From the Drug Dynamics Institute, College of Pharmacy, University of Texas, Austin, Texas 78712-1074
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ABSTRACT |
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Quinobenzoxazine A-62176, developed from the
antibacterial fluoroquinolones, is active in vitro and
in vivo against murine and human tumors. It has been
previously claimed that A-62176 is a catalytic inhibitor of mammalian
topoisomerase II that does not stabilize the cleaved complex. However,
at low drug concentrations and pH 6-7, we have found that A-62176 can
enhance the formation of the cleaved complex at certain sites. Using a
photocleavage assay, mismatched sequences, and competition experiments
between psorospermin and A-62176, we pinpointed the drug binding site on the DNA base pairs between positions +1 and +2 relative to the
cleaved phosphodiester bonds. A 2:2 quinobenzoxazine-Mg2+
self-assembly model was previously proposed, in which one drug molecule
intercalates into the DNA helix and the second drug molecule is
externally bound, held to the first molecule and DNA by two Mg2+ bridges. The results of competition experiments
between psorospermin and A-62176, as well as between psorospermin and
A-62176 and norfloxacin, are consistent with this model and provide the
first evidence that this 2:2 quinobenzoxazine-Mg2+ complex
is assembled in the presence of topoisomerase II. These results also
have parallel implications for the mode of binding of the quinolone
antibiotics to the bacterial gyrase-DNA complex.
The type II topoisomerases (topo
II)1 are enzymes that
regulate the topological state of DNA (1, 2). These enzymes function by
making transient double-stranded DNA breaks (or "gates") and allowing another DNA helix to pass through these breaks. At the DNA
cleavage step, topo II is covalently linked to the 5'-phosphoryl end of
the broken DNA by DNA-enzyme transesterification, then following strand
passage, the DNA gate is resealed by topo II. Eukaryotic topo II has
been identified as the molecular target of a number of potent
anticancer drugs, such as the anthracyclines, acridines, and
epipodophyllotoxins. Many of these topo II inhibitors, collectively
known as topo II poisons, interfere with the breakage-rejoining reactions of topo II by trapping the covalent reaction intermediate, which is called the cleavable complex or, more accurately, the cleaved
complex (3-8). Other topo II inhibitors, known as topo II suppressors,
inhibit the catalytic activity of topo II without trapping the
cleaved complex (8).
The quinobenzoxazine compounds (typified by
1-(3-aminopyrrolidin-1-yl)-2-fluoro-4-oxo-4H-quino[2,3,4-i,
j][1,4]benzoxazine5-carboxylic acid (A-62176);
Fig. 1) were initially developed from antibacterial fluoroquinolones
(typified by norfloxacin, Fig. 1) by scientists at Abbott Laboratories
(9-11). These compounds are active against a number of human and
murine cancer cell lines including the multidrug resistant P388/ADR
line in vitro and several murine and human tumors in
vivo (9). An initial study has revealed that the quinobenzoxazines
are potent inhibitors of mammalian topo II, both in vitro
and in vivo (12). According to this study, the quinobenzoxazines appear to belong to the class of topo II suppressors that interfere with the catalytic activity of the topo II at a step
prior to the formation of the cleaved complex.
The quinobenzoxazines, which are structurally related to
fluoroquinolones, possess a planar tetracyclic ring in place of the fused bicyclic ring of fluoroquinolones (Fig.
1). The extended flat aromatic ring
structure of quinobenzoxazines enables them to form a stable
intercalation complex with the DNA helix (12), while the more limited
aromaticity of the fluoroquinolone norfloxacin prevents the formation
of a stable intercalation complex with duplex DNA (13, 14). Preliminary
data from Abbott Laboratories showed that DNA binding of
quinobenzoxazine A-62176, like norfloxacin, is
Mg2+-dependent (15). Further biophysical and
electrophoretic studies on the ternary complex between the
quinobenzoxazine-Mg2+ complex and DNA have provided
evidence for a 2:2 quinobenzoxazine-Mg2+ self-assembly
complex, in which one quinobenzoxazine molecule is intercalated into
the DNA helix and the second drug molecule is externally bound, held
together by two Mg2+ bridges (17). In this proposed ternary
complex, the externally bound quinobenzoxazine molecule can be replaced
by norfloxacin to form mixed-structure dimers on DNA, an observation
that is supported by the additive effects observed on the DNA binding of quinobenzoxazines when norfloxacin is added. This 2:2
quinobenzoxazine-Mg2+ self-assembly complex has elements of
both the Shen model (13, 14), which involves a self-assembly complex,
and the Palumbo model (15, 16), which proposed a
phosphate-Mg2+-drug complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structures of the compounds used in this
study.
To further characterize the molecular basis of the anticancer activity
exhibited by the quinobenzoxazines, the interaction of quinobenzoxazine
A-62176 with the topo II-DNA complex has been investigated. In contrast
to the previous findings that A-62176 catalytically inhibits topo II
without stabilizing the cleaved complex (12), we have determined that
A-62176 possesses a dual mechanism at pH 6-7. At one topo II cleavage
site, A-62176 enhances the cleaved complex formation at low drug
concentrations but inhibits it at high drug concentrations, and at
another topo II cleavage site, A-62176 inhibits the cleaved complex
formation at both low and high drug concentrations. Use of the
intrinsic photocleavage activity of the
quinobenzoxazines,2 together
with the results from studies using mismatched sequences and
competition experiments between A-62176 and the topo II covalent reactive molecule psorospermin, has allowed us to locate the A-62176 binding site at around the 1+ and +2 positions relative to the phosphodiester bonds cleaved by topo II. Furthermore, competition experiments between psorospermin and A-62176, and between psorospermin and A-62176 and norfloxacin, are in accord with the previously proposed
2:2 quinobenzoxazine-Mg2+ model and imply that this can
also assemble on DNA in the presence of topo II.
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EXPERIMENTAL PROCEDURES |
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Materials, Enzymes, and Drugs--
Quinobenzoxazine A-62176 was
prepared according to published procedure (19). Norfloxacin was
purchased from Sigma. Psorospermin was a generous gift from Dr. John M. Cassady (Ohio State University). Electrophoretic reagents (acrylamide,
N,N'-methylenebisacrylamide, ammonium persulfate) were from
J. T. Baker, Inc., and
N,N,N',N'-tetramethylethylenediamine was from Fisher. T4
polynucleotide kinase, Drosophila topo II, and
[-32P]ATP were purchased from Amersham Pharmacia Biotech.
Preparation and End Labeling of Oligonucleotides--
The
80-base oligonucleotides were synthesized on an Expedite 8900 nucleic
acid synthesis system (PerSeptive Biosystems) using the phosphoramidite
method. The oligonucleotides were eluted out of the column using
aqueous ammonia and deprotected at 75 °C for 1 h, followed by
12% denaturing polyacrylamide gel purification. The 5'-end-labeled
single-stranded oligonucleotides were obtained by kination reaction
using T4 polynucleotide kinase and [-32P]ATP. The
labeled strands were annealed with the complementary strands and
purified on an 8% native polyacrylamide gel.
Topo II Cleavage Reactions-- The 5'-32P-labeled DNA was incubated with Drosophila topo II in 20 µl of a reaction buffer (10 mM imidazole-HCl (pH 6.0), 10 mM MgCl2, 50 mM KCl, and 1 mM App(NH)p or ATP) at 30 °C for 10 min in the presence of various amounts of A-62176. Reactions were terminated by adding SDS to 1% of the final concentration, and topo II was removed by proteinase K digestion (100 µg/ml) at 42 °C for 1 h, followed by phenol/chloroform extraction and ethanol precipitation.
Photocleavage Reactions-- The 5'-32P-labeled DNA was incubated with Drosophila topo II in 60 µl of a reaction buffer (10 mM imidazole-HCl (pH 6.0), 10 mM MgCl2, 50 mM KCl, and 1 mM App(NH)p) in the presence of 0.5 µM A-62176. The samples were loaded onto a 24-well Titertek microtiter plate (ICN) on top of a Pyrex glass shield and irradiated for 1 h with an 85-watt xenon lamp placed under the Pyrex glass. (Pyrex glass is used to filter out the UV light under 300 nm, thereby eliminating DNA damage caused directly by UV irradiation.) During the irradiation, the Titertek plate was turned three times to eliminate the light heterogeneity. Reactions were terminated by adding 10 µg of calf thymus DNA, followed by heating at 95 °C for 15 min in the presence of 0.1 M piperidine. The resulting samples were subjected to phenol/chloroform extraction followed by ethanol precipitation.
Competition Experiments-- The 5'-32P-labeled DNA was incubated with Drosophila topo II in a buffer (10 mM imidazole-HCl (pH 6.0), 10 mM MgCl2, 50 mM KCl, and 1 mM App(NH)p) containing various amounts of either A-62176, norfloxacin, or both. The incubation reactions proceeded at 30 °C for 10 min, followed by addition of psorospermin (10 µM final concentration). The reactions were continued for an additional 5 min and then terminated by adding 5 mg of calf thymus DNA, followed by heating at 95 °C for 15 min. In the presence of piperidine, this procedure induces strand breakage at the drug modification sites (20). The samples were extracted with phenol/chloroform followed by ethanol precipitation.
Gel Electrophoresis and Quantification--
The samples were
loaded onto a 12% denaturing sequencing gel. The dried gels were
exposed on both x-ray film and phosphor screen. Imaging and
quantification were performed using a PhosphorImager and ImageQuant 4.1 software from Molecular Dynamics.
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RESULTS |
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The Effect of A-62176 on the DNA Cleavage Produced by Topo II at Sites A and B of the 80-mer DNA
Experiments were designed to determine the effect of A-62176 on
the cleavage of DNA produced by topo II. The 80-mer shown in Fig.
2 was originally used in a DNase I
footprinting study, in which the enlarged region was protected by
Drosophila topo II, and only one topo II cleavage site (site
A) was detected (21). However, in the present study, it was discovered
that this fragment contains two adjacent topo II cleavage sites (sites
A and B in Fig. 3, lane 1),
although the intensity of the cleavage is much less at site B than at
site A. Under similar conditions to those used in the DNase I study (in
the presence of App(NH)p, a nonhydrolyzable ATP, and at pH 6), the
intensity of the topo II-mediated cleavage at site A decreased as the
concentrations of A-62176 were increased. Although the intensity of the
cleavage at site B was initially enhanced, it also eventually decreased
at high drug concentrations (lanes 2-7 in Fig.
3A). This reflects the typical bell-shaped curve for
intercalative topo II poisons (8), as shown in Fig. 3B. In a
previous study, it was proposed that the quinobenzoxazines are
catalytic inhibitors of mammalian topo II and do not trap the cleaved
complex (12). In accordance with the results of this study, we also
found that at pH 7.5 and above, the intensity of topo II-mediated
cleavage at both sites A and B decreased with increasing amounts of
A-62176 (data not shown). However, at a lower pH (pH 6-7) and using
App(NH)p, A-62176 can enhance Drosophila topo II-mediated
DNA cleavage at site B at low drug concentrations (Fig. 3 at pH 6, and
data at pH 7 not shown), which is typical of intercalative topo II
poisons.
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In order to determine whether the use of App(NH)p is critical for
A-62176 to behave like a topo II poison, ATP was used instead of
App(NH)p in a repetition of the topo II cleavage assay described above,
and the results are shown in Fig. 4. From
a comparison of Figs. 3 and 4, it can be seen that A-62176 behaves the
same with ATP as it does with App(NH)p, although the topo II-mediated cleavage level at site B reaches the maximum at about 0.2 µM of A-62176 with ATP instead of the 1 µM
found with App(NH)p. These results demonstrate that, at low drug
concentrations and pH levels between 6 and 7, A-62176 enhances DNA
cleavage by topo II at site B and reduces cleavage at site A,
regardless of whether ATP or App(NH)p is used.
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Evidence That A-62176 Intercalates at the +1 and +2 Sites
The Effect of Mismatched Base Pairs on Topo II Cleavage at Sites A
and B--
Recently, Bigioni and co-workers (22) observed that
mismatches introduced at the 4,
3,
2, or
1 positions reduced or abolished the topo II-mediated cleavage, whereas at +1 and +2 positions, the topo II-mediated cleavage increased (see Fig. 2 legend
for number definition). Since A-62176 can stimulate the topo
II-mediated DNA cleavage at site B, it seemed possible that A-62176
might have a similar effect at site B as that seen with the mismatched
sequences. To test this hypothesis, four sets of oligonucleotide
sequences were synthesized to contain mismatched base pairs at the +1,
+2, +3, or +4 positions of site B (Fig. 5A). The topo II cleavage
assays were performed on all four mismatched sequences (Fig.
5B). In comparison to the wild type sequence (lane W),
mismatches at either the +1 (lane M1) or +2
(lane M2) positions enhance the topo II-mediated
DNA cleavage at site B and abolish (in the case of M1) or
reduce (in the case of M2) the topo II cleavage at site A. On the other hand, mismatches at +3 (lane M3) and
+4 (lane M4) positions have little effect on topo
II-mediated DNA cleavage at site B. These results demonstrate that
mismatched base pairs at either +1 or +2 positions at site B can mimic
the A-62176 effect on topo II-induced cleavage at site A and B,
suggesting that A-62176 either introduces or stabilizes a DNA
distortion that is similar to a mismatch.
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In the Presence of Topo II, A-62176 Produces Enhanced DNA Photocleavage at Site B-- A photocleavage assay was used to determine the binding sites of A-62176 in the topo II-DNA complex. A-62176 has been shown to photocleave DNA around the drug intercalating site.2 Upon UV irradiation, DNA is cleaved through a free radical mechanism, leading to several DNA breakage products. The major products detectable in the gel electrophoresis after heating with piperidine are 3'-phosphate termini products,2 which co-migrate with the Maxam-Gilbert sequencing products in the polyacrylamide gel electrophoresis (23).
The DNA photocleavage ability of A-62176 was used to locate its binding
site in the topo II-DNA complex, as shown in Fig. 6A. In the absence of topo II,
there is a significant amount of DNA photocleavage by A-62176
(lanes 5-7 in Fig. 6A). In the presence of topo
II, the overall amount of photocleavage of DNA by A-62176 decreased
(lanes 8-10) as the amount of topo II was increased, possibly due to the increased amount of glycerol (50%) present in the
protein storage buffer, since glycerol is known to quench the free
radical reaction. However, in comparison with the control (lanes
5-7), the photocleavage of the guanine at the +2 position (arrows in Fig. 6, A and B) of
cleavage site B was enhanced (lanes 8-10). In comparison
with other photocleavage sites (i.e. bands II and III), the
enhanced site (band I) was clearly amplified 1.5-fold (or 50%
enhancement) in lanes 8-10 while the other bonds were
slightly diminished (Fig. 6D). These enhanced bands could not have come from the products generated by topo II-induced DNA cleavage, since those products associated with site B (lane
4) travel a half base farther than the enhanced photocleavage
products in the gel.3 In
fact, with an enlarged scan of this region (lane 10 in Fig. 6C), the residue of this topo II cleavage band can be
detected as a "shoulder" 5' of the guanine peak that has the
enhanced photocleavage. This result demonstrates an increased A-62176
binding to the guanine around the +2 position of cleavage site B in the
presence of topo II, indicating that topo II induces a DNA
conformational change that creates a high affinity site for binding of
A-62176.
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A-62176 Produces Enhanced Photocleavage at Site B in Mismatched Sequences M1 and M2-- To shed further light on the nature of the structural distortion of DNA induced by topo II, mismatched sequences M1 and M2 were used in the photocleavage reaction. These two sequences, which have a mismatched base pair at either the +1 or +2 position at site B, have been shown to mimic the A-62176 effect on the topo II-induced cleavage at sites A and B (see above). In the absence of topo II, A-62176 produced enhanced photocleavage at the guanine at the +2 position of site B for both M1 and M2 oligos (lanes 11 and 12 in Fig. 6, A, B, and D), which corresponds to the same guanine that showed an enhanced photocleavage by A-62176 in the presence of topo II. These results demonstrate that positioning a mismatched base pair at either the +1 or +2 position at site B mimics the structural change in DNA induced by topo II, suggesting that a similar structural perturbation, possibly a base pair opening, is introduced at the +1 and +2 positions by topo II when it binds to DNA.
Competition between Psorospermin and A-62176 for Binding at Site B-- Psorospermin, a potent DNA alkylating antitumor agent, has been shown to intercalate into the DNA helix and alkylate N-7 of guanine at the 3' side of the intercalation site (20). In the presence of topo II, the reactivity of psorospermin is enhanced more than 25-fold with the guanine at the +4' position at site B (24). A DNA strand breakage assay was used to locate the position of alkylation of DNA by the epoxide of psorospermin upon intercalation to the base pairs between the +1 and +2 positions at site B within the topo II-DNA complex (24). Psorospermin has subsequently been used to probe the binding sites for m-AMSA (24). In the present study, this same assay was performed to determine the binding site of A-62176 on DNA in the presence of topo II.
The A-62176 photocleavage studies have demonstrated that topo II
produces a structural distortion at the +1 and +2 positions at site B
that creates a binding pocket for A-62176. As an independent method for
determining the specific location of A-62176, a competition study
between psorospermin and A-62176 was carried out (Fig.
7). As demonstrated previously,
psorospermin displayed relatively weak DNA reactivity at a 10 µM concentration in the absence of topo II (Fig.
7A, lane 5). However, in the presence of topo II, the psorospermin alkylation of the guanine at the +4' position of site
B (band 2) was greatly enhanced (lane 6), while the
reactivity of psorospermin with the guanines at other positions (such
as band 1 and band 3) remained unchanged (24). As the concentrations of
A-62176 were increased (lanes 7-11), the amount of
psorospermin alkylation at site B decreased (band 2), as shown by the
reduction of the strand breakage product, while the reactivity of
psorospermin with the guanines at other position (bands 1 and 3) showed
little if any change (Fig. 7B). Since A-62176 is a
DNA-interactive intercalator, this competition effect may be due to its
nonspecific inhibition of topo II binding to DNA at site B. However, in
this experiment, nonhydrolyzable ATP was used. Under these condition, a
concentration of A-62176 (up to 10 µM) can enhance the
topo II-mediated cleavage (Fig. 3). Therefore, these results indicate
that A-62176 can specifically compete with psorospermin for
intercalation between positions +1 and +2 in the presence of topo II.
For comparison, norfloxacin was used instead of A-62176 in a parallel
experiment. In contrast to the intercalating A-62176, the
nonintercalating norfloxacin showed no competition with psorospermin
(lanes 12-16). In fact, quantitative data showed
that norfloxacin produced a small but significant enhancement of the
psorospermin alkylation at site B (Fig. 7B). Overall, these
results strongly support the idea that A-62176 specifically interacts
with DNA at the site where psorospermin binds in the presence of topo
II (i.e. between positions +1 and +2).
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The Cooperative Binding of A-62176 and Norfloxacin in the Presence of Topo II as Evidence for the Assembly of a 2:2 Drug-Mg2+ Complex on the Topo II-DNA Complex
In a previous study, it was proposed that a 2:2
drug-Mg2+ complex forms a "heterodimer complex" with
respect to DNA, in which one A-62176 molecule is intercalated into DNA
and a second A-62176 molecule is externally bound, held to the first
molecule by two Mg2+ bridges (17). In this complex, on the
basis of DNase I and viscometry studies, externally bound A-62176
molecules can be replaced by norfloxacin, demonstrating that
norfloxacin and A-62176 bind to DNA in a cooperative manner. In the
present study, competition experiments between psorospermin and
A-62176, in combination with norfloxacin, were used to determine if the
heterodimer complex is also formed in the presence of topo II. The
psorospermin alkylation of guanine at the +4' position of site B in
lane 5 (Fig. 8A) is greatly enhanced in the presence of topo II (compared with lane 4). As the concentrations of A-62176 were increased (lanes
6-11), the amount of alkylation by psorospermin at the same
positions decreased. The effects of 20 or 100 µM of
norfloxacin in combination with A-62176 on psorospermin alkylation are
shown in lanes 12-18 and 19-25, respectively.
At face value, it appears that norfloxacin has no effect on
psorospermin alkylation in combination with A-62176 (Fig.
8B, graph II). However, when the
enhancement effect of norfloxacin alone on psorospermin alkylation
(lanes 12 and 19 in Fig. 8A; Fig.
8B, graph I) is taken into account, it
is evident that norfloxacin does show an additive effect with A-62176.
If the enhancement effect by norfloxacin alone on psorospermin
alkylation is subtracted from the combination effect, as shown in Fig.
8B (graph III), A-62176, in
combination with 20 µM norfloxacin, shows an additive effect on the inhibition of psorospermin alkylation. This additive effect is even more dramatic when 100 µM of norfloxacin
is used. Hence, A-62176 and norfloxacin bind to the topo II-DNA complex in a cooperative manner, which is consistent with the 2:2
drug-Mg2+ model originally proposed for the binary complex
between the quinobenzoxazines and DNA (17).
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DISCUSSION |
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The results presented in this paper demonstrate that A-62176 possesses a dual mechanism at pH 6-7; at one topo II cleavage site (site B), A-62176 is an intercalative topo II poison, while at another topo II cleavage site (site A), A-62176 is a catalytic inhibitor of topo II. Using a combination of photocleavage assay, mismatched sequences, and competition experiments between psorospermin and A-62176, we have shown that topo II creates a high affinity intercalation site for A-62176 between the base pairs at positions +1 and +2 at site B. Our data are in accord with our previously proposed 2:2 quinobenzoxazine-Mg2+ heterodimer complex model (17).
A-62176 Has Two Mechanisms of Action: Topo II Poison at Site B and Catalytic Inhibitor at Site A-- Permana and co-workers have demonstrated that A-62176 is a strong catalytic inhibitor of mammalian topo II that does not increase topo II-mediated DNA strand breakage, suggesting that A-62176 should inhibit topo II reactions at a step prior to the formation of the cleaved complex (12). However, our results show that at pH 6-7, A-62176 can be both a topo II poison and a catalytic inhibitor. At one topo II cleavage site, A-62176 enhances the cleaved complex formation at low drug concentrations but inhibits it at high drug concentrations. At another topo II cleavage site, A-62176 inhibits the cleaved complex formation at both low and high drug concentrations. Our experimental conditions differed from Permana's study in two respects. First, in their paper, plasmid DNA was used to study overall drug effects on the DNA cleavage by topo II, whereas in our study, oligonucleotides were used in order to determine drug effects on the individual site. Second, our experiments were performed at pH 6-7, whereas in Permana's study, pH 7.5 was used. Hence, our results may not be contradictory to Permana's study, which concluded that A-62176 has an overall catalytic inhibitory effect on topo II (12).
When Binding to DNA at Site B, Topo II Induces a Conformational
Change in DNA That Creates a High Affinity Intercalation Site for
A-62176 between Base Pairs +1 and +2--
Previous studies have
suggested that topoisomerases can create high affinity DNA
intercalation sites for drugs by producing structural changes in DNA.
One example of this phenomenon is T4 topo II, which creates
preferential binding sites for m-AMSA at the 1 and +4' positions in
the immediate vicinity of the topo II cleavage gate (25). Likewise,
psorospermin shows an unusually high reactivity at the +4' position
within the gate site in the presence of Drosophila topo II
(Fig. 9C) (24). In the case
described here, both the photocleavage and topo II cleavage results
suggest that an enhanced binding of A-62176 occurs in the presence of Drosophila topo II. In addition, these results indicate that
a DNA base pair distortion, which resembles the mismatches, may occur
when topo II binds to site B, and this distortion might be recognized
and stabilized by A-62176.
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Competition experiments (Fig. 7) provide further evidence that A-62176
intercalates between the base pairs at the +1 and +2 positions in the
presence of topo II. Our results have demonstrated that the
intercalating A-62176 can compete with psorospermin for the DNA
intercalation pocket induced by topo II, while the nonintercalating analogue norfloxacin is unable to do so. Data from the competition experiments are consistent with our previous studies, which suggested that intercalating and nonintercalating topo II poisons bind to different sites in the topo II-DNA complex (24). Since psorospermin intercalates into the base pairs between the +1 and +2 positions of
site B (Fig. 9C), in order to compete with psorospermin
alkylation, A-62176 can intercalate between the same base pairs as
psorospermin (i.e. the position between +1 and +2) or, in
accordance with the nearest-neighbor exclusion principle for DNA
intercalators (26, 27), between the adjacent base pairs
(i.e. the positions between 1 and +1 or +2 and +3). Our
topo II cleavage and photocleavage studies indicate that the structural
changes induced by topo II at positions +1 and +2 create a preferential
binding site for A-62176. Therefore, it is likely that the
intercalations of both A-62176 and psorospermin into the same base
pairs between +1 and +2 may account for the competition between these
two drugs (Fig. 9). On the contrary, norfloxacin cannot compete with
psorospermin, suggesting that the planar tetracyclic ring, which is the
intercalative moiety of A-62176, is critical for the competition.
A-62176 Is Likely to Interact with the Topo II-DNA Complex at a Step prior to the Strand Cleavage-- It has been proposed that the stimulation effects of topo II poisons on topo II-mediated cleavage might be related to the cleavage-rejoining reaction of the enzyme (22, 28). A-62176, a strong DNA intercalator, appears to have the same effect, blocking the religation process of the topo II. However, subsequent studies found that base pairing within the overhang is not required for the religation process of topo II (29). In addition, a number of eukaryotic quinolones, such as CP-115,953, have been shown to enhance the topo II-mediated cleavage by increasing the forward cleavage reaction (30). Norfloxacin can stimulate the DNA binding of mutant topo IV that lacks the strand cleavage activity (31). Similarly, psorospermin alkylation is enhanced at the topo II cleavage gate without Mg2+, which is required for enzyme-mediated DNA cleavage (18). These results suggest that the DNA conformational change induced by topo II occurs preceding the strand cleavage event. Therefore, it is possible that A-62176 binds to the topo II-DNA complex at a step prior to strand cleavage and accelerates the strand cleavage by stabilizing the structural distortion so that the activation energy required for strand cleavage is reduced. A similar mechanism has been suggested for the mechanism of quinolone action (31).
The Experimental Findings Are Consistent with a 2:2 Quinobenzoxazine-Mg2+ Heterodimer Complex, Which Can Assemble in the Presence of Topo II-- The results of previous studies have led to a proposal of a 2:2 quinobenzoxazine-Mg2+ heterodimer complex model on duplex DNA, in which one quinobenzoxazine molecule serves as an intercalator and the other quinobenzoxazine molecule binds externally, held to the first drug molecule by two Mg2+ ions (Fig. 9A) (17). However, this model was proposed based on the experimental evidence on the drug-DNA binary complex in the absence of topo II. Our results (Fig. 8) show that A-62176 and norfloxacin have cooperative effects in the competition with psorospermin in the presence of topo II, indicating that this heterodimer complex may also assemble in the topo II-DNA complex (Fig. 9, A and B). In preliminary studies, in vitro experiments using norfloxacin with a quinobenzoxazine, we have also observed a dose-dependent enhancement of the cytotoxicity of the quinobenzoxazine compound by norfloxacin.4
Implications for Drug Design--
The 2:2
quinobenzoxazine-Mg2+ and 1:1:2
quinobenzoxazine-norfloxacin-Mg2+ heterodimer models have
important implications for future drug design, not only for eukaryotic
topo II but also for gyrase, since norfloxacin is known to be a gyrase
inhibitor. The proposed drug-Mg2+ heterodimers have two
moieties: one of them interacts exclusively with DNA through
intercalation and the other binds externally to DNA (Fig. 9,
A and B). The results presented in this paper suggest that topo II induces transient structural distortion, possibly
unwinding, that is captured or stabilized by the binding of A-62176 at
the topo II cleavage gate. Therefore, the binding of the 2:2
A-62176-Mg2+ heterodimer to the topo II-DNA complex
includes not only the intercalation of one A-62176 molecule to the
partially unwound DNA base pairs at the gate, but also the interaction
of the externally bound A-62176 molecule to topo II. This model
provides substantial information for drug design.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. John M. Cassady (Ohio State University) for providing the psorospermin compound. We thank Dr. Hongtao Yu (Jackson State University) for useful discussions; Dr. Robb Gardner, Maha Foote, and Frank Han for critical reading; and David Bishop for proofreading, editing, and preparing the final version of the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health (CA-49751) and the Welch Foundation (F-0890).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 correspondence should be addressed. Tel: 512-471-4841 Fax:
512-471-2746; E-mail: dg-dna{at}mail.utexas.edu.
2 H. Yu, Y. Kwok, S. M. Kerwin, and L. H. Hurley, manuscript in preparation.
3 Photoreaction with DNA alone shows no DNA cleavage under our conditions (lane 2 in Fig. 6A). In the presence of topo II, the enhanced bands detected in the gel could not have come from the products generated by topo II-induced DNA cleavage for two reasons. First, the reaction in lane 3, which is similar to that in lane 12 but without A-62176, showed a reversal of most topo II cleavage products upon heat-piperidine treatment. Second, with adenine at its 3'-end, the topo II cleavage product at site B should have traveled one base farther than the enhanced photocleavage product with cytosine at its 3'-end in the polyacrylamide gel electrophoresis (Fig. 2). However, the DNA cleavage product produced by topo II has a 3'-hydroxyl terminus, which migrates one-half base less than the 3'-phosphate product at the same position in the gel. Therefore, the topo II cleavage product of site B on the top strand travels a half base farther than the photoproduct with enhanced cleavage in the gel.
4 L. Hurley, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
topo II, topoisomerase II;
App(NH)p, ,
-imidoadenosine 5'-triphosphate;
A-62176, 1-(3-aminopyrrolidin-1-yl)-2-fluoro-4-oxo-4H-quino[2,3,4-i,j][1,4]benzoxazine-5-carboxylic
acid.
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REFERENCES |
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