Chem. Res. Toxicol. 1993,6, 813-818
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Inhibition of Topoisomerase I Function by Nitidine and Fagaronine Li-Kai Wang,? Randall K. Johnson,t and Sidney M. Hecht*p+ Departments of Chemistry and Biology, University of Virginia, Charlottesville, Virginia 22901, and Department of Biomolecular Discovery, Research and Development, SmithKline Beecham Pharmaceuticals, P.O. Box 1539, King of Prussia, Pennsylvania 19406 Received June 23,199P
The benzophenanthridine alkaloids nitidine and fagaronine were characterized as inhibitors of topoisomerase I function. In common with the antitumor agent camptothecin, both nitidine and fagaronine stabilized the covalent binary complex formed between calf thymus topoisomerase I and DNA. The effects of these compounds were readily apparent a t 0.15-0.3 pM concentrations. Both nitidine and fagaronine inhibited the topoisomerase I-mediated relaxation of supercoiled pSP64 plasmid DNA more effectively than camptothecin; unlike camptothecin, both of these benzophenanthridine alkaloids also bound directly to and mediated the unwinding of B-form DNA. Nitidine and fagaronine were also studied in comparison with camptothecin to determine the sequence specificity of DNA breaks produced from a 32P-end-labeledduplex in the presence of topoisomerase I. All three compounds produced very similar cleavage patterns. The specificity of nitidine and fagaronine for inhibiting topoisomerase I function was studied by measuring the effects of the compounds on the unknotting of P4 DNA by calf thymus topoisomerase 11. Moderate inhibition of topoisomerase II-mediated unknotting was obtained, but only in the presence of high (i.e., 40pM) concentrations of nitidine and fagaronine. In comparison, doxorubicin inhibited topoisomerase I1 to the same extent as nitidine and fagaronine when it was employed a t 2.5 pM concentration and was strongly inhibitory when employed a t 10 pM concentration. Introduction
DNA topoisomerases are enzymes that alter the topological state of DNA, which is required for certain critical cellular processes such as DNA replication and transcription (1-4). The mechanism by which topoisomerase I changes the DNA linking number is summarized in Figure 1. The key step involves the creation of a reversibly formed break in one strand of DNA; the enzyme becomes covalently bound to this DNA intermediate at the site of the strand break. Topoisomerase I1 employs a similar mechanism, but one that involves transient breakage of both strands of DNA (1-8). There are a number of low molecular weight molecules that inhibit topoisomerase-mediated DNA relaxation; some of these stabilize the covalent binary complex between topoisomerase I or I1 and DNA. As would be expected for inhibitors of an essential cell function, many topoisomerase inhibitors are cytotoxic. In fact, several topoisomerase I and I1 inhibitors have demonstrable antitumor activity, and both enzymes are presently of considerable interest as targets for the development of novel antitumor agents (9-12). Although there are many compounds that inhibit topoisomerase I-mediated DNA relaxation, the camptothecins are of particular interest in that they have been shown unequivocally to bind neither to DNA nor to topoisomerase I alone, but rather to specifically stabilize the topoisomerase I-DNA binary complex (12-18). Recently,we identified three new inhibitors of topoisomerase I-mediated DNA relaxation as constituents of Zanthox-
ylum nitidum (19). These were found to be the known (20,21) benzophenanthridine alkaloids nitidine and chel-
erythrine and a structurally novel species denoted isofagaridine. While all three compounds inhibited enzymemediated DNA relaxation, only nitidine strongly stabilized the covalent binary complex between topoisomerase I and DNA in a fashion analogous to camptothecin. The current interest in the camptothecins as antitumor agents (22-25) prompted us to compare the effects of camptothecin and nitidine on topoisomerase I-mediated DNA relaxation. Studied in parallel was the benzophenanthridine alkaloid fagaronine, which was also found to stabilize the covalent complex between topoisomerase I and DNA (Figure 2). Experimental Procedures Materials. Dithiothreitol (DTT),’ ethidium bromide, Sephacryl S-1O00, and bovine pancreatic ribonuclease were purchased from Sigma Chemical Co. (St. Louis, MO); doxorubicin was obtained from Aldrich Chemicals (Milwaukee, WI). Crystalline bovine serum albumin was obtained from American Biorganics (Niagara Falls, NY). T4 polynucleotidekinase, T4 DNA ligase, and restrictionendonucleaseHind111 were purchased from U S . Biochemicals (Cleveland,OH); PuuII was from Promega (Madison, WI). Proteinase K was purchased from Boehringer Mannheim (Indianapolis, IN). Nitidine, fagaronine, and O-methylfagaronine were obtained from the National Cancer Institute. through the courtesy of Dr. Matthew Suffness. pSP64 Plasmid DNA. Crude plasmid DNA was obtained by slight modification of a published procedure (26). Following digestion with proteinase K and bovine pancreatic ribonuclease, the DNA was purified by repeated chromatography on a
* To whom correspondence should be addressed.
t University of Virginia. 8 SmithKline Beecham Pharmaceuticals.
* Abstract published in Aduance ACS Abstracts, October 1, 1993.
~
~
Abbreviations: SDS, sodium dodecyl sulfate; DTT,dithiothreitol; PAGE, polyacrylamide gel electrophoresis. l
0893-228x/93/2706-0813$04.00/00 1993 American Chemical Society
Wang et al.
814 Chem. Res. Toxicol., Vol. 6, No. 6, 1993 Sephacryl 5-1000 column, essentially as described (27). The purified DNA was >95% supercoiled. DNA Topoisomerase I. The enzyme was purified from calf thymus by modification of a published procedure (28). The 96 OOO and 82 OOO) isolated protein exhibited two major bands (M, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had a specific activity of 1.4 X lo7 units (28)lmg protein. Topoisomerase I-Mediated DNA Cleavage. This assay was adapted from Hsiang et al. (13). Reaction mixtures consisted of 20 pL (total volume) of 40 mM Tris-HC1 (pH 7.5) containing 100 mM KC1,lO mM MgC12,0.5 mM DTT, 0.5 mM EDTA, 30 pg/mL of bovine serum albumin, and 200 ng of supercoiled pSP64 plasmid DNA. Where a test compound was present, it was added from a concentrated Me2SO solution such that the final MezSO concentration was 1%. The reactions were initiated by the addition of 68 ng of calf thymus DNA topoisomerase I and incubated at 37 "C for 30 min. The reactions were terminated by the addition of 1% SDS, then incubated with 0.75 mg/mL of proteinase K at 37 OC for 1 h, and analyzed by electrophoresis on a 1% agarose gel containing 0.5 pg/mL of ethidium bromide. Inhibition of Topoisomerase I-Mediated DNA Relaxation. Reaction mixtures consisted of 20 pL (total volume) of 50 mM Tris-HC1 (pH 7.5) containing 120 mM KCl, 10 mM MgC12, 0.5 mM DTT, 0.5 mM EDTA, 50 pg/mL of bovine serum albumin, 6% poly(ethy1ene glycol), 250 ng of supercoiled pSP64 plasmid DNA and the test compounds at the concentrations indicated in the legend to Figure 5. The reactions were initiated by the addition of 2.2 ng of topoisomerase I, incubated at 37 "C for 30 min, and then quenched by the addition of 5 pL of a concentrated gel loading solution containing 2.5% SDS, 30% glycerol, and 0.125% bromophenol blue. The reaction mixtures containing nitidine or fagaronine were extracted successively with phenol and chloroform prior to addition of loading solution, as these agents alter DNA migration. The samples were analyzed by electrophoresis on a 1%agarose gel and then stained with 0.5 pg/mL of ethidium bromide. DNA Unwinding Measurements. The DNA unwinding effects of nitidine and fagaronine were determined by slight modification of the method of Schon et al. (29). Reaction mixtures consisted of 200 pL (total volume) of 40 mM Tris-HC1 (pH 7.5) containing 10mM MgC12,10mMDTT, 5Opg/mL of bovine serum albumin, 0.5 mM ATP, 300 ng of HindIII-linearized pSP64 plasmid DNA, and the test compounds at the concentrations indicated in the legend to Figure 6. The solutions were incubated at 16 "C for 15 min, then treated with 1 unit of T4 DNA ligase (1unit is the amount of enzyme required to catalyze the exchange of 1nmol of SzP from pyrophosphate to ATP, measured as Noritabsorbablematerial, in 20 min at 37 "C), and maintained overnight at 16 "C. The reactions were terminated by addition of EDTA to a final concentration of 20 mM and then extracted successively with phenol and chloroform to remove the test compounds. The DNA was recovered by ethanol precipitation, then analyzed by 1%agarose gel electrophoresis, and stained with a 0.5 pg/mL solution of ethidium bromide. Sites of Topoisomerase I-Mediated DNA Cleavage. A DNA duplex 2767 base pairs in length was obtained by linearization of 50 pg of pSP64 plasmid DNA with restriction endonuclease HindIII. The linearized DNA was dephosphorylated at the 5'-end with calf intestinal phosphatase and then 5'end labeled in the presence of 5'-[y-S2P]ATP and T4 polynucleotide kinase, essentially as described (30).The radiolabeled DNA was further digested with restriction endonuclease PuuII and the larger DNA fragment was purified by 6% PAGE. Topoisomerase I-mediated DNA cleavage was carried out in 40 p L (total volume) of 20 mM Tris-HC1 (pH 7.5) containing 10 mM KC1,5 mM MgC12,0.5 mM EDTA, 0.5 mM DTT, 30 pg/mL of bovine serum albumin, and 3.1 X 106 cpm (-800 ng) of 5'-32P end-labeled DNA in the presence of camptothecin (10 pM), nitidine (30 pM), or fagaronine (50pM) and 340 ng of calf thymus topoisomerase I. The reaction mixture was incubated at 25 OC for 10 min and then treated with 1% SDS and 0.75 mg/mL of proteinase K at 37 "C for 60 min. The reaction mixture was
w
w supercoiled
non-covalent complex
covalent binary complex
relaxed
Figure 1. Possible mechanisms of inhibition of topoisomerase I-mediated DNA relaxation. The enzyme (0) first binds to the DNA substrate noncovalently and then becomes covalently attached via a 3'-phosphorotyrosinelinkage. Following relaxation of open circular DNA by DNA strand passage, the broken DNA strand is resealed with concomitant release of topoisomerase I. Potential points of inhibition are denoted by vertical arrows. extracted successively with phenol and chloroform and then treated with three volumes of cold ethanol to precipitate the DNA. The recovered DNA was dissolved in 25 p L of a gel-loading solution (80% aqueous formamide containing 10 mM NaOH, 1 mM EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue) and then analyzed by 7.5% denaturing PAGE.
Results and Discussion
As shown in Figure 1,t h e currently accepted mechanism for topoisomerase I-mediated DNA relaxation involves covalent attachment of t h e enzyme t o one strand of t h e DNA substrate via an active site tyrosine OH group. Following strand passage, the broken DNA strand is resealed with concomitant release of topoisomerase I (1, 5, 31). As illustrated in t h e figure, at least four steps in this mechanism are potentially susceptible to inhibition, including t h e noncovalent binding of topoisomerase I t o DNA, nicking of t h e DNA by the enzyme, passage of the broken DNA strand t o effect relaxation, a n d dissociation of the enzyme from the covalent binary complex with DNA. At present, there are a number of compounds reported to inhibit topoisomerase I function (see, for example, refs 32-36). While few of these have been studied in detail at a mechanistic level, a n d many appear t o be simple DNA binding agents (for a leading reference see ref 36), there is some data available pertinent to the mechanistic scheme in Figure 1. For example, corilagin a n d chebulagic acid, isolated from Erodium stephanianum, were shown t o effect potent inhibition both of topoisomerase I-induced DNA nicking as well as overall DNA relaxation (36). Neither compound, however, effected stabilization of t h e covalent binary complex between the enzyme and DNA (cf. Figure 1). I n comparison, t h e alkaloid camptothecin and a number of synthetic analogs stabilized t h e enzymeDNA covalent binary complex, but had a more limited effect on DNA relaxation (12,13, 15, 18). Recently, we reported t h e isolation from Zanthoxylum nitidium of t h e benzophenanthridine alkaloids nitidine, chelerythrine, and isofagaridine (19). All of these structurally related species inhibited DNA relaxation more effectively t h a n camptothecin, b u t only nitidine stabilized the covalent binary complex between toposiomerase I and DNA to a significant extent. Stabilization of the Topoisomerase I-DNA Covalent Binary Complex. I n t h e present study, we characterized t h e nature of the inhibition of topoisomerase I-mediated DNA relaxation by nitidine (1) and fagaronine (2), as well as by 0-methylfagaronine (3) whose structure is closely related t o those of 1 a n d 2 (Figure 2). As shown in Figure 3, nitidine stabilized t h e covalent topoisomerase I-DNA binary complex in a concentration-dependent fashion. T h e presence of enhanced levels of the binary complex was detectable by agarose gel electrophoresis as
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Inhibition of D N A Topoisomerase I 12
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1 nitidine
2 R = H fagaronine
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R = CH3 O-methylfagaronine
Figure 4. Effect of fagaronine on the topoisomerase I-DNA covalent binary complex: (lane 1) supercoiled pSP64 plasmid DNA alone; (lane 2) DNA + topoisomerase I; (lanes 3-20) DNA + enzyme + 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 pM fagaronine, respectively.
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camptot hecin
Figure 2. Structures of nitidine (l),fagaronine (2), O-methylfagaronine (3), and camptothecin.
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Figure 3. Effect of nitidine on the topoisomerase I-DNA covalent binary complex: (lane 1) supercoiled pSP64 plasmid DNA alone; (lane 2) DNA + 68 ng of topoisomerase I; (lanes 3-20) DNA + enzyme + 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 pM nitidine, respectively.
open circular (form 11) DNA; conversion of the binary complex to form I1 DNA was accomplished by treatment with SDS to denature the enzyme and proteinase K which effected proteolytic digestion of essentially the entire denatured protein attached to the DNA. Stabilization of the binary complex was readily apparent at nitidine concentrations as low as 0.15 p M (Figure 3, lane 17) at which 44% of the DNA migrated as form I1 DNA and 56% as form IV (relaxed circular) DNA. In comparison, 59% of the DNA migrated as form I1 in the presence of 1.2 p M nitidine (lane 14) and 71% when 9.8 p M nitidine was employed (lane 11). At higher concentrations of nitidine, the DNA not bound as an open circular complex with topoisomerase I comigrated with supercoiled (form I) plasmid DNA. While nitidine was found to inhibit topoisomerase I-mediated DNA relaxation (videinfra), under the conditions of this experiment the migration of the plasmid DNA primarily reflected binding of the nitidine to the DNA in an enzymeindependent fashion. This effect was more readily apparent for fagaronine (Figure 4) which caused pronounced alteration of DNA migration at high concentrations. Nonetheless, as is clear from the figure, fagaronine also enhanced the amount of the covalent binary complex formed between topoisomerase I and DNA and did so in a concentration-dependent fashion. Densitometry indicated, for example, that 38 and 62 % of the DNA migrated
Figure 5. Comparison of the abilities of camptothecin, nitidine and fagaronine to inhibit DNA relaxation by topoisomerase I: (lane 1)DNA alone; (lane 2) DNA + topoisomerase I; (lanes 3-7) DNA + enzyme + 55, 18, 6, 2, and 0.67 p M camptothecin, respectively; (lanes 8-12) DNA + enzyme + 55,18,6,2, and 0.67 pM nitidine, respectively; (lanes 13-17) DNA + enzyme + 55,18, 6,2, and 0.67 p M fagaronine, respectively; (lanes 18-20) DNA + 55 p M camptothecin, nitidine, and fagaronine, respectively.
as form I1 when 0.15 and 9.8 p M fagaronine, respectively, were present. For camptothecin,nitidine, and fagaronine, it was shown that omission of proteinase K from the incubation mixtures resulted in diminution of the intensity of the band that migrated as form I1 DNA and the appearance of a broad band of lesser mobility on the agarose gel, corresponding to the putative DNA-protein complex (data not shown). The changes in mobility were similar for all three compounds. Remarkably, unlike nitidine and fagaronine, O-methylfagaronine failed to stabilize the enzyme-DNA covalent binary complex to a significant extent at any tested concentration up to 2.5 mM, although it did bind to DNA at the highest concentrations tested (data not shown). Fagaronine and nitidine were found to stabilize the enzyme-DNA covalent binary complex to the same extent as camptothecin when employed at about 5-10-fold the concentration of camptothecin. Nitidine and fagaronine were also tested for their effects on calf thymus DNA topoisomerase I1 in a P4 DNA unknotting assay (37). Both compounds exhibited moderate activity, but only when tested at high concentrations; approximately 80% inhibition of DNA unknotting was achieved in the presence of 40 p M nitidine or fagaronine. In comparison, the authentic topoisomerase I1 inhibitor doxorubicin gave the same response at 2.5 p M concentration and produced a strong response at 10 p M concentration (data not shown). Inhibition of Topoisomerase I-Mediated DNA Relaxation. The abilities of nitidine and fagaronine to inhibit DNA relaxation by topoisomerase I were determined in comparison with that of camptothecin. As shown in Figure 5, both nitidine and fagaronine inhibited the relaxation of 250 ng of supercoiled pSP64 plasmid DNA in the presence of 2.2 ng of topoisomerase I. In parallel with its greater DNA affinity (cf. Figures 3 and 4), fagaroninewas more active than nitidine in inhibiting DNA relaxation, although the two compounds did not differ
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Wang et al. removal of
DNA unwinding agent linear
nicked
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Figure 6. DNA unwinding assay. HindIII-linearized DNA, prebound to a putative DNA unwinding agent, is circularized via the agency of T4 DNA ligase. Subsequent extractive removal of the test compound results in rewinding of the covalently closed circular DNA.
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Figure 7. DNA unwinding effects of nitidine and fagaronine: (lane 1) supercoiled pSP64 plasmid DNA; (lane 2) restriction endonuclease HindIII-linearized (form 111) DNA; (lanes 3-19) HindIII-linearized DNA treated with T4 DNA ligase either in the absence (lane 3) or presence (lanes 4-19) of test compounds; the test compounds were removed by extractive workup prior to gel electrophoresis (lanes 4-7, 10, 2.5, 0.63, and 0.16 p M doxorubicin, respectively; lanes 8-ll, lO, 2.5,0.63, and 0.16 pM nitidine, respectively; lanes 12-15, 10, 2.5, 0.63, and 0.16 p M fagaronine, respectively; lanes 16-19, 10,2.5,0.63, and 0.16 p M camptothecin, respectively).
dramatically in their effects. 0-Methylfagaronine was also found to inhibit relaxation, although only at much higher (167-500 pM) concentrations (data not shown). As shown in Figure 5, under these experimental conditions camptothecin did not inhibit topoisomerase I-mediated DNA relaxation. It may be noted that even the most potent inhibitors in this study, nitidine and fagaronine,were much less effective as inhibitors in this assay system than chebulagic acid (36). Further, the inhibition of topoisomerase I-mediated DNA unwinding by nitidine and fagaronine could be due predominantly to the intrinsic DNA binding capabilities of these compounds (uide infra). DNA Unwinding Studies. Also measured was DNA unwinding by nitidine and fagaronine. The assay system used was essentiallythat of Schon et al. (29),which utilized a HindIII-linearized DNA as a substrate for binding. Because each end of the linear DNA duplex has a 4-base single-stranded region, and these are complementary to each other, the doubly nicked circular DNA shown in Figure 6 should be in equilibrium with the linear DNA, if only as a minor species. Consequently, treatment of this equilibrium mixture with T4 DNA ligase effects resealing of the nicks,affordinga relaxed, covalentlyclosed circular DNA duplex. If the resealing is carried out using a linear DNA duplex already bound to some compound that has caused DNA unwinding, then subsequent removal of that agent will result in rewinding of the relaxed, closed circular DNA to afford a supercoiled species. The overall process is summarized in Figure 6. As shown in Figure 7, the known DNA intercalator doxorubicin (34,38) unwound DNA effectively at 10,2.5, and 0.63 pM concentrations. In comparison, fagaronine and nitidine effected DNA unwinding only at 10 and 2.5 pM concentrations. Consistent with the reports (13,15) that camptothecin did not bind to DNA alone,this alkaloid failed to effect DNA unwinding in the assay. Sequence Selectivity of DNA Cleavage. Topoisomerase I-mediated DNA nicks are introduced with the greatest efficiency at sites that have particular sequences in proximity to the nicks (14, 17, 18, 39-43). Some of these, in turn, are stabilized more effectively by agents
that bind to the covalent binary complexes between the enzyme and DNA. As a consequence, when a 32P-endlabeled DNA duplex is incubated with topoisomerase I and an agent capable of stabilizing the covalent enzymeDNA binary complex, subsequent treatment with SDSproteinase K produces strand breaks which give a pattern characteristic both of the enzyme and specificagent, when analyzed by polyacrylamide gel electrophoresis. The sites of cleavage produced from a linear DNA duplex by topoisomerase I in the presence of camptothecin and certain structural analogs have been described (14,17,18, 41,42). As bothnitidine (1) and fagaronine (2) were found to be capable of stabilizing the covalent binary complex formed between topoisomerase I and DNA, in analogy with camptothecin (Figures 3 and 4), it was also of interest to determine the sites of topoisomerase I-mediated DNA cleavage produced by these agents in comparison with camptothecin itself. As shown in Figure 8,this was carried out using calf thymus DNA topoisomerase I and a 5'-32P end-labeled DNA duplex 2767 base pairs in length. As is obvious from the figure, the patterns of strand breaks produced by topoisomerase I in the presence of nitidine and fagaronine were remarkably similar to those obtained with camptothecin, although each of these agents stabilized some sites particularly well in comparison with the other two. Topoisomerase I as a Locus for Expression of Antitumor Activity. That topoisomerase inhibitors can actually exert their cytotoxic effects as a consequence of their interaction with topoisomerase and DNA has been established convincingly for some agents. A particularly good example is that of camptothecin, which binds neither to DNA nor to topoisomerase I alone (13,15),but strongly stabilizes the covalent complex between the enzyme and DNA, thereby creating persistent "lesions" in the DNA which are believed to lead to its observed cytotoxic effects. In support of this thesis, it has been shown that yeast strains lacking the gene for topoisomerase I were resistant to inhibition by camptothecin (43, 44), suggesting that the presence of the enzyme is a prerequisite for expression of camptothecin-mediated cytotoxicity. It has also been shown that two mammalian cell lines resistant to camptothecin contained topoisomerase I of altered sequence and that these modified enzymes formed covalent binary complexes with DNA that were not stabilized by camptothecin (28, 45). Finally, it has been reported that camptothecin analogs that fail to inhibit topoisomerase I also lack antitumor activity (12, 25). The benzophenanthridine alkaloidshave been of interest for many years, due in no small measure to their antitumor activities in vitro and in animal tumor models (46). More than 80 naturally occurring alkaloids in this class have been isolated from plants and many more have been prepared by chemical synthesis (see, for example, refs 4654). The present findings, i.e. that both fagaronine and nitidine inhibited topoisomerase I-mediated DNA relaxation, stabilized the covalent topoisomerase I-DNA binary
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Inhibition of DNA Topoisomeruse I
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and failed to stabilize the topoisomerase I-DNA covalent complex at any tested concentration (videsupra). This observation is reminiscent of the findings made for chelerythrine and isofagaridine (19). Both of these benzophenanthridine alkaloids inhibited topoisomerase I-catalyzed DNA relaxation as effectively as nitidine, but chelerythrine was unable to stabilize the topoisomerase I-DNA covalent complex and isofagaridine did so only weakly. In this context it is worth noting that the only benzophenanthridine alkaloids found thus far to stabilize the topoisomeraseI-DNA binary complex are those which have previously been shown to have antitumor activity in experimental animal models. The substantial differences in the spectrum of biochemical activities for compounds whose chemical structures are closely related is remarkable and provides an important tool to study the biochemical loci at which the antitumor effects of the compounds are mediated.
Acknowledgment. We thank Dr. Matthew Suffness, National Cancer Institute, for authentic samples of 1-3 and Dr. Brian Rogers, University of Virginia, for helpful discussions during the course of this work. This work was supported by NIH Research grant CA50771 from the National Cancer Institute.
References
Figure 8. Effects of camptothecin, nitidine, and fagaronine on topoisomerase I-mediated cleavage of a 5'-32Pend-labeled DNA duplex: (lane 1)DNA + 50 p M fagaronine; (lane 2) DNA + 30 p M nitidine; (lane 3) DNA + 10 p M camptothecin; (lane 4)DNA alone; (lane 5) DNA + 340 ng of calf thymus topoisomerase I; (lane 6) DNA + topoisomerase I + 10 p M camptothecin; (lane 7) DNA + topoisomerase I + 30 p M nitidine; (lane 8) DNA + topoisomerase I + 50 p M fagaronine.
complex, and induced topoisomerase I-dependent DNA strand breaks at the same sequences as camptothecin is intriguing and suggests that these compounds could well exert their cytotoxic effects at the locus of topoisomerase I. It should be noted, however, that both nitidine and fagaronine have been reported to exhibit other biochemical effects not reported for camptothecin. These include an intrinsic DNA binding capability (55) and an ability to inhibit other enzymes such as DNA and RNA polymerases (56),reverse transcriptases (56-58), and topoisomerase I1 (vide supra), although the enzyme inhibitory effects obtained only at concentrations 100-fold greater than those required to produce inhibition of topoisomerase I function. Fagaronine has also been reported to inhibit nucleic acid and protein synthesis (59),althoughthe effects noted also occurred at higher concentrations than the effects on topoisomerase I described here. While the actual molecular locus or loci at which nitidine and fagaronine exert their antitumor effects remains to be established, one facet of the present work may be mentioned in this context. Unlike nitidine and fagaronine, which were found to inhibit topoisomerase I-mediated DNA relaxation and stabilize the covalent enzymeDNA complex, the closely related species O-methylfagaronine (3)was only weakly active as an inhibitor of DNA relaxation
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