancy with respect to the plane of
the aromatic moiety (not shown). The most stable structure in
vacuum was defined by the following values of the adjacent
dihedral angles starting from one of the imidazole nitrogens:
N-C-N-N = 150°; C-N-N-C = 110°; N-N-C-C =
140°;
N-C-C=C =
64°.
Steric and Electrostatic Alignment (SEA) of IHA Congeners--
For
steric and electrostatic analyses, we used optimized geometries and
atomic charges obtained for the test compounds from ab
initio calculations. In addition to bisantrene, 9-IHA and 1-IHA were used to define the physicochemical properties of monosubstituted isomers. As it could be anticipated, the similarity between 9-IHA and
bisantrene is very high, exhibiting an almost complete steric and
electronic matching (not shown). This clearly suggested a very similar
fashion of drug-receptor interaction for the two compounds. On the
contrary, the three-dimensional similarity analysis based on the steric
and electronic properties of 1-IHA and 9-IHA gave a poor correlation
coefficient (0.54). This value fully agreed with the low similitude
shown by the optimized overlapping of all stable conformations of the
two compounds (Fig. 7). In fact, to
achieve the best balance between common space occupancy and electrostatic potential, the planar moieties must be tilted and shifted
one with reference to the other, so that side chain groups are forced
to occupy distinct regions in space (Fig. 7). The different superimposition between 1-IHA and 9-IHA was corroborated with comparative charge localization analysis. As shown in Fig.
6B, the shift of the side chain from 9 to 1 position of the
anthracene moiety completely modified the electronic charge
distribution around the two molecules. Consequently, the dipole moments
of the two congeners are also different (Fig. 6B),
indicating that the topology of the electrostatic potential may
represent an important factor in the molecular recognition process.
Taken together, our results indicate that steric features and
electrostatic potential fields of minimum energy conformations common
to bisantrene, m-AMSA, and the studied 9-IHA analogs are not
shared by the structural isomer 1-IHA. This is expected to modify the
recognition of the receptor site by 1-IHA.

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Fig. 7.
Steric and electronic alignment (SEA)
analysis of the most probable overlapping between 9-IHA and 1-IHA.
The correlation coefficient is 0.54 (see "Experimental Procedures"
for details).
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DISCUSSION |
The present investigation on IHA congeners, the design of which
was prompted by earlier molecular modeling studies (17), definitely
demonstrates that, in a homogeneous series of compounds, alterations of
the shape and electron density of the drug molecule markedly affect the
sequence-specific poison interaction with topoisomerase II-DNA
complexes. Thus, this result strongly supports the idea that the
relative position of putative DNA- and enzyme-binding domains plays a
key role in determining the sequence position specificity of
topoisomerase II poisons (2).
The NOE results presented in Fig. 6A confirm the possibility
of free rotation of the side chain groups with reference to the planar
anthracene moiety. Hence, a number of conformations characterized by
similar energies are available for the molecule to interact at the
receptor site. This is confirmed by the theoretical ab initio analysis, giving small energy gaps between stable
conformations, the limiting situations being represented by a
completely planar arrangement or a perpendicular arrangement of the
imidazolyl hydrazine group versus the anthracene group. The
SEA analysis comparing the complete range of stable conformations of
9-IHA with the complete set of stable conformations of 1-IHA was not
able to find a satisfactory alignment within the two sets of
conformers. This confirms that 1-IHA and 9-IHA correspond to distinct
pharmacophores. This is further emphasized by the fact that the
observed changes in electrostatic potential are not a consequence of
the drug conformation but a result of the 9
1 shift of the side
chain group (Fig. 6B).
Our data establish that one side chain of bisantrene is sufficient to
grant poisoning activity to the compound since 9-IHA could stimulate
even higher levels of topoisomerase II DNA cleavage than bisantrene,
although the DNA affinity was maximum when two dihydroimidazolyl chains
were present. Indeed, the binding affinity dropped about 20-fold by
removing one chain and 30-fold further when replacing the bisantrene
chain with the m-AMSA side group. On the other hand, the
substitution of the anthracene ring with the acridine planar moiety in
aza-9-IHA did not appreciably alter the binding properties. This
indicates that the aza substitution at the central ring does not play
an important role in the energy balance of DNA-drug complex formation.
Only a slight decrease is observed when shifting the chain from
position 9 to position 1; hence, the location of side groups in the
IHA/amsacrine family does not significantly affect DNA affinity.
The effect of poison substituents on DNA cleavage levels cannot
be determined by a simple comparison of cleavage efficiencies. In fact,
as mentioned above, DNA binding constants of the studied compounds were
markedly different, and it is well known that a strong interaction with
free DNA by intercalating agents can fully prevent enzyme binding to
the nucleic acid and therefore enzyme-mediated DNA cleavage (2, 5, 6).
Cleavage levels stimulated by bisantrene were low, whereas they were
prominent when using the weak binder m-AMSA; IHA congeners
had intermediate effects, consistent with their intermediate
Ki values. Since the relative efficiencies in DNA
cleavage stimulation were inversely correlated to nucleic acid binding
constants for this series of compounds, the extent of cleavage
stimulation could be mostly determined by the DNA binding affinity.
Indeed, cleavage suppressive effects shown by 1,4-IHA well paralleled
its very high DNA binding activity, which even prevented drug
dissociation from the complex. The only compounds having essentially
the same affinity for DNA were 9-IHA and aza-9-IHA. In this case, it is
safe to conclude that the C
N bioisosteric substitution at the
central ring was primarily responsible for the reduced cleavage
activity. In agreement with this result, a double C
N bioisosteric
substitution in anthracenediones (including mitoxantrone) has been
shown to abolish topoisomerase poisoning activity (29).
The statistical analysis documented that an adenine(+1) requirement for
cleavage stimulation is shared by bisantrene, m-AMSA, 9-IHA,
and the hybrid drug, aza-9-IHA (data not shown). Hence, these agents
likely belong to a very similar pharmacophore class and interact
similarly in the ternary DNA-poison-topoisomerase II complex. In total
agreement with this hypothesis, the above drugs were characterized by a
similar electron density and spatial relation of drug moieties as
determined by the SEA analysis of drug conformations obtained by
ab initio theoretical calculations. In the case of 1-IHA,
which had the side chain shifted to a lateral ring, radical changes
were instead observed; the compound was still able to poison
topoisomerase II; however, cleavage was no longer preferentially
stimulated at m-AMSA sites. Therefore, we may conclude that 1-IHA
cannot share the same pharmacophore as the other congeners of the
bisantrene/m-AMSA family. Consistently, the molecular
modeling analysis showed that 1-IHA and 9-IHA were rather different
molecules, since a similarity parameter of 0.54 indicated that the two
compounds were not more alike than any two of the vast majority of
chemically unrelated DNA-interacting agents.
At this time, we do not have direct evidence of the relative
position of the 9- and 1-IHA structures within the DNA-enzyme covalent
complex; nevertheless, we can reasonably argue that the 9-IHA
derivatives are present at the enzyme/DNA interface specifically interacting with the adenine at the cleavage site in a fashion similar
to bisantrene and m-AMSA (2, 17). A simple isomerization in
the IHA structure causes dramatic modifications in the sequence specificity of poison-stimulated topoisomerase II DNA cleavage. Thus,
the present results strongly support the hypothesis that only compounds
sharing defined steric and electronic features can trap the enzyme at
the same DNA sites, suggesting a similar fit into the receptor site
(17). Taken individually, the chemical identity of the functional
groups of 1- and 9-IHA would allow the same stabilizing interactions to
occur with the receptor site. However, the different reciprocal
location of ring system and side chain impairs the contextual onset of
the key contacts with the protein and nucleic acid partners in the
covalent topoisomerase II-DNA complex. This strengthens the concept of
a cooperation between (at least) two pharmacophoric domains in
eliciting specific target recognition and, hence, in affecting drug
activity.
In conclusion, molecular modeling of poison molecules, and possibly
poison-receptor interactions, may be powerful tools in drug discovery
when combined with experimental molecular analyses of
topoisomerase-dependent DNA cleaving effects. The complete definition of the diverse pharmacophores of topoisomerase II poisons will certainly be of value for the design of new agents more effective in the treatment of human cancers.
 |
ACKNOWLEDGEMENTS |
We thank Stella Tinelli and Dr. Mariano
Stivanello for skillful technical assistance and NOE
experiments, respectively.
 |
FOOTNOTES |
*
This work was supported in part by Progetto Finalizzato
ACRO, Consiglio Nazionale delle Ricerche, Rome, Italy, and the
Associazione Italiana per la Ricerca sul Cancro, Milan, Italy.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom reprint requests should be addressed: Istituto Nazionale
Tumori, Experimental Oncology B, 20133 Milan, Italy. Tel.: 39-2-2390-203; Fax: 39-2-2390-764; E-mail:
capranico{at}istitutotumori.mi.it.
1
The abbreviations used are: m-AMSA,
amsacrine,
4-[9-acridinylamino]-N-[methanesulfonyl]-m-anisidine;
IHA, dihydro-1H-imi-dazol2-yl hydrazone; SEA, steric and
electrostatic analysis; NOE, nuclear Overhauser effect.
 |
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