Inhibition of Topoisomerase I Function by Coralyne and 5,6

Wilson, W. D., Gough, A. N., Doyle, J. J., and Davidson, M. N. (1976) Coralyne. Intercalation with DNA as a possible mechanism of antileukemic action...
1 downloads 0 Views 433KB Size
Download PDF
Chem. Res. Toxicol. 1996, 9, 75-83

75

Inhibition of Topoisomerase I Function by Coralyne and 5,6-Dihydrocoralyne† Li-Kai Wang, Brian D. Rogers, and Sidney M. Hecht* Departments of Chemistry and Biology, University of Virginia, Charlottesville, Virginia 22901 Received May 10, 1995X

The antitumor agent coralyne and a number of structural analogues were found to be inhibitors of DNA topoisomerase I and were characterized biochemically. Several of these analogues stabilized the covalent binary complex formed between calf thymus topoisomerase I and pSP64 plasmid DNA; coralyne and 5,6-dihydrocoralyne had the greatest potency as inhibitors in this assay. In common with camptothecin, the effects of coralyne and 5,6dihydrocoralyne were reversed in the presence of increasing salt concentration or temperature, consistent with the interpretation that both functioned mechanistically in a fashion analogous to camptothecin. The sequence specificity of DNA cleavage by coralyne and 5,6-dihydrocoralyne was also studied in comparison with camptothecin using a 471-bp DNA duplex as a substrate for topoisomerase I. Seven sites of cleavage were apparent, four of which were shared in common by coralyne, 5,6-dihydrocoralyne and camptothecin. Coralyne and 5,6-dihydrocoralyne produced cleavage at one sequence, 5′-TCTCVGTAA-3′, that was not apparent in the presence of camptothecin; correspondingly, two cleavage bands appeared only when camptothecin was present. Coralyne and 5,6-dihydrocoralyne also inhibited topoisomerase I-mediated relaxation of supercoiled plasmid DNA. Coralyne was the most potent inhibitor of DNA relaxation; the effects of camptothecin and 5,6-dihydrocoralyne were roughly equal. At high concentrations, coralyne completely suppressed the formation of the topoisomerase I-DNA covalent binary complex.

Introduction DNA topoisomerases are enzymes found in the nuclei of cells; they catalyze the breaking and rejoining of DNA strands, which permits control of the topological state of DNA (1-5). Two types of DNA topoisomerases are found in cells: the type I enzymes mediate the transient breakage of a single strand of DNA, while the type II topoisomerases break both strands (1-5). As a consequence, for type II enzymes, structural alterations such as the catenation and knotting of DNA are possible. Mechanistically, topoisomerase I-mediated DNA strand scission is accomplished by nucleophilic attack of an active site tyrosine OH group on an internucleotide phosphate ester linkage. As shown in Scheme 1, the transient covalent binary complex formed between topoisomerase I and DNA involves attachment of the enzyme via an oligonucleotide 3′-phosphate, with the release of a free DNA strand containing a 5′-OH group (6). In contrast, the attachment of topoisomerase II to DNA is through a 5′-phosphate linkage, with a four base pair (bp)1 stagger between the two sites of cleavage on opposite strands (7, 8). As a consequence of the involvement of topoisomerases in essential cell processes such as replication and transcription, inhibitors of the topoisomerases can be cytotoxic. In fact, several classes of compounds that inhibit eukaryotic topoisomerase I or II have antitumor activity (9). Topoisomerase II is a target for intercalative anti†

This paper is dedicated to the memory of Dr. Matthew Suffness. * Author to whom correspondence should be addressed. Abstract published in Advance ACS Abstracts, December 1, 1995. 1 Abbreviations: m-AMSA, 4′-(9-acridinylamino)methanesulfon-manisidide; EDTA, ethylenediaminetetraacetic acid; Tris, tris(hydroxymethyl)aminomethane; DTT, dithiothreitol; DMSO, dimethyl sulfoxide; SDS, sodium dodecyl sulfate; BSA, bovine serum albumin; nt, nucleotide; bp, base pair. X

0893-228x/96/2709-0075$12.00/0

tumor agents such as 4′-(9-acridinylamino)methanesulfon-m-anisidine (m-AMSA), adriamycin, and ellipticine, and also for nonintercalative compounds such as etoposide and teniposide (9, 10). Camptothecin, a plantderived antitumor alkaloid, inhibits topoisomerase I (1014). Because several classes of DNA topoisomerase inhibitors have useful antitumor activity (9), there is substantial interest in identifying novel inhibitors of these enzymes (9, 10, 15, 16). While a number of mechanisms of inhibition can be envisioned (17, 18), all of the foregoing agents function as topoisomerase inhibitors by stabilizing the covalent binary complex formed between the enzyme and DNA. Presently, we report that a number of protoberberinetype alkaloids stabilize the covalent binary complex between calf thymus DNA topoisomerase I and DNA. Among the most potent of the agents tested was coralyne, a compound known to have antitumor activity (19-21). Coralyne (1), 5,6-dihydrocoralyne (2), and camptothecin (3) (Figure 1) were compared for their abilities to inhibit DNA relaxation and stabilize the enzyme-DNA binary complex in a reversible fashion. Also reported is the topoisomerase I-dependent sequence selectivity of DNA nicking by these three compounds.

Experimental Procedures Materials. Coralyne chloride, R-coralyne, doxorubicin, dimethyl sulfate, hydrazine, and formamide were purchased from Aldrich Chemical Co. (Milwaukee, WI). m-AMSA, 5,6-dihydrocoralyne, and a number of other coralyne analogues were obtained from the National Cancer Institute through the courtesy of Dr. Matthew Suffness. Camptothecin was obtained from SmithKline Beecham Pharmaceuticals through the courtesy of Dr. Randall Johnson. Ethidium bromide, tris(hydroxy-

© 1996 American Chemical Society

76

Chem. Res. Toxicol., Vol. 9, No. 1, 1996

Figure 1. Structures of coralyne, 5,6-dihydrocoralyne, and camptothecin. methyl)aminomethane, poly(ethylene glycol), EDTA, dithiothreitol, 2-mercaptoethanol, ATP, bromphenol blue, and sodium dodecyl sulfate were obtained from Sigma Chemical Co. (St. Louis, MO). Glycerol was from Mallinckrodt (St. Louis, MO). Phenyl-Sepharose was purchased from Pharmacia (Piscataway, NJ); hydroxylapatite was from Bio-Rad (Melville, NY). Agarose was obtained from GIBCO BRL (Gaithersburg, MD); crystalline bovine serum albumin was from American Bioorganics (Niagra Falls, NY). Proteinase K was purchased from Boehringer Mannheim (Indianapolis, IN); restriction endonucleases HindIII, NheI, and PvuII, as well as AMV reverse transcriptase, T4 DNA ligase, and T4 polynucleotide kinase were obtained from United States Biochemical (Cleveland, OH); [R-32P]CTP was purchased from ICN Radiochemicals (Costa Mesa, CA). Eukaryotic topoisomerase II was purchased from TopoGen, Inc. (Columbus, OH). Methods. pSP64 plasmid DNA was prepared as described (18); knotted P4 DNA was obtained by the method of Liu and Davis (22). DNA topoisomerase I was purified from calf thymus by slight modification of published procedures (13, 23). Briefly, 168 g of calf thymus gland was cooled to 4 °C and disrupted with a Waring blender; the suspension was centrifuged at 4000 rpm for 10 min. The pellet was washed with 800 mL of 5 mM potassium phosphate (pH 7.5) containing 0.1% Triton X-100. Following an additional centrifugation (7500 rpm, 10 min), the pellet was washed with 400 mL of 5 mM potassium phosphate and again centrifuged (8000 rpm, 10 min). The pellet was treated with 60 mL of 0.35 M NaCl and stirred at 4 °C for 20 min. The resulting suspension (170 mL total volume) was treated with 170 mL of 100 mM Tris-HCl (pH 7.5) containing 2 M NaCl and then with 170 mL of 50 mM Tris-HCl (pH 7.5) containing 1 M NaCl and 18% poly(ethylene glycol). The suspension was stirred for 30 min at 4 °C and then filtered. The supernatant was treated slowly with solid ammonium sulfate to a final concentration of 0.264 g/mL. Following

Wang et al. centrifugation (8200 rpm, 15 min), the lower layer was applied to a phenyl-Sepharose column (2.5 × 6 cm) and purified as described by Gupta et al. (13). The active fractions were dialyzed against 0.2 M potassium phosphate (pH 7.0) and then applied to a hydroxylapatite column in the same buffer as described (13). The enzyme (0.77 mg, having a specific activity of 1.4 × 107 units/mg) was stored in 350 mM potassium phosphate (pH 7.0) containing 125 µg/mL of bovine serum albumin (BSA), 0.5 mM dithiothreitol (DTT), 0.1 mM EDTA, and 10% glycerol. One unit is defined as the amount of enzyme required to fully relax 1.6 µg of supercoiled pSP64 plasmid DNA at 37 °C in 30 min. Topoisomerase I-Mediated DNA Cleavage. This assay was adapted from Hsiang et al. (11). Incubation mixtures contained 100 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.5 mM EDTA, 30 µg/mL of bovine serum albumin, and 200 ng of supercoiled pSP64 plasmid DNA in 20 µL (total volume) of 40 mM Tris-HCl (pH 7.5). Potential inhibitors were added from concentrated DMSO solutions such that the final concentration of DMSO in the incubation mixtures was 1%. The reactions were initiated by the addition of 68 ng of calf thymus DNA topoisomerase I and maintained at 37 °C for 30 min. The reactions were terminated by the addition of 1% SDS and then incubated in the presence of 0.75 mg/mL of proteinase K at 37 °C for 1 h. Analysis of the reaction mixtures was carried out by 1% agarose gel electrophoresis in a gel containing 0.5 µg/mL of ethidium bromide. Inhibition of Topoisomerase I-Mediated DNA Relaxation. Incubation mixtures contained 120 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.5 mM EDTA, 50 µg/mL of bovine serum albumin, 6% poly(ethylene glycol), 250 ng of supercoiled pSP64 plasmid DNA, and 2.2 ng of DNA topoisomerase I in 20 µL (total volume) of 50 mM Tris-HCl (pH 7.5). Potential inhibitors were included at concentrations from 6 to 500 µM. Reactions were initiated by the addition of topoisomerase I, maintained at 37 °C for 30 min, and then terminated by the addition of 5 µL of a concentrated gel loading solution containing 2.5% SDS, 30% glycerol, and 0.125% bromphenol blue. Reaction mixtures containing coralyne or high (g167 µM) concentrations of camptothecin were extracted successively with phenol and chloroform prior to the addition of the gel loading solution, as these species are fluorescent. The reaction mixtures were analyzed by electrophoresis on 1% agarose gels and then stained with a solution containing 0.5 µg/mL of ethidium bromide. Inhibition of Topoisomerase II-Mediated P4 DNA Unknotting. Reaction mixtures contained 100 mM KCl, 10 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 µg/mL of BSA, 0.7 mM ATP, and 300 ng of knotted P4 DNA (22) in 20 µL (total volume) of 40 mM Tris-HCl (pH 7.5). Potential inhibitors were included at concentrations from 0.625 to 40 µM. The reactions were initiated by the addition of 2 units of calf thymus DNA topoisomerase II (1 unit is the amount of enzyme required to decatenate 0.2 µg of kinetoplast DNA in 15 min at 37 °C for 30 min). The incubation mixtures were placed on ice to terminate

Scheme 1. Putative Mechanism of DNA Nicking via a Topoisomerase I-DNA Covalent Binary Complex, and of Covalent Complex Stabilization by Binding to Camptothecin

Topo I Inhibition by Coralyne and Dihydrocoralyne the reactions, and individual reaction mixtures were extracted successively with phenol and chloroform. The samples were then treated with 5 µL of a concentrated gel loading solution containing 2.5% SDS, 30% glycerol, and 0.125% bromphenol blue. The samples were loaded on a 0.7% agarose gel and analyzed by electrophoresis at 60 V for 15 h in a 0.09 M Trisborate buffer, pH 8.3, containing 2 mM EDTA. The gel was then stained with a solution containing 0.5 µg/mL of ethidium bromide. DNA Unwinding Assay. The ability of coralyne and dihydrocoralyne to effect DNA unwinding was assayed by modification of the method of Schon et al. (24). Incubation mixtures contained 10 mM MgCl2, 10 mM DTT, 50 µg/mL of BSA, 0.5 mM ATP, 300 ng of HindIII-linearized pSP64 plasmid DNA, and the test compounds at 1-125 µM concentrations in 200 µL (total volume) of 40 mM Tris-HCl (pH 7.5). The reaction mixtures were incubated at 16 °C for 15 min, then treated with 1 unit of T4 DNA ligase (1 unit is the amount of enzyme required to catalyze the exchange of 1 nmol of 32P from pyrophosphate to ATP, measured as Norit-absorbable material, in 20 min at 37 °C), and maintained at 16 °C overnight. The reactions were quenched by the addition of EDTA to 20 mM final concentration and then extracted successively with phenol and chloroform to remove the test compounds prior to electrophoretic analysis. The DNA was recovered by precipitation with ethanol, then analyzed by electrophoresis on 1% agarose gels, and stained with a solution containing 0.5 µg/mL of ethidium bromide. Preparation of a 3′-32P End Labeled DNA Duplex. A DNA duplex having a 3′-32P end labeled strand 471 nucleotides (nt) in length was obtained by treatment of 35 µg of pSP64 plasmid DNA with restriction endonucleases NheI and PvuII. The digestions were carried out using 140 units of each enzyme in 10 mM Tris-HCl (pH 7.5), containing 10 mM MgCl2, 50 mM NaCl, and 1 mM dithiothreitol at 37 °C for 2 h. The incubation mixture was then extracted with phenol, and the DNA fragments were recovered by ethanol precipitation and centrifugation. The DNA was 3′-32P end labeled in a reaction mixture (50 µL total volume) that contained 10 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 100 mM NaCl, 1 mM 2-mercaptoethanol, 250 µCi of [R-32P]CTP, and 100 units of AMV reverse transcriptase (1 unit is the amount of enzyme required to incorporate 1 nmol of [3H]TTP into acid-insoluble product in 10 min at 37 °C). The reaction mixture was incubated at 37 °C for 3 h and then subjected to electrophoresis on a 5% native polyacrylamide gel. The band corresponding to the smaller DNA fragment was excised from the gel, and the DNA was recovered by electroelution. Topoisomerase I-Mediated Cleavage of a 3′-32P End Labeled DNA Duplex. Incubation mixtures contained 10 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 30 µg/mL of BSA, and 210 ng of 3′-32P end labeled DNA (3.1 × 105 cpm) in 40 µL (total volume) of 20 mM Tris-HCl (pH 7.5). Individual reaction mixtures contained camptothecin (10 µM final concentration), coralyne (50 µM), or 5,6-dihydrocoralyne (100 µM). Reactions were initiated by the addition of 340 ng of calf thymus DNA topoisomerase I and maintained at 37 °C for 15 min. The reaction mixtures were then treated with 1% SDS and incubated in the presence of 0.75 mg/mL of proteinase K at 37 °C for 1 h. The incubation mixture was extracted with phenol, and the DNA was recovered by ethanol precipitation and centrifugation. Because the 3′-32P end labeled DNA fragments resulting from topoisomerase I-mediated cleavage contained 5′-OH termini (6, 8), it was necessary to convert these species to the respective 5′-phosphates to facilitate comparison with the products of Maxam-Gilbert sequencing reactions. The reaction mixtures (50 µL total volume) contained 10 mM MgCl2, 5 mM DTT, 1.5 mM ATP, 5 units of T4 polynucleotide kinase (1 unit is the amount of enzyme required to incorporate 1 nmol of 32P from [32P]ATP into micrococcal nuclease-treated DNA in 30 min at 37 °C), and the 3′-32P end labeled DNA fragments. The incubation mixture was maintained at 37 °C for 30 min, and then the reaction was terminated by the addition of 2.5 µL of 200 mM

Chem. Res. Toxicol., Vol. 9, No. 1, 1996 77 EDTA and subsequent extraction with phenol. The DNA fragments were recovered by ethanol precipitation and centrifugation. The recovered DNA was dissolved in a formamide loading solution (80% formamide, 10 mM NaOH, 1 mM EDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue) for analysis on 7.5% denaturing polyacrylamide gels containing 8 M urea. Maxam-Gilbert sequencing reactions were run by slight modification of the method of Eckert (25) and used to identify the sites of topoisomerase I-mediated cleavage of the 3′-32P end labeled DNA fragment (see Figure 6).

Results Stabilization of the Topoisomerase I-DNA Covalent Binary Complex. The ability of the antitumor agent (19-21, 26) coralyne (1) and of the structurally related 5,6-dihydrocoralyne (2) (Figure 1) to stabilize the binary complex formed between topoisomerase I and DNA was studied. These species were added at varying concentrations to incubation mixtures containing supercoiled pSP64 plasmid DNA and a high concentration of calf thymus DNA topoisomerase I. The extent of stabilization of the covalent enzyme-DNA binary complex was determined by agarose gel electrophoresis following enzyme denaturation and proteolysis with SDS-proteinase K, which converted the binary complex to nicked, circular (form II) DNA. Camptothecin (3), a topoisomerase I inhibitor known (11-14, 27, 28) to stabilize the covalent binary complex, was employed as a control. As shown in Figure 2 (bottom panel), camptothecin stabilized the covalent enzyme-DNA binary complex at all tested concentrations, i.e., from 18 nM to 2.5 mM concentrations. 5,6-Dihydrocoralyne (2) (middle panel) also stabilized the covalent enzyme-DNA binary complex, although the effects of 2 were most pronounced at 19.5 µM-2.5 mM concentrations. Under the conditions of this assay, neither camptothecin nor 5,6-dihydrocoralyne inhibited topoisomerase I-mediated DNA relaxation. In comparison, coralyne (1) (top panel) inhibited DNA relaxation when employed at high (156 µM-2.5 mM) concentrations, but stabilized the enzyme-DNA covalent binary complex at somewhat lower (1.2-39 µM) concentrations. The apparent inhibition of DNA relaxation by coralyne (1) was verified by demonstrating that 1 had no effect on the mobility of relaxed DNA that had been incubated with topoisomerase I under the same conditions (not shown). There was no indication that coralyne (1) or dihydrocoralyne (2) alone bound to pSP64 plasmid DNA under these conditions, although coralyne has been reported to bind to DNA (21), an observation that we have verified under conditions different than those utilized in Figure 1. Densitometric analysis of the data in Figure 2 indicated that the extent of stabilization of the topoisomerase I-DNA binary complex was much greater in the presence of camptothecin than coralyne at all tested concentrations (Figure 3). 5,6-Dihydrocoralyne (2) produced almost as much stabilization of the binary complex as camptothecin at the highest concentrations tested, but much less stabilization at lower concentrations. Inhibition of Topoisomerase I-Mediated DNA Relaxation. The apparent inhibition of topoisomerase I-mediated DNA relaxation noted above for coralyne was studied further using a lower concentration of DNA topoisomerase I. Incubation mixtures containing 250 ng of supercoiled pSP64 plasmid DNA and 2.2 ng of topoisomerase I were incubated at 37 °C for 30 min in the presence of 6-500 µM coralyne (1), 5,6-dihydrocoralyne

78

Chem. Res. Toxicol., Vol. 9, No. 1, 1996

Wang et al.

Figure 4. Comparison of the abilities of coralyne, 5,6-dihydrocoralyne, and camptothecin to inhibit DNA relaxation by topoisomerase I. Supercoiled pSP64 plasmid DNA (250 ng) and 2.2 ng of topoisomerase I were incubated in the presence of varying concentrations of compounds 1-3 for 30 min, as described in the Experimental Procedures. Ethidium bromide staining was carried out after agarose gel electrophoresis in this case, resulting in a change in the relative mobilities of forms I and IV DNA (cf. Figures 2 and 5). (Lane 1) DNA alone; (lane 2) DNA + topoisomerase I; (lanes 3-7) DNA + topoisomerase I + 500, 167, 55, 18, and 6 µM camptothecin, respectively; (lanes 8-12) DNA + topoisomerase I + 500, 167, 55, 18, and 6 µM coralyne, respectively; (lanes 13-17) DNA + topoisomerase I + 500, 167, 55, 18, and 6 µM 5,6-dihydrocoralyne, respectively; (lane 18) 500 µM camptothecin; (lane 19) 500 µM coralyne; (lane 20) 500 µM 5,6-dihydrocoralyne. The bands in lanes 8, 9, and 19 that comigrate with form IV DNA were shown to result from treatment with phenol + coralyne.

Figure 2. Effects of coralyne, 5,6-dihydrocoralyne, and camptothecin on the topoisomerase I-DNA covalent binary complex. Supercoiled pSP64 plasmid DNA was treated with calf thymus DNA topoisomerase I in the presence of varying concentrations of coralyne (1) (top panel), 5,6-dihydrocoralyne (2) (middle panel), and camptothecin (3) (bottom panel), as described in the Experimental Procedures. (Lane 1) DNA alone (200 ng); (lane 2) DNA + 68 ng of topoisomerase I; (lanes 3-20) DNA + topoisomerase I + 2500, 1250, 625, 312, 156, 78, 39, 19.5, 9.8, 4.9, 2.4, 1.2, 0.6, 0.3, 0.15, 0.075, 0.037, and 0.018 µM coralyne, 5,6-dihydrocoralyne, or camptothecin, respectively.

Figure 3. Densitometric analysis of the extent of topoisomerase I-DNA covalent binary complex formation in the presence of coralyne (triangles), 5,6-dihydrocoralyne (squares), and camptothecin (circles). The gels illustrated in Figure 2 were analyzed by densitometry at each concentration of 1-3. The extent of covalent binary complex is expressed as a percentage of all DNA present in each reaction mixture.

(2), or camptothecin (3). As shown in Figure 4, coralyne effected completion inhibition of DNA relaxation at 167 µM concentration, while 5,6-dihydrocoralyne and camptothecin gave only partial inhibition of relaxation at this concentration but essentially complete inhibition at 500 µM concentration. Reversibility of Covalent Binary Complex Formation. The covalent topoisomerase I-DNA binary complex that is stabilized by camptothecin and certain other agents has been shown to be readily reversed upon

Figure 5. Effects of NaCl and heat on the topoisomerase I-DNA covalent binary complexes formed in the presence of coralyne, 5,6-dihydrocoralyne, and camptothecin. Supercoiled pSP64 plasmid DNA (200 ng) and 68 ng of calf thymus topoisomerase I were incubated in the presence of 5 µM camptothecin (lanes 3-5), 10 µM coralyne (lanes 6-8), or 50 µM 5,6dihydrocoralyne (lanes 9-11). The incubation mixtures were incubated at 37 °C for 30 min, then treated with SDSproteinase K prior to gel electrophoresis (lanes 1, 2, 3, 6, and 9), or else treated with 5 M NaCl to adjust the NaCl concentration to 0.5 M for 5 min (lanes 4, 7, and 10) or heated to 65 °C for 2 min (lanes 5, 8, and 11) prior to SDS-proteinase K treatment. (Lane 1) DNA alone; (lane 2) DNA + topoisomerase I.

removal of these agents, or upon treatment with increased concentrations of salt, elevated temperature, or an excess of some other DNA substrate for the enzyme (11, 14, 28-32). The reversible nature of the complexes observed in the presence of coralyne (1) and 5,6-dihydrocoralyne (2) was studied analogously. As shown in Figure 5, when an incubation mixture containing 200 ng of supercoiled plasmid DNA, 68 ng of topoisomerase I, and 10 µM coralyne was treated with 0.5 M NaCl for 5 min prior to SDS-proteinase K workup, the formation of the covalent binary complex was reversed (cf. lanes 6 and 7). The same result was obtained by incubation at 65 °C for 2 min prior to SDS-proteinase K treatment (cf. lanes 6 and 8). As shown in the figure, the covalent binary complex formed in the presence of topoisomerase I, DNA, and 50 µM 5,6-dihydrocoralyne was also reversed by treatment with NaCl or elevated temperature prior to SDS-proteinase K workup (cf. lanes 9-11). These results, which were the same as those obtained for camptothecin (lanes 3-5), are consistent with the interpretation that compounds 1 and 2 function analogously to camptothecin in stabilization of the DNA-topoisomerase I binary complex (11-14, 27, 28, 33).

Topo I Inhibition by Coralyne and Dihydrocoralyne

Inhibition of Topoisomerase II-Mediated P4 DNA Unknotting. The possible effects of coralyne and 5,6dihydrocoralyne on P4 DNA unknotting were also studied. The assay system involved the use of 300 ng of knotted P4 DNA and 2 units of calf thymus DNA topoisomerase II. Incubation at 37 °C for 30 min resulted in complete DNA unknotting, an effect that was blocked completely by 10 µM doxorubicin, a known inhibitor of topoisomerase II (34), and partially by 2.5 µM doxorubicin. In comparison, neither coralyne (1) nor 5,6-dihydrocoralyne (2) inhibited P4 DNA unknotting at concentrations up to 40 µM (supporting information, Figure 1). The use of higher concentrations of these two compounds resulted in partial inhibition of DNA unknotting by 100 µM coralyne. DNA Unwinding Measurements. Previously, it has been shown that the topoisomerase I inhibitors nitidine and fagaronine are capable of unwinding DNA (18). To investigate the possible effects of coralyne (1) and 5,6dihydrocoralyne (2) as DNA unwinding agents, HindIIIlinearized DNA was incubated with T4 DNA ligase in the presence of 1-125 µM coralyne or 5,6-dihydrocoralyne; the topoisomerase inhibitors m-AMSA (35) and camptothecin (11) were employed as controls. After quenching of the ligation reaction, any bound drug was removed by extraction and the introduction of supercoils into the cirularized DNA was measured. As shown (supporting information, Figure 2), m-AMSA produced changes in the linking number when employed at 125 µM concentration, consistent with its known (36) properties as an intercalator. Camptothecin, which is known not to bind to DNA (14), produced no effect. 5,6Dihydrocoralyne had only a minor effect in this assay system at the highest concentration tested, consistent with its apparent lack of strong DNA binding properties (vide supra). The effect of coralyne on DNA unwinding could not be assessed in this assay system because it effected complete inhibition of T4 DNA ligase at concentrations g25 µM. Sequence Selectivity of DNA Cleavage by Topoisomerase I in the Presence of Coralyne and Dihydrocoralyne. DNA topoisomerase I induces nicks in DNA at many sites; the nucleotide sequences in proximity to the nicks show substantial similarities in their sequences (29, 37-39). It has also been shown that some of these lesions are stabilized to a greater extent than others in the presence of topoisomerase I inhibitors such as camptothecin (29-31, 40), and the benzophenanthridine alkaloids nitidine and fagaronine (18). The sites of topoisomerase I-mediated DNA cleavage stabilized by coralyne and 5,6-dihydrocoralyne were determined by the use of a DNA duplex 471 nt in length. The DNA duplex was obtained by treatment of pSP64 plasmid DNA with restriction endonucleases NheI and PvuII. The recovered DNA was 3′-32P end labeled using [R-32P]CTP via the agency of AMV reverse transcriptase and used as a substrate for topoisomerase I following purification of the end labeled DNA on a polyacrylamide gel. Because the cleavage products resulting from enzymemediated cleavage had 5′-OH termini, it was necessary to introduce 5′-phosphate groups so that the derived products would comigrate on polyacrylamide gels with the products of the Maxam-Gilbert sequencing reactions; this was accomplished by the use of T4 polynucleotide kinase + ATP. As shown in Figure 6, a number of sites of cleavage of this DNA duplex were apparent after treatment of the DNA with topoisomerase I in the

Chem. Res. Toxicol., Vol. 9, No. 1, 1996 79

Figure 6. Effects of camptothecin, coralyne, and 5,6-dihydrocoralyne on topoisomerase I-mediated cleavage of a DNA duplex. A 3′-32P end labeled DNA duplex containing a radiolabeled strand 471 bp in length was treated with 340 ng of topoisomerase I and compounds 1, 2, or 3, as described in the Experimental Procedures. (Lane 1) DNA alone; (lane 2) DNA + 10 µM camptothecin; (lane 3) DNA + 50 µM coralyne; (lane 4) DNA + 100 µM 5,6-dihydrocoralyne; (lane 5) DNA + topoisomerase I; (lane 6) DNA + topoisomerase I + 10 µM camptothecin; (lane 7) DNA + topoisomerase I + 50 µM coralyne; (lane 8) DNA + topoisomerase I + 100 µM 5,6-dihydrocoralyne; (lane 9) Maxam-Gilbert G lane, (lane 10) G + A lane; (lane 11) T + C lane; (lane 12) C lane.

presence of coralyne (1), 5,6-dihydrocoralyne (2), or camptothecin (3). Seven of these sites were in a region of the gel amenable to sequence analysis. The sites of cleavage stabilized by coralyne and 5,6-dihydrocoralyne were nearly identical, and similar to those produced in the presence of camptothecin. All three agents produced lesions at sites 1, 2, 4, and 6. Both coralyne and dihydrocoralyne produced a lesion at site 3, that was not evident in the presence of camptothecin. However, camptothecin produced a nick in the DNA duplexes at two sites (5 and 7) that were not present when coralyne or dihydrocoralyne were utilized. The actual sequences that were cleaved in the presence of these agents are shown in Figure 7; a comparison of the sequences in proximity to the sites of cleavage is summarized in Table 1. Activities of Other Coralyne Analogues as Topoisomerase I Inhibitors. In an effort to define the structural features in coralyne (1) and 5,6-dihydrocoralyne (2) that contribute to their ability to stabilize the covalent binary complex between topoisomerase I and DNA, a number of structural analogues of 1 and 2 were

80

Chem. Res. Toxicol., Vol. 9, No. 1, 1996

Wang et al.

Figure 7. Nucleotide sequence of the 471 bp DNA used as a substrate for topoisomerase I in Figure 6. The cleavage sites are denoted by arrows, numbered sequentially as in Figure 6. Table 1. DNA Sequence at the Cleavage Sites Induced by Coralyne, 5,6-Dihydrocoralyne, and Camptothecin

tested. The assay system employed 200 ng of pSP64 DNA and 68 ng of calf thymus topoisomerase I, which were incubated at 37 °C in the presence of 20 µM test compound for 30 min and then treated with SDSproteinase K. The extent of conversion of form I f form II DNA, which provided a measure of the ability of each coralyne analogue to stabilize the covalent enzyme-DNA binary complex, was determined by agarose gel electrophoresis. Under these conditions, coralyne (1) and 5,6dihydrocoralyne (2) produced 58% and 54%, respectively, of form II DNA. In comparison, camptothecin produced 82% form II DNA. Of the numerous structural analogues tested, 12 compounds (4-15) (Figure 8) also promoted the formation of form II DNA when present at 20 µM concentration in the assay, but none was as potent as coralyne or 5,6-dihydrocoralyne. The most effective compound was 6, which afforded 42% form II DNA; the least effective was 14 (18%). The individual values are provided in Table 2. It is of interest that nitidine (16) and fagaronine (17), identified previously as topoisomerase I inhibitors (18, 41-43), afforded 69% and 65% form II DNA, respectively, under the same experimental conditions.

Discussion The recognition that DNA topoisomerase II may act as a locus of action for several known antitumor agents (34, 35, 44-46) prompted us to investigate an analogous role for topoisomerase I. The finding that the antitumor agent camptothecin inhibited the function of this enzyme by stabilizing the covalent topoisomerase I-DNA binary complex (11) has led to the development of several camptothecin analogues as potential anticancer agents (47-51). Also of interest has been a search for additional structural classes of compounds that may function as inhibitors of topoisomerase I. This has been carried out using whole cell (see, e.g., ref 52) and cell free assays (see, e.g., ref 17) and has resulted in the identification of numerous species that may function at this locus. While

Figure 8. Structures of 12 coralyne analogues tested as inhibitors of DNA topoisomerase I function. Table 2. Induction of Covalent Binary Complex Formation between Topoisomerase I and DNA by Coralyne Analoguesa

compound

covalent binary complex (%)b

coralyne (1) 5,6-dihydrocoralyne (2) camptothecin (3) 4 5 6 7 8 9

58 54 82 38 31 42 25 28 21

compound

covalent binary complex (%)b

10 11 12 13 14 15 nitidine (16) fagaronine (17)

35 35 26 30 18 34 69 65

a Inhibitors were employed at 20 µM final concentration as described under Experimental Procedures. b DNA present in enzyme-DNA binary complex as a percentage of all DNA.

a number of these appear to be simple DNA binding agents or compounds that act nonselectively on topoisomerase I, some of the compounds identified may well act primarily as inhibitors of topoisomerase I (ref 41 and references therein, refs 53-61).

Topo I Inhibition by Coralyne and Dihydrocoralyne

Another approach for the identification of potential topoisomerase I inhibitors is the investigation of known antitumor agents whose mechanism of action is presently unknown. In addition to camptothecin, whose mechanism was identified in this fashion (11), the validity of this approach is also supported by the recent finding that certain benzophenanthridine alkaloids, long known to have antitumor activity (62-67), also inhibit topoisomerase I function in a fashion similar to camptothecin (18, 41-43). These include nitidine (16) and fagaronine (17). In the present study, we have found that a number of protoberberine-type alkaloids, including the known antitumor agent (19-21, 26) coralyne (1) whose mechanism of action has not been reported, also inhibited topoisomerase I function. As shown in Figure 2, and quantified in Figure 3, coralyne (1) and 5,6-dihydrocoralyne (2) both stabilized the topoisomerase I-DNA binary complex in the same fashion as camptothecin. 5,6-Dihydrocoralyne was almost as effective as camptothecin at stabilization of the binary complex when employed at high concentration; coralyne was more effective than 5,6dihydrocoralyne at lower concentrations but simply inhibited DNA relaxation when employed at high (156 µM-2.5 mM) concentrations. The inhibition of DNA relaxation noted for coralyne in Figure 2 was studied in greater detail using a lower concentration of enzyme. As shown in Figure 4, in the presence of 2.2 ng of DNA topoisomerase I, inhibition of relaxation by coralyne was complete at 167 µM concentration. Although neither camptothecin nor 5,6-dihydrocoralyne inhibited relaxation in the experiments shown in Figure 2, both were almost as inhibitory as coralyne at the lower concentration of topoisomerase I utilized for the experiment in Figure 4. It may be noted that the behavior of coralyne and 5,6-dihydrocoralyne in the DNA relaxation assay was quite different than that noted previously for the benzophenanthridine alkaloids nitidine (16) and fagaronine (17) (18). While 16 and 17 stabilized the topoisomerase I-DNA covalent binary complex in a fashion analogous to compounds 1-3, these benzophenanthridine alkaloids also bound to DNA strongly enough to alter topoisomerase I-mediated DNA relaxation and caused DNA unwinding (18). As noted in Scheme 1, camptothecin is believed to bind non-covalently to the covalent binary complex formed between topoisomerase I and DNA. This scheme is supported by the ready reversibility of camptothecinmediated inhibition when camptothecin is removed from the assay system, and by the observation of a timedependent loss in reversibility of complex formation when certain electrophilic analogues of camptothecin were employed (32). Logically, alteration of experimental conditions might also be expected to change the ratio of products shown in Scheme 1; in fact, the addition of high concentrations of some other DNA substrate, increased salt concentration, and increased temperature have all been shown to shift the equilibrium toward free DNA (11, 14, 28-32). The effect of such changes in experimental conditions on the covalent binary complexes stabilized by coralyne and 5,6-dihydrocoralyne was studied. As shown in Figure 5, the binary complexes stabilized by coralyne, 5,6-dihydrocoralyne and camptothecin were all disrupted by treatment with 0.5 M NaCl for 5 min at 37 °C, or by heating to 65 °C for 2 min. Essentially the same results were obtained for all three compounds at shorter NaCl treatment times (g15 s) at 37 °C.

Chem. Res. Toxicol., Vol. 9, No. 1, 1996 81

One further comparision involved the sites of topoisomerase I-mediated DNA cleavage stabilized by camptothecin, coralyne, and 5,6-dihydrocoralyne. Because the enzyme is capable of nicking DNA at many sites, only some of which are stabilized at the level of covalent binary complex, the comparison has the potential to provide rather detailed comparative information about the behavior of a number of agents believed to act in the same fashion. This comparison was carried out using a DNA duplex 32 P-end labeled on one strand 471 nt in length. As shown in Figure 6, seven sites of cleavage stabilized by one or more of the agents could be sequenced on this gel; the results are summarized in Figure 7 and in Table 1. Coralyne, 5,6-dihydrocoralyne, and camptothecin all stabilized four of the seven cleavage sites; a comparison of the sequences of these sites reveals that all four had rather similar sequences in proximity to the actual site of cleavage. Two sites in the DNA (5 and 7) were stabilized only by camptothecin; both had the sequence TGGT on the 5′-side of the site of cleavage. Cleavage at site 3 was stabilized only by coralyne and 5,6-dihydrocoralyne; the nucleotide sequence at this cleavage site was rather different than those at the other six cleavage sites. Mapping of topoisomerase I cleavage sites has indicated a loose consensus sequence, with the most prominent preference being for a T 5′ to the cleavage site with no specificity observed on the 3′ side of the site of cleavage (37, 68). In contrast, a bias for the 3′ base was induced in the topoisomerase I cleavage sites enhanced by camptothecin, with the most usual being for G (40, 69). Studies mapping drug-stimulated cleavage of additional topoisomerase I and II inhibitors have demonstrated a similar bias for a particular base immediately flanking the site of cleavage (ref 5 and references therein), suggesting a direct interaction of the drugs with the flanking bases (70). The ability of coralyne and 5,6dihydrocoralyne to induce cleavage at the majority of camptothecin sites supports a similar mechanism of stabilization of the topoisomerase I-DNA complex by these agents. Further, while coralyne is capable of binding to DNA at high concentrations (21), we have not found this to be true for 5,6-dihydrocoralyne. The fact that both exhibit the same sequence specificity of DNA cleavage argues that the sites stabilized by coralyne do not result primarily from its ability to bind to DNA in the absence of topoisomerase I. It is believed that topoisomerase I first binds noncovalently to its DNA substrate and then mediates cleavage of one strand by the use of an active site tyrosine hydroxyl group, which becomes covalently attached to the enzyme (Scheme 1). Following DNA relaxation, which presumably occurs by passage of the broken strand not covalently attached to the enzyme, the DNA strand is religated with concomitant release of free enzyme. As noted previously, several steps in this process are potentially amenable to inhibition (17, 18), and examples of some of these types of inhibitors have been described. In spite of the existence of topoisomerase I inhibitors that function by different molecular mechanisms, the evidence available at the present time suggests strongly that stabilization of the covalent enzyme-DNA binary complex is the mechanism responsible for the cytotoxic effects of topoisomerase I inhibitors that function as antitumor agents. In this context it is interesting to note that while coralyne and 5,6-dihydrocoralyne both stabilized the DNA-topoisomerase I covalent binary complex

82

Chem. Res. Toxicol., Vol. 9, No. 1, 1996

(vide supra), only coralyne exhibited antileukemic activity when tested comparatively in P388 and L1210 (19). This may reflect the greater potency of coralyne as a topoisomerase I inhibitor when used at low concentrations or may indicate that stabilization of the covalent binary complex is necessary, but not sufficient, for agents that function at the locus of DNA topoisomerase I.

Acknowledgment. We thank Dr. Matthew Suffness, National Cancer Institute, for providing many of the coralyne analogues studied as inhibitors of DNA topoisomerase I, Dr. Randall Johnson, SmithKline Beecham Pharmaceuticals, for a sample of camptothecin, and Ms. Xiangyang Wang, University of Virginia, for assistance with some of the assays. Supporting Information Available: Two figures showing the effects of doxorubicin, coralyne, 5,6-dihydrocoralyne, and camptothecin on the unknotting of P4 DNA by topoisomerase II and a comparison of the DNA unwinding effects of m-AMSA, 5,6-dihydrocoralyne, and camptothecin (3 pages). Ordering information is given on any current masthead page.

References (1) Gellert, M. (1981) DNA topoisomerases. Annu. Rev. Biochem. 50, 879-910. (2) Gupta, M., Fujimori, A., and Pommier, Y. (1995) Eukaryotic DNA topoisomerases I. Biochim. Biophys. Acta 1262, 1-14. (3) Wang, J. C. (1987) Recent studies of DNA topoisomerases. Biochim. Biophys. Acta 909, 1-9. (4) Wang, J. C. (1987) DNA topoisomerases: nature’s solution to the topological ramifications of the double-helix structure of DNA. Harvey Lect. 81, 93-110. (5) Ralph, R. K., Judd, W., Pommier, Y., and Kohn, K. W. (1994) DNA N-alkylating agent-DNA interaction. In Topics in Molecular and Structural Biology, Molecular Aspects of Anticancer Drug-DNA Interactions (Neidle, S., and Waring, M., Eds.) Vol. 2, pp 1-95, Macmillan Press, London. (6) Champoux, J. J. (1977) Strand breakage by the DNA untwisting enzyme results in covalent attachment of the enzyme to DNA. Proc. Natl. Acad. Sci. U.S.A. 74, 3800-3804. (7) Liu, L. F., Rowe, T. C., Yang, L., Tewey, K. M., and Chen, G. L. (1983) Cleavage of DNA by mammalian DNA topoisomerase II. J. Biol. Chem. 258, 15365-15370. (8) Schneider, E., Hsiang, Y.-H., and Liu, L. F. (1990) DNA topoisomerases as anticancer drug targets. Adv. Pharmacol. 21, 149-183. (9) Liu, L. F. (1989) DNA topoisomerase poisons as antitumor drugs. Annu. Rev. Biochem. 58, 351-375. (10) D’Arpa, P., and Liu, L. F. (1989) Topoisomerase-targeting antitumor drugs. Biochim. Biophys. Acta 989, 163-177. (11) Hsiang, Y.-H., Hertzberg, R. P., Hecht, S., and Liu, L. F. (1985) Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J. Biol. Chem. 260, 14873-14878. (12) Andoh, T., Ishii, K., Suzuki, Y., Ikegami, Y., Kusunoki, Y., Takemoto, Y., and Okada, K. (1987) Characterization of a mammalian mutant with a camptothecin-resistant DNA topoisomerase I. Proc. Natl. Acad. Sci. U.S.A. 84, 5565-5569. (13) Gupta, R. S., Gupta, R., Eng, B., Lock, R. B., Ross, W. E., Hertzberg, R. P., Caranfa, M. J., and Johnson, R. K. (1988) Camptothecin-resistant mutants of Chinese hamster ovary cells containing a resistant form of topoisomerase I. Cancer Res. 48, 6404-6410. (14) Hertzberg, R. P., Caranfa, M. J., and Hecht, S. M. (1989) On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme-DNA complex. Biochemistry 28, 4629-4638. (15) Ross, W. E. (1985) DNA topoisomerases as targets for cancer therapy. Biochem. Pharmacol. 34, 4191-4195. (16) Hertzberg, R. P., Caranfa, M. J., Kingsbury, W. D., Smith, B., Johnson, R. K., and Hecht, S. M. (1991) The biochemistry of camptothecin-topoisomerase I interaction. In DNA Topoisomerases in Cancer (Potmesil, M., and Kohn, K. W., Eds.) pp 103-120, Oxford University Press, New York. (17) Hecht, S. M., Berry, D. E., MacKenzie, L. J., Busby, R. W., and Nasuti, C. A. (1992) A strategy for identifying novel, mechanistically unique inhibitors of topoisomerase I. J. Nat. Prod. 55, 401413.

Wang et al. (18) Wang, L.-K., Johnson, R. K., and Hecht, S. M. (1993) Inhibition of topoisomerase I function by nitidine and fagaronine. Chem. Res. Toxicol. 6, 813-818. (19) Zee-Cheng, K.-Y., Paull, K. D., and Cheng, C. C. (1974) Experimental antileukemic agents. Coralyne, analogs, and related compounds. J. Med. Chem. 17, 347-351. (20) Zee-Cheng, R. K.-Y., and Cheng, C. C. (1976) Tetramethoxydibenzoquinolizinium salts. Preparation and antileukemic activity of some positional and structural isomers of coralyne. J. Med. Chem. 19, 882-886. (21) Wilson, W. D., Gough, A. N., Doyle, J. J., and Davidson, M. N. (1976) Coralyne. Intercalation with DNA as a possible mechanism of antileukemic action. J. Med. Chem. 19, 1261-1263. (22) Liu, L. F., and Davis, J. L. (1981) Novel topologically knotted DNA from bacteriophage P4 capsids: studies with DNA topoisomerases. Nucleic Acids Res. 9, 3979-3989. (23) Liu, L. F., and Miller, K. G. (1981) Eukaryotic DNA topoisomerases: two forms of type I DNA topoisomerases from HeLa cell nuclei. Proc. Natl. Acad. Sci. U.S.A. 78, 3487-3491. (24) Schon, E., Evans, T., Welsh, J., and Efstraiadis, A. (1983) Conformation of promoter DNA: fine mapping of S1-hypersensitive sites. Cell 35, 837-848. (25) Eckert, R. L. (1995) in Current Protocols in Molecular Biology (Albright, L. M., Coen, D. M., and Varki, A., Eds.) pp 7.5.1-7.5.11, John Wiley & Sons, New York. (26) Weimar, C., Angerer, S. V., and Wiegrebe, W. (1991) Methoxyand acetoxy-8-oxoberbinesssynthesis, antitumor activity, and interaction with DNA. Arch. Pharm. (Weinheim, Ger.) 324, 509518. (27) Mattern, M. R., Mong, S.-M., Bartus, H. F., Mirabelli, C. K., Crooke, S. T., and Johnson, R. K., (1987) Relationship between the intracellular effects of camptothecin and the inhibition of DNA topoisomerase I in cultured L1210 cells. Cancer Res. 47, 17931798. (28) Hsiang, Y.-H., and Liu, L. F. (1988) Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin. Cancer Res. 48, 1722-1726. (29) Thomsen, B., Mollerup, S., Bonven, B. J., Frank, R., Blocker, H., Nielsen, O. F., and Westergaard, O. (1987) Sequence specificity of DNA topoisomerase I in the presence and absence of camptothecin. EMBO J. 6, 1817-1823. (30) Kjeldsen, E., Mollerup, S., Thomsen, B., Bonven, B., Bolund, L., and Westergaard, O. (1988) Sequence-dependent effect of camptothecin on human topoisomerase I DNA cleavage. J. Mol. Biol. 202, 333-342. (31) Champoux, J. J., and Aronoff, R. (1989) The effects of camptothecin on the reaction and the specificity of the wheat germ type I topoisomerase. J. Biol. Chem. 264, 1010-1015. (32) Hertzberg, R. P., Busby, R. W., Caranfa, M. J., Holden, K. G., Johnson, R. K., Hecht, S. M., and Kingsbury, W. D. (1990) Irreversible trapping of the DNA-topoisomerase I covalent complex. Affinity labeling of the camptothecin binding site. J. Biol. Chem. 265, 19287-19295. (33) Hsiang, Y.-H., Lihou, M. G., and Liu, L. F. (1989) Mechanism of cell killing by camptothecin: arrest of replication forks by drugstabilized topoisomerase I-DNA cleavable complexes. Cancer Res. 49, 5077-5082. (34) Tewey, K. M., Rowe, T. C., Chen, G. L., Yang, L., Halligan, B. D., and Liu, L. F. (1984) Adriamycin-induced DNA damage-mediated by mammalian DNA topoisomerase II. Science 226, 466-468. (35) Nelson, E. M., Tewey, K. M., and Liu, L. F. (1984) Mechanism of antitumor drug action: poisoning of mammalian DNA topoisomerase II on DNA by 4′-(9-acridinylamino)methanesulfon-manisidide. Proc. Natl. Acad. Sci. U.S.A. 81, 1361-1365. (36) Waring, M. J. (1981) DNA modification and cancer. Annu. Rev. Biochem. 50, 159-192. (37) Been, M. D., Burgess, R. R., and Champoux, J. J. (1984) Nucleotide sequence preference at rat liver and wheat germ type 1 DNA topoisomerase breakage sites in duplex SV40 DNA. Nucleic Acids Res. 12, 3097-3114. (38) Bonven, B. J., Gocke, E., and Westergaard, O. (1985) A high affinity topoisomerase I binding sequence is clustered at DNase I hypersensitive sites in tetrahymena R-chromatin. Cell 41, 541551. (39) Stevnsner, T., Mortensen, U. H., Westergaard, O., and Bonven, B. J. (1989) Interactions between eukaryotic DNA topoisomerase I and a specific binding sequence. J. Biol. Chem. 264, 1011010113. (40) Jaxel, C., Capranico, G., Kerrigan, D., Kohn, K. W., and Pommier, Y. (1991) Effect of local DNA sequence on topoisomerase I cleavage in the presence or absence of camptothecin. J. Biol. Chem. 266, 20418-20423. (41) Fang, S. D., Wang, L.-K., and Hecht, S. M. (1993) Inhibitors of DNA topoisomerase I isolated from the roots of Zanthoxylum nitidum. J. Org. Chem. 58, 5025-5027.

Topo I Inhibition by Coralyne and Dihydrocoralyne (42) Janin, Y. L., Croisy, A., Riou, J.-F., and Bisagni, E. (1993) Synthesis and evaluation of new 6-amino-substituted benzo[c]phenanthridine derivatives. J. Med. Chem. 36, 3686-3692. (43) Larsen, A. K., Grondard, L., Couprie, J., Desoize, B., Comoe, L., Jardillier, J.-C., and Riou, J.-F. (1993) The antileukemic alkaloid fagaronine is an inhibitor of DNA topoisomerases I and II. Biochem. Pharmacol. 46, 1403-1412. (44) Chen, G. L., Yang, L., Rowe, T. C., Halligan, B. D., Tewey, K. M., and Liu, L. F. (1984) Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II. J. Biol. Chem. 259, 13560-13566. (45) Ross, W., Rowe, T., Glisson, B., Yalowich, J., and Liu, L. (1984) Role of topoisomerase II in mediating epipodophyllotoxin-induced DNA cleavage. Cancer Res. 44, 5837-5860. (46) Tewey, K. M., Chen, G. L., Nelson, E. M., and Liu, L. F. (1984) Intercalative antitumor drugs interfere with the breakagereunion reaction of mammalian DNA topoisomerase II. J. Biol. Chem. 259, 9182-9187. (47) Giovanella, B. C., Stehlin, J. S., Wall, M. E., Wani, M. C., Nicholas, A. W., Liu, L. F., Silber, R., and Potmesil, M. (1989) DNA topoisomerase I-targeted chemotherapy of human colon cancer in xenografts. Science 246, 1046-1048. (48) Hertzberg, R. P., Caranfa, M. J., Holden, K. G., Jakas, D. R., Gallagher, G., Mattern, M. R., Mong, S. M., O’Leary-Bartus, J., Johnson, R. K., and Kingsbury, W. D. (1989) Modification of the hydroxy lactone ring of camptothecin: inhibition of mammalian topoisomerase I and biological activity. J. Med. Chem. 32, 715720. (49) Ohno, R., Okada, K., Masaoka, T., Kuramoto, A., Arima, T., Yoshida, Y., Ariyoshi, H., Ichimaru, M., Sakai, Y., Oguro, M., Ito, Y., Morishima, Y., Yokomaku, S., and Ota, K. (1990) An early phase II study of CPT-11: a new derivative of camptothecin, for the treatment of leukemia and lymphoma. J. Clin. Oncol. 8, 1907-1912. (50) Kingsbury, W. D., Boehm, J. D., Jakas, D. R., Holden, K. G., Hecht, S. M., Gallagher, G., Caranfa, M. J., McCabe, F. L., Faucette, L. F., Johnson, R. K., and Hertzberg, R. P. (1991) Synthesis of water-soluble (aminoalkyl)camptothecin analogues: inhibition of topoisomerase I and antitumor activity. J. Med. Chem. 34, 98-107. (51) Potmesil, M., and Pinedo, H., Eds. (1995) Camptothecins: New Anticancer Agents, CRC Press, Boca Raton. (52) Eng, W.-K., Faucette, L., Johnson, R. K., and Sternglanz, R. (1988) Evidence that DNA topoisomerase I is necessary for the cytotoxic effects of camptothecin. Mol. Pharmacol. 34, 755-760. (53) Beerman, T. A., McHugh, M. M., Sigmund, R. D., Lown, J. W., Rao, K. E., and Bathini, Y. (1992) Effects of analogs of the DNA minor groove binder Hoechst 33258 on topoisomerase II and I mediated activities. Biochim. Biophys. Acta 1131, 53-61. (54) Chen, A. Y., Yu, C., Bodley, A., Peng, L. F., and Liu, L. F. (1993) A new mammalian DNA topoisomerase I poison Hoechst 33342: cytotoxicity and drug resistance in human cell cultures. Cancer Res. 53, 1332-1337. (55) Chen, A. Y., Yu, C., Gatto, B., and Liu, L. F. (1993) DNA minor groove-binding ligands: a different class of mammalian DNA topoisomerase I inhibitors. Proc. Natl. Acad. Sci. U.S.A. 90, 81318135. (56) Fujii, N., Yamashita, Y., Chiba, S., Uosaki, Y., Saitoh, Y., Tuji, Y., and Nakano, H. (1993) UCE6, a new antitumor antibiotic with

Chem. Res. Toxicol., Vol. 9, No. 1, 1996 83

(57)

(58)

(59)

(60)

(61)

(62)

(63)

(64) (65)

(66)

(67)

(68)

(69)

(70)

topoisomerase I mediated DNA cleavage activity, from actinomycetes. J. Antibiot. 46, 1173-1174. Fujii, N., Yamashita, Y., Saitoh, Y., and Nakano, H. (1993) Induction of mammalian DNA topoisomerase I-mediated DNA cleavage and DNA winding by bulgarein. J. Biol. Chem. 268, 13160-13165. Fujii, N., Yamashita, Y., Ando, K., Agatsuma, T., Saitoh, Y., Gomi, K., Nishiie, K., and Nakano, H. (1994) UCE1022, a new antitumor antibiotic with topoisomerase I mediated DNA cleavage activity, from Paecilomyces. J. Antibiot. 47, 949-951. Li, C. J., Averbouk, L., and Pardee, A. B. (1993) Beta-lapachone, a novel DNA topoisomerase I inhibitor with a mode of action different from camptothecin. J. Biol. Chem. 268, 22463-22468. Yoshinari, T., Yamada, A., Uemura, D., Nomura, K., Arakawa, H., Kojiri, K., Yoshida, E., Suda, H., and Okura, A. (1993) Induction of topoisomerase I-mediated DNA cleavage by a new indolocarbazole, ED-110. Cancer Res. 53, 490-494. Zhao, J., and Benbow, R. M. (1993) An inhibitor of DNA topoisomerase I from Xenopus laevis ovaries. Biochemistry 32, 1062210628. Stermitz, F. R., Gillespie, J. P., Amoros, L. G., Romero, R., Stermitz, T. A., Larson, K. A., Earl, S., and Ogg, J. E. (1975) Synthesis and biological activity of some antitumor benzophenanthridinium salts. J. Med. Chem. 18, 708-713. Zee-Cheng, R. K.-Y., and Cheng, C. C. (1975) Preparation and antileukemic activity of some alkoxybenzo[c]phenathridinium salts and corresponding dihydro derivatives. J. Med. Chem. 18, 66-71. Caolo, M. A., and Stermitz, F. R. (1979) Benzophenanthridinium salt equilibria. Heterocycles 12, 11-15. Phillips, S. D., and Castle, R. N. (1981) A review of the chemistry of the antitumor benzo[c]phenanthridine alkaloids nitidine and fagaronine and of the related antitumor alkaloid coralyne. J. Heterocycl. Chem. 18, 223-232. Cushman, M., and Mohan, P. (1985) Synthesis and antitumor activity of structural analogues of the anticancer benzophenanthridine alkaloid fagaronine chloride. J. Med. Chem. 28, 10311036. Ishii, H., Chen, I. S., and Ishikawa, T. (1987) Studies on the chemical constituents of rutaceous plants. Part 62. Efficient synthesis of fagaronine, a phenolic benzo[c]phenanthridine alkaloid with antileukaemic activity. J. Chem. Soc., Perkin Trans. 1, 671-676. Edwards, K. A., Halligan, B. D., Davis, J. L., Nivera, N. I., and Liu, L. F. (1982) Recognition sites of eukaryotic DNA topoisomerase I: DNA nucleotide sequencing analysis of topo I cleavage sites on SV40 DNA. Nucleic Acids Res. 10, 2565-2576. Porter, S. E., and Champoux, J. J. (1988) Mapping in vivo topoisomerase I sites on simian virus 40 DNA: asymmetric distribution of sites on replicating molecules. Mol. Cell. Biol. 9, 541-550. Pommier, Y., Capranico, G., Orr, A., and Kohn, K. A. (1991) Local base sequence preferences for DNA cleavage by mammalian topoisomerase II in the presence of amsacrine or teniposide. Nucleic Acids Res. 19, 5973-5980.

TX950080Y