©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Mutation in Yeast TOP2 Homologous to a Quinolone-resistant Mutation in Bacteria
MUTATION OF THE AMINO ACID HOMOLOGOUS TO Ser OF ESCHERICHIA COLI gyrA ALTERS SENSITIVITY TO EUKARYOTIC TOPOISOMERASE INHIBITORS (*)

(Received for publication, March 14, 1995; and in revised form, June 5, 1995)

Yuchu Hsiung (1) (2) Sarah H. Elsea (3)(§) Neil Osheroff (3) John L. Nitiss (1) (2) (4)(¶)

From the  (1)Developmental Therapeutics Section, Division of Hematology/Oncology, Children's Hospital Los Angeles, Los Angeles, California 90027, the (2)Department of Biochemistry and Molecular Biology, University of Southern California Medical School, Los Angeles, California 90089, the (3)Departments of Biochemistry and Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, and (4)St. Jude Children's Research Hospital, Molecular Pharmacology Department, Memphis, Tennessee 38101

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In prokaryotic type II topoisomerases (DNA gyrases), mutations that result in resistance to quinolones frequently occur at Ser or Ser of the gyrA subunit. Mutations to Trp, Ala, and Leu have been identified, all of which confer high levels of quinolone resistance. Extensive segments of DNA gyrase are homologous to eukaryotic topoisomerase II, and Ser of yeast TOP2 is homologous to Ser of prokaryotic DNA gyrA. Introduction of the Ser Trp mutation into yeast TOP2 confers resistance to 6,8-difluoro-7-(4`-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid (CP-115,953), a fluoroquinolone with substantial activity against eukaryotic topoisomerase II, whereas changing Ser to either Leu or Ala does not change sensitivity to quinolones. Interestingly, Ser Trp in the yeast TOP2 also confers hypersensitivity to etoposide. Sensitivity to intercalating anti-topoisomerase II agents such as amsacrine is not changed by any of the three mutations. The topoisomerase II protein carrying the Ser Trp mutation was overexpressed and purified. The purified mutant enzyme had enhanced levels of etoposide stabilized covalent complex as compared with the wild type enzyme and reduced cleavage with CP-115,953. Unlike the wild type enzyme, etoposide-stabilized cleavage is not readily reversible by heat. We suggest that Ser is near a binding site for both quinolones and etoposide and that the Ser Trp mutation leads to a more stable ternary complex between etoposide, DNA, and the mutant enzyme.


INTRODUCTION

DNA topoisomerases play essential roles in a wide range of DNA metabolic processes(1) . Studies in both prokaryotic and eukaryotic cells have demonstrated their importance in transcription, DNA replication, and chromosome segregation(2) . The type II topoisomerases, which make transient double strand breaks and change the linking number of DNA in steps of two, play key roles in chromosome structure. In eukaryotic cells, these enzymes are essential for the chromosome condensation/decondensation process critical for the normal progression through mitosis(3, 4) . The ability of the enzyme to pass two DNA strands and change linking number in steps of two uniquely allows this class of enzyme to segregate fully replicated DNA molecules prior to cell division (reviewed in (5) and (6) ).

Type II topoisomerases have also been identified as a major target of chemotherapeutic agents that are specifically active against prokaryotic cells (7) or against rapidly proliferating cancer cells (8) . Fluoroquinolone antibiotics, such as norfloxacin and ciprofloxacin, along with their less active congeners nalidixic and oxolinic acid are antibacterial agents that target DNA gyrase(9) , whereas a variety of DNA-intercalating agents such as anilinoacridines, ellipticines, and mitoxantrone and nonintercalating agents such as epipodophyllotoxins are active against eukaryotic topoisomerase II and are clinically important anti-cancer agents(10, 11) .

Recently, it has been shown that 6,8-difluoro-7-(4`-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid (CP-115,953), (^1)a fluoroquinolone closely related to ciprofloxacin, is highly toxic to mammalian cells in culture and active against topoisomerase II in vitro(12, 13, 14) . Genetic studies in yeast demonstrated that topoisomerase II is the primary physiological target for quinolone cytotoxicity(15) . Unlike etoposide, which stabilizes cleavage mainly by inhibiting the religation reaction of topoisomerase II, CP-115,953 stabilizes cleavage by enhancing the forward rate of cleavage(12) . Therefore, fluoroquinolones are nonintercalating anti-topoisomerase II agents with a different biochemical mechanism of action than etoposide. Although the action of quinolone-based drugs differs from agents such as etoposide, the ultimate mechanism of cell-killing, enhanced levels of the covalent complex is the same(15, 16) .

In Escherichia coli, mutations that lead to quinolone resistance are most often found in gyrA, the structural gene for the DNA gyrase A subunit, although changes that lead to quinolone resistance have also been found in the gyrB subunit. Ser of gyrA is the amino acid most frequently mutated in strains with high levels of quinolone resistance(17) . Among the substitutions noted in quinolone-resistant mutants are mutations of Ser to Ala, Leu, or Trp, with higher levels of resistance in strains carrying Ser Trp or Ser Leu mutations. It has recently been reported that gyrase protein with the Ser Trp mutation in gyrA has greatly reduced binding of ciprofloxacin compared with the wild type protein, further demonstrating the importance of this region of the protein(18) .

These findings have led us to examine the effects of mutations in the yeast TOP2 gene that change Ser, the amino acid that is homologous to Ser in gyrA of E. coli, on the yeast type II enzyme for sensitivity to quinolones as well as other topoisomerase II inhibitors. We have found that the Ser Trp mutation results in resistance to quinolones that act against eukaryotic topoisomerase II. In addition, this mutation causes hypersensitivity to etoposide. Overexpression, purification, and characterization of yeast topoisomerase II carrying the Ser Trp demonstrated that the protein is relatively insensitive to fluoroquinolones and is hypersensitive to etoposide. Our results demonstrate that Ser of yeast TOP2 plays a critical role in the action of some anti-topoisomerase II agents and may be directly involved in the interactions of the eukaryotic enzyme with etoposide.


MATERIALS AND METHODS

Yeast Strains

The yeast strains used to examine drug sensitivity were all derived from JN362a (a ura3-52 leu2 trp1 his7 ade1-2 ISE2). Isogenic derivatives of JN362a carrying TOP2 mutations were constructed as described previously(19) . Briefly, the strain was transformed with Asp-digested pMJ2 carrying mutations constructed by oligonucleotide directed mutagenesis, and the transformants were selected on SC-URA medium(20) . pMJ2 carries the 3` portion of the yeast TOP2 gene (4.1-kilobase BglII/BglII fragment) in a yeast integrating plasmid, which has the yeast URA3 gene as a selectable marker. Strains carrying mutations of Ser Trp, Ser Leu, and Ser Ala were constructed. The strains carrying top2 mutations were then converted to rad52 by one-step gene disruption using pSM20, which carries a LEU2 disruption of the RAD52 gene(21) . All yeast transformations were carried out using the modified lithium acetate protocol of Schiestl and Gietz(22) .

Plasmids

The plasmid pMJ2 has been previously described (19) . Point mutations were constructed in pMJ2 using the method of Kunkel et al.(23) with the Muta-Gene kit (Bio-Rad) following the supplier's instructions. E. coli strain DH5alpha was used for propagation of plasmids. The mutations at Ser of the yeast TOP2 gene were constructed in pMJ2 using the following mutagenic oligonucleotides: GGTGAGCAGTGGTTGGCACAA for Ser Trp, GGTGAGCAGGCGTTGGCACAA for Ser [arrow] Ala, and GGTGAGCAGCTGTTGGCACAA for Ser Leu. The bold letters indicate the changes from the wild type yeast TOP2 sequence(24) . All mutations were verified by DNA sequencing.

In order to construct a plasmid for overexpressing the Ser Trp mutant topoisomerase II, plasmid pMJ2-S*W and YEpTOP2-PGAL1 (25) were digested with KpnI and AvrII. The 2.2-kilobase fragment from pMJ2-S*W and the 11.5-kilobase fragment from YEpTOP2-PGAL1 were gel purified, ligated, and transformed into E. coli DH5alpha competent cells. Plasmids that had an identical restriction pattern to YEpTOP2-PGAL1 were identified, and the presence of the Ser Trp mutation in YEpTOP2-PGAL1 was confirmed by DNA sequencing. The plasmid carrying the mutation was designated YEptop2-S*W-PGAL1.

Measurements of Drug Sensitivity in Yeast

Etoposide was obtained from Sigma, amsacrine was a gift from Dr. Steve Member of Bristol Myer Laboratories, and CP-115,953 was the gift of Drs. P. R. McGuirk and T. D. Gootz of Pfizer Laboratories. Etoposide and amsacrine were dissolved in dimethyl sulfoxide, and 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-HCl, pH 8.0. All drugs were stored at -80 °C in dark. Drug sensitivity measurements were carried out as described previously(19) . Briefly, cells growing in yeast extract/peptone/dextrose/adenine (YPDA) medium at were adjusted to 2 10^6 cells/ml. Drugs or drug solvent was added, and aliquots of samples were removed at the indicated times, diluted, and plated to YPDA medium solidified with 1.6% agar. The minimum lethal concentrations shown in Table 1were determined by examining drug sensitivity as described above and determining the drug concentration required to reduce viability below 100% (i.e. the number of viable cells at T = 0) at 8 and 24 h. Drug concentrations from 1 to 100 µg/ml (or 1-50 µM for CP-115,953) were examined. We also determined IC concentrations by growing the cells to stationary phase in the presence of various drug concentrations and then plating dilutions to YPDA plates. The IC is the drug concentration that reduces the number of colonies by 50% compared with cells grown to stationary phase in the absence of drug.



Overexpression and Purification of Yeast Topoisomerase II

Wild type yeast TOP2 and Ser Trp proteins were overexpressed using YEpTOP2-PGAL1 or YEptop2-S*W-PGAL1 using yeast strain JEL1 (26) and purified to homogeneity by a modification of the procedure of Worland and Wang(25) . The detailed procedure has been described elsewhere(27) . Topoisomerase II reactions were carried out as described previously using either supercoiled pBR322 to monitor ATPdependent relaxation or kinetoplast DNA isolated from Crithidia fasiculata to monitor decatenation(19, 27) .

Quantitation of Drug-stabilized Cleavage

Quantitation of DNA cleavage was determined using a modified version of the K/SDS method(28) . The substrate was pUC18 DNA that was end-labeled by filling in the EcoRI-digested plasmid with alpha-P-labeled dATP using the Klenow fragment of E. coli DNA polymerase I. The specific activity of the labeled DNA was typically 1-3 10^6 cpm/µg of DNA, and 5-10 10^5 cpm were added per reaction. The amount of drug-stabilized cleavage was determined in triplicate for each drug concentration. Each cleavage reaction contained 8 units (80 ng) of topoisomerase II. Independent determination of cleavage with the same batch of labeled substrate showed levels of cleavage with a standard error of no more than 10%.


RESULTS

Strains Carrying the Ser Trp Mutation in Yeast Topoisomerase II Are Resistant to CP-115,953 and Hypersensitive to Etoposide in Vivo

We constructed mutations in the yeast TOP2 gene by oligonucleotide-directed mutagenesis at Ser, the amino acid homologous to Ser of gyrA. Because mutations to tryptophan, alanine, and leucine have been described in prokaryotes as leading to quinolone resistance, we changed Ser to each of these amino acids. Replacement of wild type TOP2 with each of the mutant TOP2 genes produced viable colonies, so none of the mutations inactivated TOP2. The strains were then converted to rad52, and the resulting strains were tested for sensitivity to the fluoroquinolone CP-115,953.

We determined the IC of cells carrying either wild type or the Ser Trp top2 mutation. The IC of wild type cells is about 1 µM, whereas the IC of cells carrying the Ser Trp top2 mutation is about 10 µM. The results in Fig. 1show that strains carrying the Ser Trp mutation grow in medium containing 20 µM CP-115,953, although the growth is considerably less than in drug-free medium. However, 10 µM CP-115,953 is very cytotoxic for cell-carrying wild type TOP2 (Fig. 1). At a drug concentration of 20 µM, the viability of TOP2 cells is reduced to less than 0.1% after 24 h of exposure to CP-115,953, whereas the Ser Trp mutant shows no reduction in viability compared with the time of drug addition. The presence of the Ser Leu or the Ser Ala mutation did not have significant effects on sensitivity to CP-115,953, compared with TOP2 (Table 1).


Figure 1: Mutation of Ser Trp of yeast TOP2 results in resistance to the fluoroquinolone CP-115,953. Strain JN394 or an isogenic derivative carrying the Ser Trp (Ser741Trp) mutation in TOP2 was treated with CP-115,953 for the indicated times. Samples were removed, diluted, and plated to YPDA medium to determine the number of viable colony-forming units. Results are expressed relative to T = 0.



Because the Ser Trp mutation conferred resistance to CP-115,953, we examined whether the sensitivity to other anti-topoisomerase II agents was affected. Cells carrying either the Ser Trp mutation or wild type TOP2 have the same minimum lethal concentration as the intercalating topoisomerase II inhibitors amsacrine and mitoxantrone (Table 1).

A very different result was obtained with the nonintercalating topoisomerase II inhibitor etoposide. Strains carrying the Ser Trp mutation are hypersensitive to etoposide. The IC for etoposide is approximately 10 µg/ml. By contrast the IC for etoposide for cells carrying the Ser Trp mutation is less than 1 µg/ml. Mutant cells are efficiently killed by 20 µg/ml etoposide, whereas the isogenic wild type strain is able to grow at this drug concentration (Fig. 2). At 100 µg/ml of etoposide, the viability of cells carrying the Ser Trp mutation is approximately 0.1% after 24 h drug exposure compared with about 3% for cells carrying wild type TOP2. The minimum lethal concentration for etoposide for cells carrying the Ser Trp mutant TOP2 is 5 µg/ml, versus 50 µg/ml for cells carrying wild type TOP2 (Table 1). The mutant strain also shows hypersensitivity to another epipodophyllotoxin teniposide; the minimum lethal concentration for the mutant strain is about 10 µg/ml teniposide, compared with 50 µg/ml teniposide for strains carrying wild type topoisomerase II (Table 1).


Figure 2: The Ser Trp mutation confers hypersensitivity to etoposide. Strain JN394 or an isogenic derivative carrying the Ser Trp mutation in TOP2 was treated with etoposide for the indicated times. Samples were removed, diluted, and plated to YPDA medium to determine the number of viable colony-forming units. Results are expressed relative to T = 0.



Purification and Characterization of the Ser Trp Mutant Protein

In order to more fully characterize the effects of etoposide and quinolones on the Ser Trp mutant topoisomerase II, we subcloned a part of the TOP2 gene from pMJ2S*W into YEptop2-PGAL1, which carries the yeast TOP2 gene under the control of the GAL1 promoter. The resulting plasmid YEptop2-S*W-PGAL1 was introduced into the yeast strain JEL1, and after induction with galactose, TOP2 protein was purified to apparent homogeneity (Fig. 3A). We also purified the wild type TOP2 protein expressed from YEptop2-PGAL1 in JEL1. We compared the activity of wild type and Ser Trp proteins by titration of ATP-dependent relaxation activity. For both proteins, a concentration of 3 nM of TOP2 protein effects 50% relaxation of 0.3 µg of supercoiled pBR322 DNA in 15 min, and 15 nM will accomplish 90% relaxation with both proteins (Fig. 3B). Hence, the Ser Trp protein has essentially the same level of catalytic activity as wild type, and differences in drug sensitivity are unlikely to be due to differences in activity between the wild type and mutant proteins.


Figure 3: Purification and catalytic activity of yeast topoisomerase II protein carrying Ser Trp mutation. A, yeast topoisomerase II (TopoII) was expressed from the yeast GAL1 promoter in strain JEL1. Protein samples were analyzed by SDS-polyacrylamide gel electrophoresis. The samples were: lanes 1 and 6, molecular mass markers (Std); lane 2, the JEL1 homogenate (Homo) from cells expressing wild type (WT) yeast topoisomerase II; lane 3, purified wild type yeast topoisomerase II; lane 4, the JEL1 homogenate from cells expressing Ser Trp (S741W) topoisomerase II; and lane 5, purified topoisomerase II carrying the Ser Trp mutation. B, catalytic activity of wild type (WT) topoisomerase II or the Ser Trp (Ser741Trp) mutant was determined by monitoring ATP-dependent relaxation of pBR322. The reaction mixtures were analyzed by agarose gel electrophoresis, and a negative of the gel was scanned by densitometry to determine the percentage of the input substrate DNA that was relaxed by the enzyme.



Ser Trp Protein Is Fluoroquinolone-resistant and Etoposide Hypersensitive in Vitro

We next examined the stabilization of cleavage with the Ser Trp mutant protein. In order to obtain quantitative estimates of the levels of cleavage stabilized by different drugs, we used a modified version of the K/SDS assay. In this assay, end-labeled DNA is treated with 8 units of topoisomerase II, and the reaction is terminated with SDS. The addition of K quantitatively precipitates proteins, and labeled DNA will only be precipitated if it is covalently bound to protein. The results obtained with wild type and Ser Trp proteins in the presence of different concentrations of CP-115,953 are shown in Fig. 4. The results shown are the average of triplicate determinations at each drug concentration. Variation between samples with the same drug concentration and the same batch of substrate was <10%. Even though the Ser Trp protein has the same catalytic activity as the wild type enzyme, the level of drug-independent cleavage is reduced to about 70% compared with the wild type enzyme. Wild type TOP2 protein produces a dose-dependent increase in precipitated counts with increasing CP-115,953 concentration. At a concentration of about 1-2 µM CP 115,953, a 2-fold increase in precipitated counts is seen, compared with samples without drugs. At 10 µM CP-115,953, there is a 4-fold increase in precipitated counts. Although some increase in precipitated counts is also seen with the Ser Trp protein, there is less than a 2-fold increase in precipitated counts at 10 µg/ml CP-115,953. Hence, the Ser Trp protein is less sensitive to the fluoroquinolone CP-115,953 than the wild type protein.


Figure 4: K/SDS assay of cleavage stabilized by the fluoroquinolone CP-115,953. Covalent complex formation stabilized by CP-115,953, and purified yeast topoisomerase II was measured using the K/SDS assay as described in the text. Drug concentrations are indicated on the figure. WT, wild type; Ser741Trp, Ser Trp mutation.



We then examined the sensitivity of the Ser Trp protein to etoposide using the K/SDS assay. An etoposide concentration of 1 µg/ml is required to effect a 2-fold increase in precipitated counts with wild type TOP2 protein, whereas the same level of precipitated counts occurs at <0.1 µg/ml for the Ser Trp protein (Fig. 5). At 1 µg/ml etoposide, the Ser Trp protein has greater than a 5-fold increase in precipitated counts. The etoposide hypersensitivity is less pronounced at higher drug concentrations; at 5 µg/ml etoposide there is a 2-fold difference in the level of cleavage between Ser Trp and wild type TOP2 protein (Fig. 5).


Figure 5: K/SDS assay of cleavage stabilized by etoposide. Covalent complex formation stabilized by etoposide and purified yeast topoisomerase II was measured using the K/SDS assay as described in the text. Drug concentrations are indicated on the figure. WT, wild type; Ser741Trp, Ser Trp mutation.



Cleavage Complexes Stabilized by Etoposide with the Ser Trp Protein Have Enhanced Heat Stability

If the interaction between etoposide and TOP2 is enhanced by the Ser Trp mutation, it might be expected that the ternary complex formed between protein, drug, and DNA would have enhanced stability. For wild type TOP2, the ternary complex is readily reversed by exposure to heat or high salt(29, 30, 31) . This has been interpreted to mean that the equilibrium favoring the ternary complex at low temperature (or low salt concentration) favors dissociation at higher temperatures. We examined whether we could detect a difference in the stability of the etoposide-DNA-TOP2 cleavage complex with the Ser Trp mutant protein by examining the heat reversibility of the ternary complex formed with drug, protein, and DNA.

The K+/SDS cleavage reaction was carried out with wild type TOP2 or the Ser Trp protein as above except that the reaction was incubated at 65 °C for various times prior to the addition of SDS. In the absence of the drug, the 65 °C treatment reduces the amount of precipitated counts to about 25% of the level observed without heat for both the wild type and Ser Trp proteins (Fig. 6A). Because the cleavage complex formed in the absence of drugs is heat reversible, it is likely that the Ser Trp protein does not form a complex that is qualitatively different than the wild type protein. Similarly, for both the wild type and Ser Trp proteins, the complexes formed in the presence of 10 µM CP-115,953 were completely reversed by 1 min of incubation at 65 °C prior to the addition of SDS.


Figure 6: Heat reversibility of covalent complexes formed by the Ser Trp mutant protein. A, wild type and Ser Trp (Ser741Trp) proteins (8 units) were used in a K/SDS assay under the same conditions as in Fig. 4. The samples treated with heat were incubated at 65° for 1 min prior to the addition of SDS. B, wild type (WT) and Ser Trp proteins (8 units) were used in a K/SDS assay in the presence of 10 µg/ml etoposide. Samples were placed at 65 °C, and at the indicated times, SDS was added to an aliquot of the reaction. The percentage of precipitable counts relative to unheated samples is shown.



In the presence of etoposide, a different result is seen. At 10 µg/ml etoposide, the cleavage complexes formed with the wild type protein are rapidly reversed by heat treatment, as indicated by a 50% reduction in precipitable counts after 1 min at 65 °C. However, little reversal is seen when the Ser Trp protein treated with etoposide is exposed to 65 °C prior to SDS addition. An incubation of 20 min is required to reduce the precipitated counts by 50% under these conditions (Fig. 6B). These results taken together strongly suggest that the etoposide-DNA-Ser Trp protein ternary complex has enhanced stability compared with the wild type protein, which is consistent with a more stable interaction between etoposide and the Ser Trp mutant protein.


DISCUSSION

Acquired drug resistance by mutation of the drug target likely represents a contributor to the failure of drug treatment for both antibacterial and anti-cancer drugs. This has been clearly demonstrated with quinolone antibiotics, where mutations in the structural genes of bacterial DNA gyrase are frequently observed in quinolone-resistant clinical isolates(7) . In this report, we have demonstrated that mutations in yeast TOP2 at a position homologous to Ser of gyrA gives rise to fluoroquinolone resistance. The same mutation also generates hypersensitivity to another class of drugs, epipodophyllotoxins. Our results suggest that in some contexts, it may be possible to overcome acquired drug resistance due to a mutation in the drug target by use of a different class of drugs that act against the same target.

We constructed several changes at Ser in the yeast TOP2 gene of Saccharomyces cerevisiae and have demonstrated that mutation of Ser Trp of yeast topoisomerase II confers hypersensitivity to etoposide and resistance to quinolone CP-115,953 in vivo. In agreement with the in vivo results, the Ser Trp protein has increased drug-stabilized cleavage in response to etoposide and decreased drug stabilized cleavage when treated with the fluoroquinolone CP-115,953. The results of Maxwell and Willmott strongly suggest that Ser of gyrA is critical for the binding of quinolones to gyrase(18) . We reasoned that the homology between gyrase and eukaryotic topoisomerases (32) suggested that the equivalent amino acid would be important, at least for the action of fluoroquinolones that have activity against eukaryotic topoisomerase II. However, the homology between yeast TOP2 and gyrA does not result in a perfect correspondence in sensitivity to fluoroquinolones. Most notably, the Ser Leu mutant is not resistant to CP-115,953, nor is it etoposide-hypersensitive. By contrast, a Ser Leu mutation in gyrA results in almost the same level of quinolone resistance as Ser Trp.

Although there are likely to be several ways that alterations in TOP2 lead to a drug-resistant protein, it would seem that there are only a limited number of ways where mutations could generate drug hypersensitivity. Because the Ser Trp protein has essentially wild type activity and because the drug hypersensitivity is specific for epipodophyllotoxins (i.e. sensitivity to intercalating drugs is not affected by the mutant protein), the simplest explanation is that the protein's affinity for epipodophyllotoxins is affected. The increased stability of the etoposide-stabilized covalent complex at 65 °C also supports the hypothesis that the drug hypersensitivity is due to a more stable interaction between the mutant TOP2 protein and etoposide. The nature of the covalent interaction is unlikely to be qualitatively different with the Ser Trp protein because the covalent complex formed in the absence of drug or in the presence of CP-115,953 is fully heat-reversible. Because gyrA protein carrying a Ser Trp mutation has reduced fluoroquinolone binding(17, 18) , Ser either is part of a domain of gyrase that binds to quinolones or is fairly close to the quinolone binding site. Our results would suggest that the domain including Ser in yeast TOP2 interacts with both fluoroquinolones and epipodophyllotoxins. This result is consistent with recently reported results that suggest that fluoroquinolones and etoposide can compete for binding to topoisomerase II(33) . A simple interpretation is that the two agents share (at least in part) the domain required for drug/protein interactions. Our results suggest that Ser may be a key part of that domain. Drug binding assays with eukaryotic topoisomerase II have yet to be reported, however, so we have not been able to directly demonstrate the roles of specific domains in interaction with etoposide or other topoisomerase II agents.

It should be possible to design etoposide derivatives that generate covalent complexes that behave like the TOP2 Ser Trp-etoposide-DNA complexes with the wild type protein. Because other agents such as clerocidin have been described that can generate heat stable complexes(34) , we suggest that such agents have a high enough affinity for topoisomerase II such that at 65 °C the covalent complex is still favored. It will be of considerable interest to determine whether agents that form such stable cleavage complexes might result in more effective antibacterial or anti-cancer agents.


FOOTNOTES

*
This work was supported by Grants NP-812 and FRA-370 (to N. O.) from the American Cancer Society, Grants GM33944 (to N. O.) and CA52814 (to J. L. N.) from the National Institutes of Health, and the Martell Foundation for Leukemia, Cancer, and AIDS. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported in part by Training Grant 5 T32 CA09582 from the National Institutes of Health.

To whom correspondence should be addressed: St. Jude Children's Research Hospital, Molecular Pharmacology Dept., 332 N. Lauderdale, Memphis TN 38101. Tel.: 901-495-2794; Fax: 901-495-2176.

(^1)
The abbreviations used are: CP-115,953, 6,8-difluoro-7-(4`-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid; YPDA, yeast extract/peptone/dextrose/adenine medium; SC-URA, synthetic complete medium minus uracil.


ACKNOWLEDGEMENTS

We thank Dr. Steve Member of Bristol Myer Laboratories for amsacrine and Drs. P. R. McGuirk and T. D. Gootz of Pfizer Laboratories for CP-115,953.


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