effects of quinolone derivatives on eukaryotic topoisomerase i1

THE JOURNAL Q 1991 by The American Society for Biochemistry and

OF BIOLOGICAL CHEMISTRY Molecular Biology, Inc

Vol. 266, No. 22, Issue of August 5, pp. 14585-14592,1991 Printed in U. S A .

Effects of Quinolone Derivatives on Eukaryotic Topoisomerase I1 A NOVEL MECHANISM FOR ENHANCEMENT OF ENZYME-MEDIATED DNA CLEAVAGE*

(Received for publication, February 1, 1991)

Megan J. Robinson$#, Barbara Anne MartinlI, Thomas D. Gootzv, Paul R. McGuirkll, Melinda Moynihanll, Joyce A. Sutcliffev, and Neil Osheroff$# From the $Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and the TDepartment of Immunology and Infectious Diseases, Pfizer Central Research, Pfizer, Inc., Groton, Connecticut 06340

The effects of two novel quinolone derivatives, CP- 67,804 and CP-115,953 (the 1-ethyl and l-cyclopropyl derivatives of 6,8-difluoro-7-(4-hydroxyphenyl)- 4-quinolone-3-carboxylic acid, respectively), on the enzymatic activities of Drosophila melanogaster topoisomerase I1 were examined. Both drugs enhanced the enzyme’s pre- and post-strand passage DNA cleavage activities. CP-67,804 was nearly as potent an en- hancer as etoposide, while CP-115,953 was -2 times more potent than this topoisomerase 11-targeted antineoplastic drug. In contrast to etoposide, which stabi- lizes enzyme-DNA cleavage complexes primarily by inhibiting topoisomerase 11-mediated DNA religation, neither quinolone impaired the enzyme’s ability to religate cleaved DNA. To further assess the character- istics of these unusual quinolone derivatives, the cytotoxic effects of CP-67,804 and CP-115,953 toward wild-type Chinese hamster ovary cells and VpmR-5 cells (an epipodophyllotoxin-resistant Chinese hamster ovary line) were examined. Both quinolones were cytotoxic to the wild-type cells. CP-115,953 was the more potent agent and displayed a level of cytotoxicity similar to that of etoposide. Finally, the VpmR-5 line showed cross-resistance to CP-67,804 (-3.7-fold) and CP-115,953 (-1.3-fold). Although quinolone cross-resistance was less pronounced than observed for etoposide (-12-fold), it indicates that topoisomerase I1 is a physiological target for CP-67,804 and CP- 115,953 in mammalian cells. These findings strongly suggest that these quinolone derivatives represent a novel class of topoisomerase 11-targeted drugs which have potential as antineoplastic agents.

The ability to modulate the topological state of DNA is essential to the survival of both eukaryotic and prokaryotic cells (1). The enzymes responsible for this critical function are known as DNA topoisomerases (1-3). Most cell types contain at least two different topoisomerases. Eukaryotic topoisomerase I1 and its prokaryotic counterpart, DNA gyrase, are required for proper chromosome structure, segrega-

* This work was supported by National Institutes of Health Grant GM-33944, North Atlantic Treaty Organization Grant 5-2-05/RG 0157188, and funds from Pfizer Central Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I Trainee under Grant T32 CA-09582 from the National Cancer Institute.

11 Supported by Faculty Research Award FRA-370 from the Amer- ican Cancer Society. To whom correspondence and reprint requests should be addressed.

tion, and condensation (4-16). They also play important roles in DNA replication, transcription, and recombination (1, 2). Both enzymes alter nucleic acid topology by creating a tran- sient double-stranded break in the DNA backbone and pass- ing a second DNA helix through the break (1-3).

In addition to their normal cellular functions, the eukaryotic and prokaryotic type I1 enzymes are targets for a wide variety of clinically relevant chemotherapeutic drugs (17-22). Eukaryotic topoisomerase I1 is the target for intercalative (with respect to DNA) antineoplastic agents such as m- AMSA,’ adriamycin, and ellipticine, as well as nonintercalative agents such as etoposide and teniposide (17-19, 23-26). The clinical efficacies of these drugs correlate with their abilities to enhance enzyme-mediated DNA cleavage (17-19). Recent work indicates that m-AMSA and etoposide stabilize the topoisomerase 11-DNA cleavage complexes formed both prior to and following strand passage and, in both cases, do so primarily by inhibiting the enzyme’s ability to religate cleaved DNA (27-29).

DNA gyrase is the target for nonintercalative (30) quinolone-based drugs such as ciprofloxacin and norfloxacin (19- 22). These quinolones represent two of the most potent antimicrobial agents currently in clinical use (21, 22, 31). Al- though their mechanism of action is not as well characterized as that of the eukaryotic topoisomerase 11-targeted drugs, quinolone efficacy also correlates with the ability to enhance enzyme-mediated DNA cleavage (19-22, 31). Moreover, pre- liminary evidence indicates that some members of this drug class inhibit gyrase-mediated DNA religation (61).

A number of studies have found that, at high concentrations, quinolone-based drugs can inhibit the catalytic DNA strand passage activity of eukaryotic topoisomerase I1 (32- 39). In addition, some of the most potent quinolones, such as ciprofloxacin and CP-67,015, stimulate DNA cleavage mediated by the eukaryotic enzyme (36). Considering the wide clinical use of quinolones as antimicrobial agents, it is important to determine their potential for interactions with eukaryotic topoisomerase 11. To this end, the effects of CP-67,804 and CP-115,953, two novel quinolone derivatives (and more potent analogs of CP-67,015), on the enzymatic activities of Drosophila melanogaster topoisomerase I1 were characterized. Both drugs enhanced pre- and post-strand passage DNA

The abbreviations used are: m-AMSA, 4’-(9-acridinylam- ino)methanesulfon-m-anisidide; etoposide, 4’-demethylepipodophyl- lotoxin 9-[4,6-0-ethylidene-@-~-glucopyranoside]; CP-67,015, 6,8-difluoro-7-pyridyl-l-ethyl-4-quinolone-3-carboxylic acid; CP-67,804, 6,8-difluoro-7-(4-hydroxyphenyl)-l-ethyl-4-quinolone-3-carboxylic acid; CP-115,953, 6,8-difluoro-7-(4-hydroxyphenyl)-l-cyclopropyl-4- quinolone-3-carboxylic acid CHO, Chinese hamster ovary; APP(NH)P, adenyl-5”yl @,y-imidodiphosphate; SDS, sodium dode- cy1 sulfate.

14585

14586 Effects of Novel Quinolones on Eukaryotic Topoisomerase I1

cleavage with efficacies similar to or greater than that of etoposide. However, in contrast to etoposide, neither quinolone inhibited the ability of Drosophila topoisomerase I1 to religate cleaved DNA. These results strongly suggest that CP- 67,804 and CP-115,953 represent a novel class of topoisomerase 11-targeted drugs.

EXPERIMENTAL PROCEDURES

Materials

DNA topoisomerase I1 was purified from the nuclei of D. melanogaster Kc tissue culture cells or 6- to 12-h-old embryos by the procedure of Shelton et al. (40). Calf thymus topoisomerase I1 was the generous gift of Dr. P. S. Jensen and Dr. 0. Westergaard and was purified by the procedure of Andersen et al. (41). Negatively supercoiled bacterial plasmid pBR322 DNA was obtained from Escherichia coli DH1 by a Triton X-100 lysis followed by a double banding in cesium chloride-ethidium bromide gradients (42). Quinolone derivatives CP-67,804 and CP-115,953 were synthesized at Pfizer Central Research by the procedure described in Ref. 62. CP-67,804 was dissolved as a 15 mM solution in 0.1 N NaOH and diluted to a 1 mM stock with 10 mM Tris-HC1, pH 8.0. CP-115,953 was dissolved as a 25 mM solution in 0.1 N NaOH and diluted to a 5 mM stock with 10 mM Tris-HC1, pH 8.0. Quinolone stock solutions were stored in the dark at -80 "C. Etoposide (VePesid, VP-16-23) was purchased from Bristol Laboratories as a sterile 20 mg/ml solution in etoposide diluent (2 mg/ml citric acid, 30 mg/ml benzyl alcohol, 80 mg/ml polysorbate 80/Tween 80, 650 mg/ml polyethylene glycol 300, 30.5% (v/v) ethanol). The drug was stored at room temperature as per the manufacturer's instructions. Tris, ethidium bromide, and APP(NH)P were obtained from Sigma; SDS and proteinase K were from E. Merck Biochemicals; ATP was from Pharmacia LKB Biotechnology Inc.; and calf thymus topoisomerase I was from Bethesda Research Laboratories. All other chemicals were analytical reagent grade.

Topoisomerase II-mediated DNA Cleavage

All DNA cleavage reactions contained 25-100 nM topoisomerase I1 and 5 nM negatively supercoiled pBR322 DNA in a total volume of 20 p1 of cleavage buffer (10 mM Tris-HC1, pH 7.9, 25 mM NaC1, 50 mM KC1, 5 mM MgCl,, 0.1 mM EDTA, and 2.5% glycerol). Reactions that monitored the DNA cleavage/religation equilibrium established prior to the enzyme's strand passage event contained no ATP analog. Reactions that monitored the DNA cleavage/religation equilibrium established after enzyme-mediated strand passage contained 1 mM APP(NH)P. Samples containing Drosophila topoisomerase I1 were incubated at 30 "C for 6 min. Samples containing calf thymus topoisomerase I1 were incubated at 37 "C for 6 min. Cleavage products were trapped by the addition of 2 pl of 10% SDS (43). One microliter of 250 mM EDTA and 2 p1 of an 0.8 mg/ml solution of proteinase K were added, and samples were incubated at 45 "C for 30 min to digest the topoisomerase 11. Final products were mixed with 2.5 pl of loading buffer (60% sucrose, 0.05% bromphenol blue, 0.05% xylene cyano1 FF, and 10 mM Tris-HC1, pH 7.9), heated at 70 "C for 1 min, and subjected to electrophoresis in 1% agarose (MCB) gels in 40 mM Tris- acetate, pH 8.3, and 2 mM EDTA. Following electrophoresis, DNA bands were stained in a 1 pg/ml solution of ethidium bromide and visualized by transillumination with ultraviolet light (300 nm). In experiments which contained APP(NH)P, ethidium bromide (0.5 pg/ ml) was added to both the gel and the running buffer prior to electrophoresis in order to facilitate the separation of DNA cleavage products from covalently closed relaxed topoisomers. DNA bands were photographed through Kodak 23A and 12 filters using Polaroid type 665 positive/negative film. The amount of DNA was quantitated by scanning negatives with an EC 910 scanning densitometer using HSI 370 software. Under the conditions employed, the intensity of the bands in the negative was directly proportional to the amount of DNA present.

The effects of drugs on both the pre- and post-strand passage DNA cleavage/religation equilibria were examined over a range of 0 to 150 p ~ . An amount of diluent equal to that in drug-containing samples was added to all control samples. None of the diluents affected the topoisomerase 11-mediated DNA cleavage/religation equilibrium.

Topoisomerase II-mediated DNA Religation Reactions contained 25-100 nM topoisomerase I1 and 5 nM nega-

tively supercoiled pBR322 DNA in a total of 20 pl of cleavage buffer. Initial DNA cleavage/religation equilibria were established at 30 "C for 6 min. As described above, pre-strand passage cleavage/religation equilibria were established in the absence of a nucleotide triphosphate and post-strand passage equilibria were established in the presence of 1 mM APP(NH)P. Topoisomerase 11-mediated religation of cleaved DNA was induced by the following methods.

Calcium Religation-Cleavagelreligation equilibria were established as described above except that CaCL replaced MgC1, as the divalent cation (44,45). Kinetically competent topoisomerase 11-DNA cleavage complexes were trapped by the addition of 0.8 pl of 250 mM EDTA. NaCl(0.6 pl of a 5 M solution) was added to prevent recleavage of the DNA, and samples were placed on ice to slow reaction rates. Religation was initiated by the addition of cold MgCl, (8.5 mM final concentration). Religation was terminated by the addition of SDS (1% final concentration) at various time points up to 30 s.

Heat-induced Religation-Samples were rapidly shifted from 30 "C to 55 "C (29, 44, 46, 47). Religation was terminated by the addition of SDS (1% final concentration) at various time points up to 30 s.

Cold-induced Religation-Samples were rapidly shifted from 30 "C to 0 "C (29, 48). Religation was terminated by the addition of SDS (1% final concentration) at various time points up to 120 s.

Following all procedures, samples were treated with EDTA and proteinase K as described above. Reaction products were resolved by agarose gel electrophoresis and quantitated as described above.

The effects of drugs on topoisomerase 11-mediated DNA religation were examined by the addition of 100 p~ etoposide, 150 p M CP- 67,804, or 50 p M CP-115,953 to reactions. Drugs were added during the initial enzyme-DNA incubation in religation protocol 1 and prior to the temperature shifts in protocols 2 and 3. Control reactions contained amounts of diluent equivalent to those in drug-containing samples.

Topoisomerase II-mediated DNA Relaxation Assays were carried out as previously described (33). Briefly, re-

actions contained 0.3 nM topoisomerase 11 and 5 nM negatively supercoiled pBR322 DNA in a total of 20 pl of relaxation buffer (10 mM Tris-HC1, pH 7.9, 50 mM NaCl, 50 mM KCl, 5 mM MgC12, 0.1 mM EDTA, and 2.5% glycerol) that contained 1 mM ATP. DNA relaxation was carried out at 30 "C for 15 min, and reactions were stopped by the addition of 2.5 pl of stop solution (0.77% SDS and 77 mM EDTA, pH 8.0). Samples were mixed with 2.5 p1 of loading buffer, heated 1 min at 70 "C, and subjected to electrophoresis in 1% agarose gels in 40 mM Tris-borate, pH 8.3, and 2 mM EDTA. Gels were stained and visualized, and bands were quantitated as described above. The effects of drugs were examined over a range of 0-150 pM.

Cytotoxicity of Quinolone Derivatives toward Mammalian Cells Wild type CHO cells and VpmR-5, a mutant CHO cell line selected

for resistance against epipodophyllotoxins (49), were the generous gift of Dr. R. Gupta and Dr. D. M. Sullivan. Cells were cultured as monolayers at 37 "C under 5% COz in a-minimal essential medium (Gibco Grand Island Biologicals) (without antibiotics) supplemented with 5% fetal calf serum. Drug cytotoxicity was determined by a colony-forming assay (50). Assays employed log phase, trypsinized cells that were tested for viability by trypan blue exclusion. Cells were seeded, 1,000 per well, in 35-mm plates for 18 h prior to drug treatment. Both wild type CHO and VpmR-5 cells were treated with CP-67,804, CP-115,953, or etoposide (as a control) for 1 h at 37 "C. Cells were rinsed twice with phosphate-buffered saline and incubated in fresh medium for 5 days. Culture medium was removed, and colonies were stained with 2% crystal violet in methanol and counted on an Artek Systems plate reader. Plating efficiencies ranged from 20 to 30%.

RESULTS

A number of quinolone derivatives have been shown to affect the catalytic activities of eukaryotic topoisomerase I1 (32-39). In particular, ciprofloxacin and CP-67,015 enhance the enzyme's ability to mediate double-stranded DNA cleavage (36). Despite the wide clinical usage of quinolone-based gyrase inhibitors as antimicrobial agents (20-22, 31), their interactions with eukaryotic topoisomerase I1 have not been

Effects of Novel Quinolones on Eukaryotic Topoisomerase 11 14587 0 0

CP-67,804 CP-115,953 FIG. 1. Structures of the 4-quinolone derivatives.

1 2 3 4 5 FIG. 2. Effects of CP-67,804, CP-115,953, and etoposide

on the pre-strand passage DNA cleavage/religation equilibrium of D. melanogaster topoisomerase 11. An agarose gel is shown. Lane I, DNA standard lane 2, reaction contained topoisomerase I1 but no drug; lane 3, reaction contained 100 pM CP-67,804; lane 4, reaction contained 100 p~ etoposide; lane 5, reaction contained 100 p~ CP-115,953. The positions of fully supercoiled DNA (form I, FI) , nicked circular plasmid molecules (form 11, FZZ), and linear molecules (form 111, F I I I ) are indicated.

well characterized. In order to elucidate the mechanism by which these drugs interact with the eukaryotic type I1 enzyme, the effects of two novel quinolones, CP-67,804 and CP- 115,953, on the activity of D. melanogaster topoisomerase I1 were examined. The structures of these compounds are shown in Fig. 1. CP-67,804 and CP-115,953 were originally developed as gyrase-targeted antimicrobial drugs. They show potent activity against the E. coli enzyme and stimulate gyrase- mediated DNA cleavage (36) at minimal concentrations of 0.58 and 0.14 pM drug, respectively.’ The effects of CP-67,804 and CP-115,953 on the DNA cleavage/religation and relaxation activities of Drosophila topoisomerase I1 are described below. In all cases, the properties of these quinolones are compared to those of etoposide, a clinically relevant topoisomerase 11-targeted antineoplastic agent (17-19).

Effects of CP-67,804 and CP-115,953 on the DNA Cleavage/ Religation Reaction Mediated by Topoisomerase 11 Prior to Strand Passage-The ability to cleave and religate double- stranded DNA is central to the physiological functions of topoisomerase I1 (1-3). The enzyme establishes DNA cleavage/religation equilibria both before and after its double- stranded DNA passage event. Recent studies indicate that both of these equilibria are targets for the actions of antineoplastic drugs (27-29, 51, 52). Since strand passage requires ATP binding (2, 3, 33), the enzyme’s pre- and post-strand passage DNA cleavage/religation equilibria can be separated experimentally. All of the assays described in this section were carried out in the absence of ATP. Thus, only pre-strand passage equilibria were established.

As determined by either the increase in linear (FIII) or the decrease in supercoiled (FI) DNA, both CP-67,804 and CP- 115,953 enhanced the ability of Drosophila topoisomerase I1 to mediate the formation of double-stranded breaks in DNA (Fig. 2). The effect of quinolone concentration on the en-

’ These values are as compared to 0.60 pM for ciprofloxacin (36), one of the most potent gyrase-targeted antimicrobial agents currently in clinical use (31).

zyme’s pre-strand passage DNA cleavage/religation equilibrium is shown in Fig. 3. In this experiment, relative DNA cleavage (double-stranded) was determined by the increase in linear DNA in drug-containing assays relative to assays which contained no drug. Clearly, CP-67,804 and CP-115,953 are potent stimulators of DNA cleavage as compared with etoposide. At 150 p~ CP-115,953, a slight decrease in DNA cleavage was observed (Fig. 3). This decrease in linear DNA was caused by the formation of multiple topoisomerase II- DNA cleavage complexes per plasmid molecule (which con- verted unit length to subunit length FIII molecules) (see Fig. 2) rather than an inhibition of enzyme activity. As calculated from the linear range (550 pM drug) of the drug concentration curves CP-67,804 was -80% as potent and CP-115,953 was -200% more potent than etoposide at enhancing enzyme- mediated DNA cleavage.

To further characterize the efficacies of these quinolone derivatives, the effects of CP-67,804 and CP-115,953 on the DNA cleavage reaction of calf thymus topoisomerase I1 were determined (not shown). If anything, the quinolones were considerably more potent against the mammalian enzyme than they were for Drosophila topoisomerase 11.

Since CP-67,804 and CP-115,953 are novel compounds, it is important to ensure that all of the DNA breakage generated in their presence was produced by topoisomerase 11. The data presented in Fig. 4 demonstrate that this is the case. All DNA cleaved in the presence of CP-67,804 or CP-115,953 was covalently attached to the enzyme. Reaction products had to be digested with proteinase K in order for cleaved DNA to be released. The covalent topoisomerase 11-cleaved DNA linkage is a hallmark of the type I1 enzyme (1-3, 44, 48, 53). Finally, in the absence of topoisomerase 11, neither quinolone derivative produced any DNA cleavage.

Effects of CP-67,804 and CP-115,953 on the Ability of To- poisomerase IZ to Religate Cleaved DNA Prior to Strand Pas- sage-Previous studies demonstrated that two structurally disparate topoisomerase 11-targeted antineoplastic drugs, etoposide (27) and m-AMSA (28), stimulate DNA breakage before strand passage primarily by inhibiting the enzyme’s ability to religate cleaved DNA. In order to determine the

1 r FIG. 3. Effects of quinolone concentration on the pre-strand

passage cleavage/religation equilibrium of topoisomerase 11. An etoposide titration is shown for comparison. Results are plotted as the relative amount of double-stranded DNA cleavage versus drug concentration. The relative level of DNA cleavage was arbitrarily set to 1 in the absence of drug. Reactions were carried out in the presence of CP-67,804 (O), CP-115,953 (O), or etoposide (W). Data represent the average of 2-4 independent experiments. The average standard deviation (or standard error where appropriate) for the data shown was less than 0.9.

14588

F I I - F 111 -

FI- 1

Effects of Novel Quinolones on Eukaryotic Topoisomerase II

-

Bound DNA

d

2 3 4 5 6 7 8 FIG. 4. Double-stranded DNA cleavage stimulated by CP-

67,804 and CP-115,953 is mediated by topoisomerase 11. DNA cleavage assays were carried out in the presence of 150 PM CP-67,804 (lanes 1-4) or 50 PM CP-115,953 (lanes 5-8). Lanes J and 5, DNA standards; lanes 2 and 6, DNA cleavage products were digested with proteinase K prior to electrophoresis; lanes 3 and 7, DNA cleavage products were not digested with proteinase K; lanes 4 and 8, assays were carried out in the absence of topoisomerase 11. The positions of topoisomerase 11-bound DNA as well as those of form I, 11, and 111 plasmid molecules are indicated.

mechanism by which CP-67,804 and CP-115,953 enhance DNA breakage, their effects on the pre-strand passage religation reaction of topoisomerase I1 were examined.

Three independent DNA religation assays were employed for this purpose. One assay (calcium religation) takes advan- tage of the fact that calcium can be used to trap the pre- strand passage topoisomerase 11-DNA cleavage complex in an active form (44, 45). This allows the enzyme's religation reaction to be uncoupled from its cleavage activity. The other two assays rely on the finding that the religation activity of topoisomerase I1 is less sensitive to variations in temperature than is its cleavage activity (29,44,46-48). While the enzyme displays a decreased ability to cleave double-stranded DNA at extremes of temperature (0 "C or 55 "C for the Drosophila type I1 enzyme), it can still religate cleaved nucleic acids a t these temperatures. Therefore, following a shift from 30 "C (the optimal reaction temperature for the Drosophila enzyme (33)) to either 55 "C (heat religation) or 0 "C (cold religation), DNA molecules cleaved by topoisomerase I1 are reconverted to their original supercoiled state in a time-dependent fashion. In all of the drug-containing assays, 150 pM CP-67,804, 50 ~ L M CP-115,953, or 100 p~ etoposide was included. These drug concentrations were employed because they stimulated enzyme-mediated DNA cleavage to a similar extent (see Fig. 3).

The effects of CP-67,804 and CP-115,953 on the pre-strand passage DNA religation reaction of Drosophila topoisomerase I1 as monitored by the calcium religation assay are shown in Fig. 5. Neither quinolone derivative significantly inhibited DNA religation. Indeed, CP-67,804 and CP-115,953 decreased the apparent first order rate of religation by only 20 and 40%, respectively, as compared to etoposide which decreased this apparent rate by -3-fold. Because of this unexpected finding, the effects of quinolones on pre-strand passage religation were also monitored by the heat religation assay (Fig. 6). This latter assay is more sensitive to the effects of topoisomerase 11-targeted antineoplastic drugs. As can be seen in Fig. 6, etoposide inhibited the apparent first order rate of religation in the heat religation system by -9-fold. In contrast, CP- 67,804 and cP-115,953 still inhibited religation rates by less than 40%. Finally, to further confirm the above data, the effect of CP-67,804 on topoisomerase 11-mediated DNA reli-

5 0 10 20

Time (SI FIG. 5. Effects of 150 p~ CP-67,804 (0) and 50 p~ CP-

115,953 (0) on the ability of topoisomerase I1 to mediate pre- strand passage DNA religation as monitored by the calcium assay. Religation was performed as described under "Experimental Procedures." Results obtained in the absence of drug (0) or in the presence of 100 PM etoposide (M) are shown for comparison. Data are plotted in a semilogarithmic fashion as the loss of linear DNA uersus time. The percent linear DNA for each assay was arbitrarily set a t 100% a t time zero. Plots represent the average of 2-3 independent experiments. The average standard deviation (or standard error where appropriate) for the data shown was less than 5%.

F L J -0 10 20 30

Time (SI FIG. 6. Effects of 150 p~ CP-67,804 (0) and 50 pM CP-

115,953 (0) on the ability of topoisomerase I1 to mediate pre- strand passage DNA religation as monitored by the heat assay. Religation was initiated by shifting assay mixtures from 30 'C to 55 "C as described under "Experimental Procedures." Results obtained in the absence of drug (0) or in the presence of 100 pM etoposide (W) are shown for comparison. Data are plotted in a semilogarithmic fashion as the loss of linear DNA uersus time. The percent linear DNA for each assay was arbitrarily set a t 100% a t time zero. Plots represent the average of 2-3 independent experiments. The average standard deviation (or standard error where appropriate) for the data shown was less than 5%.

gation was monitored by the cold religation assay (not shown). A result similar to that found with the heat religation assay was obtained.

As determined by three independent assay systems, neither

Effects of Novel Quinolones on Eukaryotic Topoisomerase 11 14589

CP-67,804 nor CP-115,953 impairs the ability of Drosophila topoisomerase I1 to religate DNA substrates cleaved prior to strand passage. This conclusion is in marked contrast to previous findings obtained with etoposide (27) and m-AMSA (28) and suggests that these quinolone derivatives increase topoisomerase 11-mediated DNA breakages by enhancing the enzyme's forward rate of cleavage.

Actions of CP-115,953 and Etoposide Are Not Synergistic- The finding that CP-115,953 and etoposide stimulate DNA cleavage by different (and complementary) mechanisms opens the possibility that these drugs may have a synergistic effect on topoisomerase 11-mediated DNA breakage. Therefore, the enzyme's pre-strand passage DNA cleavage/religation equilib- 0 50 100 150 rium was examined in the simultaneous presence of CP- 115,953 and etoposide (not shown). Three independent experiments were carried out. All contained equimolar levels of the strand passage cleavage,religation equilibrium of topoiso,,,-

FIG. 7. Effects of quinolone concentration on the post-

two drugs over a range of subsaturating concentrations (5100 erase 11. ~ 1 1 assays contained 1 mM ~ p p ( ~ ~ ) p . An etoposide pM drug). At all drug concentrations employed, the effects of titration is shown for comparison. Results are plotted as the amount CP-115,953 and etoposide were at most additive. No synergis- of double-stranded DNA cleavage versus the concentration of drug. tic enhancement of DNA breakage was observed in any of the The relative level of DNA cleavage was arbitrarily set to 1 in the assays performed. This result indicates that the actions of absence of drug. Reactions were carried out in the presence of CP-

67,804 (.), CP-115,953 (O), or etoposide (W). Data represent the

plementary, are mutually exclusive. viation (or standard error where appropriate) for the data shown was

Religation Reaction Mediated by Topoisomerase ZZ following Strand Passage-Topoisomerase I1 also establishes a DNA cleavage/religation equilibrium following its strand passage event (2, 3). This post-strand passage equilibrium can be isolated from its pre-strand passage counterpart by the inclu- sion in assays of APP(NH)P, a nonhydrolyzable ATP analog which supports strand passage but will not allow enzyme turnover (33,34,54). Levels of enzyme-mediated DNA cleavage observed in the absence of drugs are -4 times higher following strand passage (29, 55). A previous study on the mechanism of action of etoposide and m-AMSA demonstrated ae that both drugs stimulated DNA breakage after strand passage, albeit to a lesser relative extent than observed before strand passage, and did so primarily by inhibiting topoisom- 0 10 20 30 erase 11-mediated DNA religation (29).

duced a minimum of 75% of the topoisomerase 11-DNA com- FIG. 8. Effects of 150 PM CP-67,804 (0) and 60 p~ CP- plexes to undergo strand passage (29). Over a wide concentra- 115,953 (0) on the ability of topoisomerase I1 to mediate post- tion range, CP-67,804 and CP-115,953 enhanced the ability strand Passage DNA religation as monitored by the heat assay.

Religation was initiated by shifting assay mixtures from 30 "C to

4- Q

[Drug] (pM)

cp-115,953 and etoposide On topoisomerase ' ' 9 average of 2-4 independent experiments. The average standard de-

Effects of CP-67,804 and CP-115,953 on the DNA Cleavage/ less than 0.8.

Under the reaction conditions employed, APP(NH)P in- Time (SI

Of Drosophila topoisomerase I1 to produce 55 "C as described under "Experimental Procedures." All assays con- breaks in DNA following strand passage (Fig. 7). The order tained 1 mM APP(NH)P. Results obtained in the absence of drug (0) of drug potency (CP-115,953 > etoposide > CP-67,804) was or in the presence of 100 PM etoposide (W) are shown for comparison. the same as that found prior to strand passage (see Fig. 3). Data are plotted in a semilogarithmic fashion as the loss of linear However, as shown for other topoisomerase 11-targeted drugs DNA V m u s time. The percent linear DNA for each assay was

(29), the relative abilities of these quinolone derivatives to independent experiments. The average standard deviation (or arbitrarily set at 100% at time zero. Plots represent the average of 2-

enhance enzyme-mediated DNA decreased standard error where appropriate) for the data shown was less than strand passage. 5%.

poisomerase IZ to Religate DNA following Strand passage- found for 100 p~ etoposide, which inhibited the apparent rate Effects of CP-67,804 and CP-115,953 on the Ability of To-

Because of their unexpected Properties in Pre-strand Passage of DNA religation -&fold. Thus, as found for the pre-strand religation assays, the effects of 150 PM Cp-677804 and 50 PM passage reaction, the novel quinolone derivatives do not im- Cp-1159953 on the enzyme's Post-strand Passage DNA reli- pair the ability of Drosophila topoisomerase 11 to religate gation reaction were examined. The calcium religation assay DNA substrates cleaved after strand passage. could not be employed for this Purpose, since Calcium-ATP Effects of CP-67,804 and CP-115,953 on the DNA Relaxation Will not support the DNA strand Passage event mediated by Reaction Catalyzed by Topoisomerase ZZ-Many topoisomer- Drosophila topoisomerase 11 (33). Therefore, quinolone effects ase II-targeted antineoplastic drugs have been shown to in- on post-strand passage DNA religation were monitored by the hibit the enzyme's catalytic strand passage reaction as moni- heat religation assay described earlier. As seen in Fig. 8, tored by its DNA relaxation, unknotting, catenation, or de- neither quinolone derivative inhibited religation. In fact, CP- catenation activities (17-19). With few exceptions (55), drugs 67,804 and CP-115,953 both stimulated the apparent first which are potent inducers of topoisomerase 11-mediated DNA order rate of enzyme-mediated post-strand passage DNA recleavage are also potent inhibitors of these activities. ligation by -40%. This result is in sharp contrast to that Since a number of quinolone derivatives have been found

14590 Effects of Novel Quinolones on Eukaryotic Topoisomerase 11

to inhibit the catalytic strand passage reaction of eukaryotic topoisomerase I1 (32-39), the effects of CP-67,804 and CP- 115,953 on the DNA relaxation activity of the Drosophila enzyme were examined. A quinolone concentration range similar to that employed for DNA cleavage/religation experiments was used for these studies. As seen in Fig. 9, CP- 67,804 and CP-115,953 were less inhibitory than etoposide. While 50% inhibition was observed at -10 p~ etoposide, -50 p~ CP-115,953 was required to achieve this level of inhibition. This is despite the fact that CP-115,953 is -2-fold more potent than etoposide in stimulating topoisomerase 11-mediated DNA cleavage (see Figs. 3 and 7). In addition, CP-67,804 (which stimulated DNA cleavage nearly as well as etoposide) showed little ability to inhibit DNA relaxation. At a concentration of 100 p ~ , CP-67,804 inhibited relaxation less than 10%. Fifty percent inhibition was not observed until the drug concentration was increased to -325 pM (not shown). There- fore, CP-67,804 and CP-115,953 are less potent inhibitors of enzyme-catalyzed DNA relaxation than they are enhancers of DNA cleavage.

CP-67,804 and CP-115,953 Are Nonintercalative with Re- spect to DNA-Topoisomerase 11-targeted drugs fall into two broad classes: those which are intercalative with respect to DNA (such as rn-AMSA) (23, 24), and those which are nonintercalative (such as etoposide) (25, 26). Quinolone derivatives previously examined appear to be nonintercalative in nature (30). Due to the novel properties of CP-67,804 and CP-115,953, their abilities to intercalate in DNA were determined by an unwinding assay (52) (not shown). In this assay, pBR322 plasmid DNA was reIaxed by topoisomerase I in the presence of either 150 pM CP-67,804 or 150 pM CP-115,953. This was possible as neither quinolone derivative affected the DNA relaxation or cleavage activities of the eukaryotic type I enzyme (not shown). Following relaxation, samples were phenol-extracted and subjected to electrophoresis on an agarose gel. No unwinding was observed with either drug. Thus, as found for other quinolones, CP-67,804 and CP-115,953 appear to be nonintercalative in nature.

Cytotoxicity of CP-67,804 and CP-115,953-In order to further characterize the properties of CP-67,804 and CP-115,953, their cytotoxicities toward mammalian cells were examined. Two tissue culture lines were employed for this purpose. The first was a wild type CHO cell line. The second was VpmR-5, a CHO cell line selected for resistance against epipodophyl-

lot! 25 50 75 loo’ [Drug] (pM)

FIG. 9. Effect of quinolones on the DNA relaxation activity of topoisomerase 11. Assays were carried out as described under “Experimental Procedures.” Results with etoposide are shown for comparison. Data are plotted in a semilogarithmic fashion as percent DNA relaxed versus drug concentration. DNA relaxation was arbitrarily set to 100% in the absence of drug. Reactions were carried out in the presence of CP-67,804 (O), CP-115,953 (0), or etoposide (HI. Data represent the average of 2-3 independent experiments. The average standard deviation (or standard error where appropriate) for the data shown was less than 7%.

lotoxins (49). This latter line is 10- to 20-fold resistant to etoposide (a representative epipodophyllotoxin) and shows cross-resistance to a number of other topoisomerase II-targeted antineoplastic drugs (49). Drug resistance in the VpmR- 5 cell line does not result from a decrease in drug uptake, but rather correlates with the presence of a mutant, resistant form of topoisomerase I1 (49,56).

Both CP-67,804 and CP-115,953 were cytotoxic to wild type CHO cells (Fig. 10; please note that the drug concentration scales used in the figure are different for each drug employed). CP-115,953 was the more potent of the two quinolone derivatives, with an EC50 (effective concentration required to kill 50% of the cells) of -9 p~ as compared to -70 p~ for CP- 67,804. The EC5o for CP-115,953 was similar to that obtained for etoposide (EC5o = 9 p ~ ) (Fig. 10) and was -10-fold lower than that reported for ciprofloxacin with mouse lymphoblast cells (34).

As previously determined (49), the VpmR-5 line was -12- fold resistant (EC50 = 110 PM) to the cytotoxic effects of etoposide (Fig. 10). While this line showed some degree of cross-resistance to the quinolone derivatives, resistance was not as pronounced as that found for etoposide. The EC50 values for CP-67,804 (-265 pM) and CP-115,953 (-12 pM) correspond to a 3.7- and 1.3-fold resistance, respectively. This decreased resistance may reflect a mechanism of quinolone- induced cytotoxicity which does not involve topoisomerase 11. Alternatively, it could reflect a differential sensitivity of the mutated VpmR-5 topoisomerase I1 toward these quinolone derivatives. In support of this latter point, a mutant human type I1 enzyme (57) and a mutant bacteriophage T4 enzyme (58) which show differential cross-resistance (or even ultra- sensitivity) toward different classes of topoisomerase 11-tar-

;%H ETOPOSIDE

60 VpmA-s

0 50 100 150 200

0 4 . ~ ’ ” ~ J b 0 100 200 300 400

0 2 4 6 8 1 0 1 2 1 4 [DRUG] ( p )

FIG. 10. Cytotoxicity of CP-67,804 and CP-115,953 in CHO cell lines. Assays were carried out as described under “Exper- imental Procedures.” Results for the wild type CHO line (0) and the drug-resistant VpmR-5 line (H) are shown. Cells were incubated with etoposide (top panel), CP-67,804 (middle panel), or CP-115,953 (bot- tom panel). Data represent the average of 3 independent experiments. Standard deviations are indicated by the vertical bars.

Effects of Novel Quinolones on Eukaryotic Topoisomerase 11 14591

geted drugs have been isolated. Regardless, the fact that VpmR-5 shows cross-resistance to the quinolones suggests that topoisomerase I1 is a physiological target for CP-67,804 and CP-115,953 in mammalian cells.

DISCUSSION

Quinolone-based drugs represent a potent class of antimicrobial agents (19-22, 31). Although these drugs are highly specific for the bacterial type I1 topoisomerase, DNA gyrase, many alter the catalytic functions of eukaryotic topoisomerase I1 at high drug concentrations (32-39). Because of the wide clinical use of quinolones, it is important to characterize their interactions with the eukaryotic type I1 enzyme. The present study describes the effects of two novel quinolone derivatives, CP-67,804 and CP-115,953, on the enzymatic activities of Drosophila topoisomerase 11. These compounds are structurally identical except for the substitution of the 1-ethyl group in CP-67,804 by the 1-cyclopropyl group in CP-115,953.

As found for DNA gyrase, CP-67,804 and CP-115,953 effec- tively enhanced DNA cleavage mediated by eukaryotic topoisomerase 11. Prior to the enzyme’s strand passage event, CP- 67,804 was -80% as potent and CP-115,953 was -200% more potent than etoposide at stimulating double-stranded DNA cleavage, Following strand passage, a similar order of potency was observed. Thus, CP-115,953 is the first quinolone-based drug found to enhance enzyme-mediated DNA cleavage with an efficacy greater than that of a clinically important topoisomerase 11-targeted antineoplastic agent such as etoposide.

In contrast to etoposide and m-AMSA which stabilize enzyme-DNA cleavage complexes primarily by inhibiting the rate of topoisomerase 11-mediated DNA religation (27-29), neither CP-67,804 nor CP-115,953 impaired the ability of the Drosophila enzyme to religate cleaved DNA (either prior to or following strand passage). Therefore, these quinolones represent a novel class of topoisomerase 11-targeted agents. Although their mechanism of action has yet to be established, the inability of CP-67,804 and CP-115,953 to inhibit religation strongly suggests that both drugs increase levels of cleavage intermediates primarily by enhancing the enzyme’s forward rate of DNA cleavage. Recently, the 2-nitroimidazole Ro15- 0216 also was found to stimulate topoisomerase 11-mediated pre-strand passage DNA cleavage without significantly inhibiting DNA religation (55). Despite major structural differences between the quinolone derivatives and the 2-nitroimidazole, this latter finding makes it likely that these compounds share a common mode of action.

The ability of a drug to enhance topoisomerase 11-mediated DNA cleavage is usually accompanied by a similar ability to inhibit the enzyme’s catalytic strand passage cycle (17-19). This was not the case for the two quinolone derivatives. CP- 115,953 was only 20% and CP-67,804 was only 3% as potent as etoposide at inhibiting the DNA relaxation reaction catalyzed by Drosophila topoisomerase 11. A decreased ability to inhibit DNA relaxation also has been reported for Ro15-0216 (55). Thus, stimulation of enzyme-mediated DNA cleavage does not necessarily require that a drug also inhibit the overall catalytic cycle of topoisomerase 11.

Both quinolone derivatives were cytotoxic to CHO cells. CP-115,953 was the more effective compound, showing a potency equivalent to that of etoposide. The increased cytotoxicity of CP-115,953 relative to CP-67,804 (-7.8-fold for CHO cells and -22.0-fold for VpmR-5 cells) was dispropor- tionally high in comparison to its increased ability to enhance topoisomerase 11-mediated DNA cleavage (-2.6-fold). This difference in toxicity may reflect differences in the pharma- cokinetic properties of these two compounds or may be related

to the increased ability of CP-115,953 to inhibit the enzyme’s catalytic strand passage reaction (see Fig. 9). Regardless of mechanism, the cytotoxic nature of CP-115,953 and CP- 67,804 together with their effects on eukaryotic topoisomerase I1 indicate that these compounds may have potential as antineoplastic agents. This potential is increased by the finding that the VpmR-5 cell line which shows broad resistance to a number of topoisomerase 11-targeted antineoplastic drugs displayed decreased cross-resistance to these two quinolone derivatives.

Although topoisomerase 11-targeted drugs all bind DNA, they also are believed to interact with the enzyme (17-19,58, 59). Unfortunately, the site(s) of drug binding have not been determined. A number of studies which characterized drug- resistant mutant forms of topoisomerase I1 (56-58,60) raised the question of whether agents from different structural classes share a common interaction domain on the enzyme. Results of the present work indicate that quinolones and etoposide probably share overlapping, but nonidentical sites on topoisomerase 11. This conclusion is based on three lines of evidence. First, the effects of CP-115,953 and etoposide on enzyme-mediated DNA breakage were mutually exclusive rather than synergistic. This argues for a common (or at least overlapping) site of drug action. Second, quinolone derivatives and etoposide increased levels of topoisomerase 11-DNA cleavage complexes by different mechanisms. This finding makes it unlikely that the sites of quinolone and etoposide action are identical. Third, the etoposide-resistant VpmR-5 cell line displayed a decreased cross-resistance toward CP-67,804 and CP-115,953. This last result also points to differences between drug binding sites. The proposal that structurally disparate classes of topoisomerase 11-target drugs can share overlapping (but different) sites of interaction on the enzyme is consistent with the results of previous mutagenesis studies (56-58,60).

In summary, the quinolone derivatives CP-67,804 and CP- 115,953 represent a novel class of agents targeted to eukaryotic topoisomerase 11. This conclusion indicates that quinolone-based drugs, which have long been employed strictly as antimicrobial agents, may in the future play a role in the treatment of human cancers.

Acknowledgments-We are grateful to Dr. P. S. Jensen and Dr. 0. Westergaard for their generous gift of calf thymus topoisomerase 11, to Dr. R. Gupta and Dr. D. M. Sullivan for generously providing tissue culture lines, to C. Brewer for her assistance in preparing plasmid DNA and Drosophila topoisomerase 11, to J . Rule for her expert help with photography, to A. H. Corbett for her critical reading of the manuscript and her expert assistance with the figures, and to S. Heaver for her conscientious preparation of the manuscript.

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