Mechanism of topoisomerase II inhibition by staurosporine and other protein kinase inhibitors.

Topoisomerase II is an essential enzyme for proliferation of eukaryotic cells. It is also a target for many antineoplastic drugs that promote stabilization of covalent complexes between topoisomerase II and DNA. Topoisomerase II and protein kinases both catalyze the transfer of phosphoester bonds from nucleotides to proteins. This similarity suggests that inhibitors may affect both classes of enzymes. In the present study, we have examined the mechanism of topoisomerase II inhibition by three different classes of protein kinase inhibitors. We report that staurosporine inhibited the catalytic activity of topoisomerase II by blocking the transfer of phosphodiester bonds from DNA to the active tyrosine site, a mechanism of inhibition not previously reported for this enzyme. In contrast, other kinase inhibitors, such as methyl 2,5-dihydroxycinnamate, most likely inactivated topoisomerase II by alkylation of essential amino acids, whereas the mechanism of inhibition of bis-indolylmaleimide possibly involved a direct interaction with DNA.

Topoisomerase II is an essential enzyme for proliferation of eukaryotic cells. It is also a target for many antineoplastic drugs that promote stabilization of covalent complexes between topoisomerase II and DNA. Topoisomerase II and protein kinases both catalyze the transfer of phosphoester bonds from nucleotides to proteins. This similarity suggests that inhibitors may affect both classes of enzymes. In the present study, we have examined the mechanism of topoisomerase II inhibition by three different classes of protein kinase inhibitors. We report that staurosporine inhibited the catalytic activity of topoisomerase II by blocking the transfer of phosphodiester bonds from DNA to the active tyrosine site, a mechanism of inhibition not previously reported for this enzyme. In contrast, other kinase inhibitors, such as methyl 2,5-dihydroxycinnamate, most likely inactivated topoisomerase II by alkylation of essential amino acids, whereas the mechanism of inhibition of bis-indolylmaleimide possibly involved a direct interaction with DNA.
Topoisomerase II is a nuclear enzyme that regulates the topology of DNA by passing an intact double strand of DNA through transient double-stranded breaks created in an adjacent DNA segment (1)(2)(3). This allows for resolution of topological perturbations that occur during transcription (4), DNA replication, and separation of chromosomes (5). In the past decade, a number of clinically important and structurally diverse antineoplastic agents have been found to exert their cytotoxic mechanism by stabilizing the covalent complex formed between topoisomerase II and DNA (e.g. etoposide, mitoxantrone, and Adriamycin; reviewed in Ref. 6). Accumulation of these covalent protein-DNA intermediates activates apoptosis, resulting in cell death (7)(8)(9). Newer inhibitors have recently been described that do not stabilize the covalent complex but nevertheless inhibit the function of this essential enzyme. Some of these inhibitors were shown to have a different spectrum of antitumor activity (10 -14) compared to etoposide or Adriamycin. For example, a 2,6-dioxopiperazine found to be effective against adult T-cell leukemia/lymphoma was not cross-resistant to other antitumor drugs and was recently approved for clinical use in Japan (15). Detailed mechanistic studies revealed that 2,6-dioxopiperazines inhibited the catalytic activity of topoisomerase II by interfering with enzyme turnover (16). These findings have renewed interest in identi-fying topoisomerase II inhibitors with new mechanisms of action.
The catalytic cycle of topoisomerase II can be separated into six discrete steps as reviewed by Osheroff et al. (17): 1) noncovalent binding of topoisomerase II to DNA; 2) establishing pre-strand passage cleavage/religation equilibrium; 3) DNA strand passage upon binding of ATP; 4) establishing cleavage/ religation equilibrium following strand passage; and 5) ATP hydrolysis that results in the 6) dissociation of the enzyme from the DNA (i.e. enzyme turnover). Topoisomerase II maintains the integrity of the cleaved DNA (steps 2-4) by forming covalent, O 4 -phosphotyrosyl bonds with each newly created 5Ј-phosphate termini of the cleaved DNA segment. This transfer of phosphodiester bonds from DNA to topoisomerase II is similar to the autophosphorylation reaction of tyrosine kinases, where the enzyme forms an O 4 -phosphotyrosyl bond between its tyrosine and the ␥-phosphate of ATP. It is possible, therefore, that kinase inhibitors that interact with the active tyrosine site of the kinase may also inhibit topoisomerase II through a similar mechanism. It was also noticed that topoisomerase II shares the second of its two consensus ATP binding motifs, GXGXXG, with protein kinases (18). When an initial observation was made that some tyrosine kinase inhibitors (e.g. erbstatin, tyrphostin, and genistein) also inhibit topoisomerase II, it was suggested that these compounds inhibit both classes of enzymes by interacting with their ATP sites (18,19). Subsequent experiments (20) indicated, however, that yeast topoisomerase II uses the first of its two consensus ATP binding motifs, which is not shared with kinases, and the mechanism whereby these compounds inhibit topoisomerase II remained unelucidated. To clarify the mechanism of topoisomerase II inhibition by protein kinase antagonists, we have studied three of them in detail, including an ATP competitive inhibitor (bisindolylmaleimide) (21); a substrate/ATP-competitive tyrosine kinase inhibitor (methyl-2,5-dihydroxycinnamate) (22), and staurosporine (not competitive with either substrate or ATP) (23).

EXPERIMENTAL PROCEDURES
Enzymes and Chemicals-Topoisomerase II (from Drosophila melanogaster) was purchased from Amersham Corp. Staurosporine, etoposide, NaCl (molecular biology grade), N-lauroylsarcosine, and polyethyleneimine-impregnated thin layer cellulose plates were purchased from Sigma. Methyl-2,5-dihydroxycinnamate, genistein, EDTA, CsCl (optical grade), ethidium bromide, and all buffers were obtained from Life Technologies, Inc. Proteinase K, pBR322 plasmid DNA, AMP-PNP, 1  DNA from Crithidia fasciculata was isolated according to a modified procedure of Englund (24). One liter of culture (6 ϫ 10 6 cells/ml) was labeled with 2.5 mCi of [ 3 H] thymidine for 24 h at 30°C. Cells were collected by centrifugation, washed with 500 ml of ice-cold phosphatebuffered saline, and resuspended in 32 ml of the lysis buffer (10 mM Tris/HCl, pH 8.0, 100 mM NaCl, and 200 mM EDTA). After the addition of 100 mg of proteinase K in 5 ml of the lysis buffer, followed by 32 ml of the lysis buffer containing 6% sodium N-lauroylsarcosine, cells were lysed for 45 min at 37°C. The very viscous lysate was applied on ice-cold CsCl layers prepared in 10 mM EDTA, pH 8.0 (12 ml lysate, 20 ml of 0.5 g/ml CsCl, and 4 ml of the 1 g/ml CsCl containing 10 g/ml ethidium bromide, per tube). Tubes were centrifuged in the SW 28 rotor for 20 min at 72,000 ϫ g at 4°C. KDNA aggregates banded on the lower CsCl layer and were collected as a viscous solution by dripping through a hole in the bottom of each tube. Ethidium bromide was removed by repeated extractions with 1-butanol, KDNA was dialyzed once against 4 liters of 10 mM Tris/HCl, pH 8.0 containing 1 mM EDTA, and the CsCl purification step was repeated. KDNA was then extensively dialyzed and concentrated by 1 h centrifugation at 4°C and 140,000 ϫ g. After careful removal of the supernatant, thick, clear KDNA aggregates were resuspended in 0.5 ml of 10 mM Tris/HCl, pH 8.0, containing 1 mM EDTA. The DNA yield, calculated from absorption at 260 nm, was 850 g (28,000 dpms/g).
Topoisomerase II-mediated DNA Decatenation-Topoisomerase IIcatalyzed decatenation of Crithidia fasciculata KDNA was assayed according to Sahai and Kaplan (25). Topoisomerase II (2.5 ng) and [ 3 H]thymidine-labeled KDNA (0.4 g) were incubated for 1 h at 30°C in 40 l of total volume of the reaction buffer (10 mM Tris/HCl, pH 7.8, 5 mM MgCl 2 , 0.1 mM EDTA, and 15 g/ml BSA) containing 150 mM NaCl, 200 M ATP, and various concentrations of inhibitors, ranging from 0.1 to 200 M. Reactions were stopped by the addition of 10 l of a stop solution containing 50% glycerol, 50 mM EDTA, pH 8.0, 2.5% SDS, and 0.1% bromphenyl blue. After gentle mixing, samples were centrifuged at 15,000 ϫ g at 20°C for 5 min, and 25 l of each supernatant were counted in the scintillation counter. Concentrations causing 50% inhibition of topoisomerase II decatenating activity (IC 50 ) were determined from detailed 7-point experiments, where at least two drug concentrations were below and one above the IC 50 . Each experiment was repeated at least once.
Inhibition of the Topoisomerase II Decatenating Activity: Competition with ATP-Reactions were performed as described above, and concentrations causing 50% inhibition of topoisomerase II decatenating activity were determined for each compound at 50 M, 500 M, and 5 mM ATP. A nonhydrolyzable ATP analogue, AMP-PNP, was used as a control inhibitor that was competitive with ATP, and mitoxantrone was used as an inhibitor not competitive with ATP. All experiments were repeated three times.
Topoisomerase II-mediated, ATP-independent DNA Cleavage, and Stabilization of the Pre-strand Passage Topoisomerase II-cleaved DNA Complex-Topoisomerase II (0.5 g) was incubated with 1 g of [ 3 H]KDNA and various drugs, in the the reaction buffer (10 mM Tris/ HCl, pH 7.8, 5 mM MgCl 2 , 0.1 mM EDTA, and 15 g/ml BSA) containing 25 mM NaCl and no ATP (total reaction volume, 20 l) at 30°C for 10 min. Reactions were terminated by the addition of SDS (final concentration, 1%), and topoisomerase II was digested for 30 min at 37°C with 50 g of proteinase K. After digestion, samples were mixed with loading buffer (5% glycerol, 0.017% bromphenyl blue, 100 mM Tris, 90 mM borate, and 1 mM EDTA, final concentrations), and they were subjected to electrophoresis at 1.2 V/cm for 16 h in 1.2% agarose gels in 100 mM Tris, 90 mM borate, 1 mM EDTA, and 0.5 g/ml ethidium bromide. DNA was visualized by transillumination with ultraviolet light (312 nm) and photographed using the Eagle Eye II imaging system (Stratagene). Intensity of DNA bands was measured using an Eagle Eye II software, and the amount of linear DNA (directly reflecting the quantity of topoisomerase II-cleaved DNA complex) was calculated using linearized KDNA as a standard. The intensity of bands was linearly proportional to the amount of DNA through the range of concentrations tested (correlation coefficient, 0.999).
Quantitation of the Pre-strand Passage Topoisomerase II-cleaved DNA Complex-Reactions were performed as described above, except that after proteinase K digestion and addition of a stop solution as described for decatenation, samples were centrifuged at 15,000 ϫ g at 20°C for 5 min, and 20 l of each supernatant were counted in the scintillation counter.
Inhibition of Etoposide-stabilized Pre-strand Passage Topoisomerase II-cleaved DNA Complex-Topoisomerase II (0.5 g) was incubated with 1 g KDNA in 20 l of the reaction buffer (10 mM Tris/HCl, pH 7.8, 5 mM MgCl 2 , 0.1 mM EDTA, and 15 g/ml BSA) that contained 25 mM NaCl, 250 M etoposide, and various concentrations of inhibitors. In these experiments, reaction components were added in the following sequence: DNA, etoposide, inhibitor, and topoisomerase II. Me 2 SO concentration was adjusted to a final concentration of 1% in all samples. After reaction termination with SDS (1% final) and 30 min digestion with 50 g of proteinase K at 37°C, reaction products were resolved in 1.2% agarose gels, and the amount of linear DNA was quantitated using the Eagle Eye II imaging system.
Binding of Topoisomerase II to DNA-Binding of topoisomerase II to DNA was determined using gel mobility shift as described by Osheroff (26). Topoisomerase II (0.5 g) was incubated with 0.15 g of supercoiled pBR322 DNA at 30°C for 2 min in 10 mM Tris/HCl, pH 7.8, 50 mM NaCl, 50 mM KCl, 0.1 mM EDTA, and 15 g/ml BSA. Samples were subjected to electrophoresis in 1.2% agarose gels. For 200 M staurosporine, experiments were repeated three times, each time in duplicate. Free DNA was quantitated using the Eagle Eye II imaging system, and results were presented with standard deviations.
ATPase Activity of Topoisomerase II-ATP hydrolysis by topoisomerase II was measured essentially as described by Osheroff et al. (1). Reaction mixture (4 l) contained 10 mM Tris/HCl, pH 7.8, 150 mM NaCl, 5 mM MgCl 2 , 0.1 mM EDTA, 15 g/ml BSA, 0.74 g of KDNA, 200 M [␥-33 P]ATP (4 Ci), 5 ng of topoisomerase II, and various drug concentrations. After incubation at 30°C for 15 min, 2 l from each reaction were applied on the polyethyleneimine-impregnated thin layer cellulose plates, and samples were chromatographed in freshly prepared 0.4 M ammonium bicarbonate. Inorganic phosphate was visualized by autoradiography, and areas containing inorganic phosphate were cut out of the chromatograms and quantitated in a Packard liquid scintillation counter. The amount of inorganic phosphate generated by topoisomerase II in the absence of DNA was small and was subtracted from the total to give the DNA-dependent ATP hydrolysis.

Inhibition of the Topoisomerase II-mediated Decatenation by
Protein Kinase Inhibitors-The catalytic activity of topoisomerase II was determined using a [ 3 H]KDNA decatenation assay that quantitates the formation of the final products of this multistep reaction. The effect of various protein kinase inhibitors on the catalytic activity of topoisomerase II was evaluated using this assay. Several kinase inhibitors were more effective than etoposide at inhibiting the catalytic activity of topoisomerase II. Staurosporine was the most potent (IC 50 , 17 M), followed by methyl-2,5-dihydroxycinnamate (IC 50 , 32 M), bisindolylmaleimide (IC 50 , 84 M), and genistein (IC 50 , 160 M). By comparison, the IC 50 of etoposide was 196 M.
Stabilization of Topoisomerase II-cleaved DNA Complexes-Stabilization of the covalent topoisomerase II⅐DNA complexes was assayed by measuring the amount of linear DNA generated from KDNA in the presence of topoisomerase II and ATP. Except for genistein, none of the protein kinase inhibitors promoted the stabilization of the topoisomerase II•DNA complex.
Inhibition of the Topoisomerase II-mediated Decatenation: Competition with ATP-Many protein kinase inhibitors, including bis-indolylmaleimide, inhibit kinases by competing with ATP for binding to the enzyme (20). Since ATP binding is necessary for activity of both kinases and topoisomerase II, these compounds may inhibit both enzymes through a similar mechanism. To address this possibility, we have carried out a series of competition experiments to calculate the IC 50 values for protein kinase inhibitors using the topoisomerase II decatenation assay at different ATP concentrations. ATP saturated the reaction at 20 M, and there was no significant substrate inhibition effect at 5 mM (Fig. 1). The IC 50 values for protein kinase inhibitors were calculated at 50 M, 500 M, and 5 mM ATP. The nonhydrolyzable ATP analog, AMP-PNP, was used as a positive control, while the intercalating topoisomerase II inhibitor, mitoxantrone served as a negative control (Fig. 2). IC 50 values were expressed relative to the IC 50 obtained at 50 M ATP to facilitate comparison between compounds (Table I).
A 100-fold increase in the ATP concentration resulted in a more than 100-fold increase in the IC 50 of AMP-PNP, in agreement with an ATP-competitive mode of action. A 100-fold increase in the ATP concentration produced less than a 2-fold increase in the IC 50 for mitoxantrone and less than a 3-fold increase in the IC 50 values for staurosporine and methyl-2,5-dihydroxycinnamate. For genistein, bis-indolylmaleimide, and N-ethylmaleimide, the increase in IC 50 values was higher but was still less than 7-fold. These results suggest that staurosporine does not inhibit topoisomerase II by competing at the ATP site and possibly interacts with a different domain and inhibits a different step of the catalytic cycle of the enzyme.
Inhibition of the Pre-strand Passage Topoisomerase IIcleaved DNA Complex-In the absence of ATP, topoisomerase II can bind to DNA and establish a cleavage-religation equilibrium prior to strand passage (26). Etoposide shifts the equilibrium toward cleaved DNA (27), resulting in elevated levels of the topoisomerase II⅐DNA complex. DNA present in the complex is in the form of a broken circle, held together by phosphodiester bonds, linking each terminal 5Ј-phosphate to a tyrosine residue of topoisomerase II (17). Digestion of topoisomerase II with proteinase K results in release of the cleaved (linear) DNA. The amount of released linear DNA corresponds to the quantity of the topoisomerase II⅐DNA complex. Inhibitors of topoisomerase II that interfere with any of the reaction steps preceding the binding of ATP would decrease the amount of etoposide-stabilized topoisomerase II⅐DNA complex.
The effect of staurosporine on the etoposide-induced topoisomerase II⅐DNA complex is shown on Fig. 3. At equimolar concentrations, staurosporine reduced the amount of linear DNA to background levels. Bis-indolylmaleimide was less potent than staurosporine but also substantially inhibited formation of the complex (78% inhibition at equimolar concentrations). In contrast, inhibitors that interfere with the topoisomerase II-catalyzed reaction at or after the ATP-binding step (ICRF 193, see Table II) had no effect on the level of etoposide-induced complex.
One molecule of the topoisomerase II dimer generates one molecule of topoisomerase II⅐DNA complex. This contrasts with the decatenation reaction where each topoisomerase II dimer catalyzes the liberation of several molecules of circular DNA. Therefore, more enzyme is required to generate a measurable amount of the topoisomerase II⅐DNA complex than to obtain a comparable amount of decatenated reaction products. Consequently, higher concentrations of inhibitors are necessary to affect the formation of the topoisomerase II⅐DNA complex. For example, a 5-fold higher concentration of staurosporine (80 M) was necessary to inhibit, by 50%, formation of the topoisomerase II⅐DNA complex than was required to inhibit the decatenation reaction (17 M).
These findings indicate that staurosporine and bis-indolylmaleimide either directly inhibited formation of the topoisomerase II⅐DNA complex, or prevented the initial noncovalent binding of the enzyme to DNA. However, staurosporine and bis-indolylmaleimide may simply neutralize etoposide by directly interacting with it. To address that possibility, we have examined the effect of staurosporine on the formation of the pre-strand passage topoisomerase II⅐DNA complex in the absence of etoposide. In the absence of etoposide, staurosporine inhibited the pre-strand passage topoisomerase II⅐DNA complex in a dose-dependent manner (Fig. 3). This indicates that the block in the etoposide-stabilized pre-strand passage com-  plex caused by staurosporine was due to inhibition of the enzyme and not staurosporine-etoposide interactions. In the same assay, methyl-2,5-dihydroxycinnamate tested at 250 and 500 M caused a 70 and 97% inhibition, respectively. Inhibition of the Initial Noncovalent Binding of DNA to Topoisomerase II-Formation of the ATP-independent topoisomerase II-cleaved DNA complex is preceded by the initial recognition and noncovalent binding of the DNA substrate by the enzyme. This occurs in the absence of any cofactors and can be followed by the DNA gel mobility shift method (26). As shown on Figs. 4 and 5, staurosporine did not inhibit noncovalent DNA-topoisomerase II binding at concentrations that totally inhibit formation of the pre-strand passage topoisomerase II-DNA complex. In contrast, aurintricarboxylic acid, a compound recently shown to disrupt noncovalent DNA-topoisomerase II interactions (28) totally inhibited binding at 5 M concentration (Fig. 4). Results similar to that for Staurosporine were obtained for methyl-2,5-dihydroxycinnamate and bis-indolylmaleimide. However, the latter one was found to interact strongly with DNA, as demonstrated by a shift in mobility of both closed circular and nicked DNA (Fig. 5). Taken together, our results suggest that staurosporine, methyl-2,5-dihy-droxycinnamate, and bis-indolylmaleimide directly inhibited the formation of the pre-strand passage topoisomerase II-DNA complex through different mechanisms.
Effect of Dithiothreitol on Inhibition of Topoisomerase II by Protein Kinase Inhibitors-Methyl-2,5-dihydroxycinnamate represents a class of chemically reactive protein kinase inhibitors called erbstatins (29). These compounds are capable of reacting with sulfhydryls and may inactivate enzymes by alkylating their cysteines (30). Topoisomerase II contains essential cysteines, and reaction with N-ethylmaleimide leads to loss of catalytic activity (31). We have examined the possibility that some protein kinase inhibitors inhibit topoisomerase II through that mechanism. Preincubation with dithiothreitol totally abolished inhibition of topoisomerase II by methyl-2,5dihydroxycinnamate. Similar results were obtained for the standard sulfhydryl reagent, N-ethylmaleimide. Surprisingly, bis-indolylmaleimide showed some reactivity with sulfhydryls, as judged from a reproducible, 2.3-fold increase in IC 50 upon treatment with dithiothreitol. In contrast, staurosporine inhibited topoisomerase II with the same potency before (IC 50 , 16.5 M) and after (IC 50 , 15.5 M) preincubation with dithiothreitol.
Inhibition of the ATPase Activity of Topoisomerase II by Protein Kinase Inhibitors-All topoisomerase II inhibitors we have investigated interfere with catalytic steps of the enzyme FIG. 3. Inhibition of the topoisomerase II-mediated, ATP-independent DNA cleavage by staurosporine. E, inhibition of the cleavage in the absence of etoposide. q, inhibition of the etoposide-enhanced cleavage. In both cases, the amount of linear DNA produced in the absence of staurosporine was set to 100%. Reactions were carried out as described under "Experimental Procedures" without or with 250 M etoposide. All linear DNA was covalently associated with topoisomerase II, and the enzyme was digested with proteinase K prior to electrophoresis.  4. Effect of staurosporine and aurintricarboxylic acid (ATA) on the initial, noncovalent binding of topoisomerase II to DNA. Shown are results of a gel mobility shift assay analogous to that shown on Fig. 5. The amount of free DNA in the absence of topoisomerase II was set to 100%. The gel mobility shift experiment was repeated for 200 M staurosporine three times, each time in duplicate, and the amount of free DNA was quantitated using the Eagle Eye II imaging system; standard deviation was calculated. In the absence of topoisomerase II, the amount of free DNA was 100 Ϯ 6%. In the presence of topoisomerase II (regardless of whether it was with or without staurosporine), there was only 5 Ϯ 6% free DNA. Results similar to that for staurosporine were obtained for methyl-2,5-dihydroxycinnamate and bis-indolylmaleimide. prior to the ATP hydrolysis. Therefore, we expected all of them to indirectly inhibit the topoisomerase II ATPase. Fig. 6 shows inhibition of the DNA-dependent topoisomerase II ATPase by staurosporine, aurintricarboxylic acid, and a nonhydrolyzable ATP analog, AMP-PNP, used as a positive control. Similar data were obtained for methyl-2,5-dihydroxycinnamate and bis-indolylmaleimide with IC 50 values of 77 and 89 M, respectively. Our results are also in accord with those published by Osheroff's group, where they showed that all seven topoisomerase II inhibitors, selected to represent several structurally and mechanistically diverse classes, inhibited enzyme-catalyzed ATP hydrolysis (32). DISCUSSION Genistein inhibits protein kinases by competing at the ATP binding site (33) and was the first tyrosine kinase inhibitor shown to also inhibit topoisomerase II (18). Binding of ATP is required for activity of both protein kinases and topoisomerase II, and it was initially suggested that genistein inhibits both enzymes by interfering with their ATP cassettes (18). We have shown that the inhibition of topoisomerase II by genistein was partially competitive with ATP (Table I). However, this proposed mode of action is not consistent with the observation that genistein stabilized the topoisomerase II-DNA complex (18). The binding site for genistein apparently overlaps that of etoposide, but not novobiocin, a compound known to interfere with the ATP cassette of topoisomerase II (34).
These observations suggest that genistein inhibited topoisomerase II by a mechanism different than interaction with the ATP binding site. Both topoisomerase II and tyrosine kinase catalyze a phosphoesterification reaction between the phosphate group of a nucleotide and a tyrosine residue. Therefore, it is possible that tyrosine kinase inhibitors, which interfere with the formation of phosphomonoester bonds, may also inhibit the formation of phosphodiester bonds by topoisomerase II. Such compounds would inhibit the catalytic activity of the enzyme without stabilizing the topoisomerase II⅐DNA complexes.
It was recently shown that the tyrosine kinase inhibitors (e.g. erbstatin and tyrphostin), thought to be partially competitive at both substrate and ATP binding sites, also inhibited topoisomerase II catalytic activity (19). The erbstatins and tyrphostins are chemically reactive Michael acceptors and as such are capable of forming covalent adducts with nucleofile centers on proteins, preferentially cysteines, but also histidines and ⑀-amino groups (30). Topoisomerase II can be inhibited by exposure to N-ethylmaleimide (31), another Michael acceptor reacting with nucleofiles. It is, therefore, possible that the mechanism of topoisomerase II inhibition by erbstatin and tyrphostin is identical to that of N-ethylmaleimide. To test this hypothesis, we have studied the inhibition of topoisomerase II by the erbstatin methyl-2,5-dihydroxycinnamate (22) and compared it to N-ethylmaleimide. Both compounds blocked formation of the ATP-independent topoisomerase II⅐DNA complex. 2 Preincubation of these compounds with an excess of dithiothreitol totally abolished inhibition of the enzyme by N-ethylmaleimide as well as by methyl-2,5-dihydroxycinnamate. In both cases, inhibition of the catalytic activity was partially competitive with ATP. It is known that ATP binding to topoisomerase II changes the conformation of the enzyme from "open" to "closed" (35), possibly rendering target nucleofiles less accessible to N-ethylmaleimide or methyl-2,5-dihydroxycinnamate. Thus, ATP may protect topoisomerase II from inactivation by an allosteric effect. These results strongly suggest that 2,5dihydroxycinnamate inactivates topoisomerase II by alkylating residues essential for the enzyme activity, most likely cysteines.
Staurosporine was originally thought to be an ATP-competitive inhibitor specific for protein kinase C. More recent studies showed that it is a broad, potent inhibitor of various kinases, including tyrosine kinases, and does not compete with ATP (23,36,37). We report here that staurosporine is also a potent inhibitor of the catalytic activity of topoisomerase II, does not seem to compete with ATP, and that it inhibits the ATPindependent transfer of phosphodiester bonds from DNA to the active site tyrosine residues of the enzyme. Staurosporine does not react with thiols, which makes its mechanism of action different from that of the erbstatins. Staurosporine is the first topoisomerase II inhibitor shown to directly interfere with the transfer of phosphodiester bonds from DNA to the enzyme. Although staurosporine is not selective for topoisomerase II, it may be possible to develop analogs that are selective for this enzyme. Similar efforts to optimize the specificity of staurosporine toward protein kinase C were successful, resulting in the synthesis of bis-indolylmaleimide, a selective and potent, ATP-competitive inhibitor of protein kinase C, that only modestly affects other kinases (21). This compound inhibited topoisomerase II but was less potent than staurosporine and was partially competitive with ATP. Surprisingly, it was shown to interact directly with DNA, suggesting that it may inhibit topoisomerase II by more than one mechanism.
It was recently shown that compounds that inhibit the catalytic activity of topoisomerase II but that do not stabilize the formation of topoisomerase II⅐DNA complexes are clinically useful in the treatment of cancer (15). Our results indicate that the ATP-independent transfer of the phosphodiester bond from the DNA to the enzyme can constitute a target for the design and development of novel anticancer drugs.