Mechanism of action of ferrocene derivatives on the catalytic activity of topoisomerase IIalpha and beta--distinct mode of action of two derivatives

Topoisomerase II is found to be present in two isoforms alpha and beta, and both the isoforms are regulated in cancerous tissue. Development of isoform-specific topoisomerase II poisons has been of great interest for cancer-specific drug targeting. In the present investigation using quantitative structure-activity analysis of ferrocene derivatives, we show that two derivatives of ferrocene, azalactone ferrocene and thiomorpholide amido methyl ferrocene, can preferentially inhibit topoisomerase IIbeta activity. Thiomorpholide amido methyl ferrocene shows higher inhibition of catalytic activity (IC(50) = 50 microM) against topoisomerase IIbeta compared to azalactone ferrocene (IC(50) = 100 microM). The analysis of protein DNA intermediates formed in the presence of these two compounds suggests that azalactone ferrocene readily induces formation of cleavable complex in a dose-dependent manner, in comparison with thiomorpholide amido methyl ferrocene. Both the compounds show significant inhibition of DNA-dependent ATPase activity of enzyme. These results suggest that azalactone ferrocene inhibits DNA passage activity of enzyme leading to the formation of cleavable complex, while thiomorpholide amido methyl ferrocene competes with ATP binding resulting in the inhibition of catalytic activity of enzyme. In summary, thiomorpholide amido methyl ferrocene and azalactone ferrocene show distinctly different mechanisms in inhibition of catalytic activity of topoisomerase IIbeta.     

Synthesis, cytotoxicity, and DNA topoisomerase II inhibitory activity of benzofuroquinolinediones

Benzofuroquinolinediones (7c and 7d) were synthesized by base-catalyzed condensation of dichloroquinolinediones with phenolic derivatives. Their dialkylaminoalkoxy derivatives (8i-8p) were prepared by reaction with various dialkylaminoalkyl chlorides. The cytotoxicity of the synthesized compounds was evaluated against eight types of human cancer cell lines, and their topoisomerase II inhibition was assessed. In general, the cytotoxicity of benzofuroquinolinediones (8i-8p) was similar or superior to that of doxorubicin and showed more potent inhibitory activity than naphthofurandiones (8a-8h). Also, most of the compounds exhibited excellent topoisomerase II inhibitory activity at a concentration of 5 microM and two compounds, 8d and 8i, showed IC50 values of 1.19 and 0.68 microM, respectively, and were much more potent than etoposide (IC50=78.4 microM), but similar to doxorubicin (IC50=2.67 microM). However their inhibitory activity on topoisomerase I was lower, and 8d and 8i showed IC50 values of 42.0 and 64.3 microM, respectively.     

An insight into the mechanism of inhibition of unusual bi-subunit topoisomerase I from Leishmania donovani by 3,3'-di-indolylmethane, a novel DNA topoisomerase I poison with a strong binding affinity to the enzyme

DIM (3,3'-di-indolylmethane), an abundant dietary component of cruciferous vegetables, exhibits a wide spectrum of pharmacological properties. In the present study, we show that DIM is a potent inhibitor of Leishmania donovani topoisomerase I with an IC50 of 1.2 microM. Equilibrium dialysis shows that DIM binds strongly to the free enzyme with a binding constant of 9.73x10(-9) M. The binding affinity of DIM to the small subunit is 8.6-fold more than that of the large subunit of unusual LdTOP1LS (bi-subunit L. donovani topoisomerase I). DIM stabilizes topoisomerase I-DNA cleavage complexes in vitro and also in vivo. Like CPT (camptothecin), DIM inhibits the religation step when the drug was added to preformed topoisomerase I-DNA binary complex. Hence, DIM is similar to CPT with respect to its ability to form the topoisomerase I-mediated 'cleavable complexes' in vitro and in vivo. But unlike CPT, DIM interacts with both free enzyme and substrate DNA. Therefore DIM is a non-competitive class I inhibitor of topoisomerase I. DIM also inhibits the relaxation activity of the CPT-resistant mutant enzyme LdTOP1Delta39LS (N-terminal deletion of amino acids 1-39 of LdTOP1LS). The IC50 values of DIM in simultaneous and enzyme pre-incubation relaxation assays were 3.6 and 2.9 muM respectively, which are higher than that of wild-type topoisomerase I (LdTOP1LS), indicating that the affinity of DIM to LdTOP1Delta39LS is less than that for LdTOP1LS. This is the first report on DIM as an L. donovani topoisomerase I poison. Our study illuminates a new mode of action of enzyme inhibition by DIM that might be exploited for rational drug design in human leishmaniasis.     

Some benzoxazoles and benzimidazoles as DNA topoisomerase I and II inhibitors

Some novel fused heterocyclic compounds of 2, 5-disubstituted-benzoxazole and benzimidazole derivatives, which were previously synthesized by our group, were investigated for their inhibitory activity on both eukaryotic DNA topoisomerase I and II in a cell free system. 2-Phenoxymethylbenzimidazole (17), 5-amino-2-(p-fluorophenyl)benzoxazole (3), 5-amino-2-(p-bromophenyl)benzoxazole (5), 5-nitro-2-phenoxymethyl-benzimidazole (18), 2-(p-chlorobenzyl)benzoxazole (10) and 5-amino-2-phenylbenzoxazole (2) were found to be more potent as eukaryotic DNA topoisomerase I poisons than the reference drug camptothecin having IC(50) values of 14.1, 132.3, 134.1, 248, 443.5, and 495 microM, respectively. 5-Chloro-2-(p-methylphenyl)benzoxazole (4), 2-(p-nitrobenzyl)benzoxazole (6) and 5-nitro-2-(p-nitrobenzyl)benzoxazole (8) exhibited significant activity as eukaryotic DNA topoisomerase II inhibitors, having IC(50) values of 22.3, 17.4, 91.41 microM, respectively, showing higher potency than the reference drug etoposide.     

DNA topoisomerases as targets for the anticancer drug TAS-103: DNA interactions and topoisomerase catalytic inhibition

TAS-103 is a novel anticancer drug that kills cells by increasing levels of DNA cleavage mediated by topoisomerase II. While most drugs that stimulate topoisomerase II-mediated DNA scission (i.e., topoisomerase II poisons) also inhibit the catalytic activity of the enzyme, they typically do so only at concentrations above the clinical range. TAS-103 is unusual in that it reportedly inhibits the catalytic activity of both topoisomerase I and II and does so at physiologically relevant concentrations [Utsugi, T., et al. (1997) Jpn. J. Cancer Res. 88, 992-1002]. Without a topoisomerase activity to relieve accumulating torsional stress, the DNA tracking systems that promote the action of TAS-103 as a topoisomerase II poison would be undermined. Therefore, the effects of TAS-103 on the catalytic activity of topoisomerase I and II were characterized. DNA binding and unwinding assays indicate that the drug intercalates into DNA with an apparent dissociation constant of approximately 2.2 microM. Furthermore, DNA strand passage assays with mammalian topoisomerase I indicate that TAS-103 does not inhibit the catalytic activity of the type I enzyme. Rather, the previously reported inhibition of topoisomerase I-catalyzed DNA relaxation results from a drug-induced alteration in the apparent topology of the nucleic acid substrate. TAS-103 does inhibit the catalytic activity of human topoisomerase IIalpha, apparently by blocking the DNA religation reaction of the enzyme. The lack of inhibition of topoisomerase I catalytic activity by TAS-103 explains how the drug is able to function as a topoisomerase II poison in treated cells.     

Solid phase combinatorial synthesis of benzothiazoles and evaluation of topoisomerase II inhibitory activity

To investigate one possible mechanism of action of the cytotoxic activity of benzothiazoles, we synthesized 2-(substituted-phenyl)benzothiazoles and evaluated their ability to inhibit topoisomerase II activities. Solid phase combinatorial method using trityl resin was employed and benzothiazole derivatives with various substitution on 2'-, 3'-, or 4'-position of phenyl group were obtained in ca. 30 mg scale (7-96% yield). Most of the compounds synthesized exhibited topoisomerase II inhibitory activity at 100 microM. 2-(3-Amino-4-methylphenyl)benzothiazole showed high activity (IC(50) = 71.7 microM), comparable to etoposide (IC(50) = 78.4 microM).     

Human cells express two differentially spliced forms of topoisomerase II beta mRNA.

Screening of a human B-cell cDNA library with a topoisomerase II beta gene-specific probe revealed the presence of two distinct forms of topoisomerase II beta cDNA. One form (designated topoisomerase II beta-1), representing the majority of the clones, would encode the topoisomerase II beta amino acid sequence reported recently [Jenkins, J.R. et al. (1992) Nucleic Acids Res., 20, 5587-5592]. The second form (designated topoisomerase II beta-2) would encode a protein containing an additional 5 amino acids inserted after Valine-23 of the topoisomerase II beta-1 protein sequence. The topoisomerase II beta-1 and beta-2 mRNAs were both widely expressed in human cell lines and tissues. Topoisomerase II beta-2 mRNA was expressed at a lower level than that of the beta-1 form, but the relative expression of the two forms varied in different cell types. Analysis of genomic DNA clones revealed that the two forms of topoisomerase II beta mRNA arose via differential splicing. These data indicate that in addition to the closely related topoisomerase II alpha and beta isozymes, there are two forms of topoisomerase II beta mRNA widely expressed in human cells.     

SUMO-1 conjugation to human DNA topoisomerase II isozymes

Topoisomerase I-mediated DNA damage induced by camptothecin has been shown to induce rapid small ubiquitin-related modifier (SUMO)-1 conjugation to topoisomerase I. In the current study, we show that topoisomerase II-mediated DNA damage induced by teniposide (VM-26) results in the formation of high molecular weight conjugates of both topoisomerase IIalpha and IIbeta isozymes in HeLa cells. Immunological characterization of these conjugates suggests that both topoisomerase IIalpha and IIbeta isozymes are conjugated to SUMO-1. The involvement of SUMO-1/UBC9 in the modification of topoisomerase II isozymes is also supported by the demonstration of physical interaction between topoisomerase II and SUMO-1/UBC9. Surprisingly, ICRF-193, which does not induce topoisomerase II-mediated DNA damage but traps topoisomerase II into a circular clamp conformation, is also shown to induce similar SUMO-1 conjugation to topoisomerase II isozymes. In addition, we show that both oxidative and heat shock stresses, which can cause protein damage, rapidly increase nuclear SUMO-1 conjugates. These studies raise the question on whether SUMO-1 conjugation to topoisomerases is an indirect result of a DNA damage response or a direct result because of protein conformational changes.     

Inhibitory activities against topoisomerase I and II by isoaurostatin derivatives and their structure-activity relationships

Isoaurostatin A (IAS-A) isolated from Thermomonospora alba showed weak inhibition against topoisomerase (topo) I (IC(50)=307microM). To get more strong inhibition, derivatives of IAS-A were prepared and their structure-activity relationships against topo I and II were investigated. The addition of hydroxyl group on aromatic rings increased the activities, 3-(3,4,5-trihydroxybenzylidene)-5-hydroxy-3H-benzofuran-2-one (IAS-9) showed strong inhibition (IC(50)=3microM) against topo I. And also, the increasing of hydroxyl group increased growth inhibition against a variety of cancer cells, and IAS-9 showed most potent inhibition. Unlike camptothecin and etoposide, IAS-9 neither stabilized DNA-topo cleavable complex nor intercalated into DNA, and it inhibited topo I and II noncompetitively. The inhibitory activities also increased by opening of lactone ring in the molecule of IAS-9.     

Vaccinia virus DNA ligase recruits cellular topoisomerase II to sites of viral replication and assembly

Vaccinia virus replication is inhibited by etoposide and mitoxantrone even though poxviruses do not encode the type II topoisomerases that are the specific targets of these drugs. Furthermore, one can isolate drug-resistant virus carrying mutations in the viral DNA ligase and yet the ligase is not known to exhibit sensitivity to these drugs. A yeast two-hybrid screen was used to search for proteins binding to vaccinia ligase, and one of the nine proteins identified comprised a portion (residue 901 to end) of human topoisomerase IIbeta. One can prevent the interaction by introducing a C(11)-to-Y substitution mutation into the N terminus of the ligase bait protein, which is one of the mutations conferring etoposide and mitoxantrone resistance. Coimmunoprecipitation methods showed that the native ligase and a Flag-tagged recombinant protein form complexes with human topoisomerase IIalpha/beta in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase IIalpha and IIbeta antigens are recruited to cytoplasmic sites of virus replication and that less topoisomerase was recruited to these sites in cells infected with mutant virus than in cells infected with wild-type virus. Immunoelectron microscopy confirmed the presence of topoisomerases IIalpha/beta in virosomes, but the enzyme could not be detected in mature virus particles. We propose that the genetics of etoposide and mitoxantrone resistance can be explained by vaccinia ligase binding to cellular topoisomerase II and recruiting this nuclear enzyme to sites of virus biogenesis. Although other nuclear DNA binding proteins have been detected in virosomes, this appears to be the first demonstration of an enzyme being selectively recruited to sites of poxvirus DNA synthesis and assembly.     

Curcumin as a DNA topoisomerase II poison

Curcumin, the major active component of the spice turmeric, is recognised as a safe compound with great potential for cancer chemoprevention and cancer therapy. It induces apoptosis, but its initiation mechanism remains poorly understood. Curcumin has been assessed on the human cancer cell lines, TK-10, MCF-7 and UACC-62, and their IC50 values were 12.16, 3.63, 4.28 microM respectively. The possibility of this compound being a topoisomerase II poison has also been studied and it was found that 50 microM of curcumin is active in a similar fashion to the antineoplastic agent etoposide. These results point to DNA damage induced by topoisomerase II poisoning as a possible mechanism by which curcumin initiates apoptosis, and increase the evidence suggesting its possible use in cancer therapy.     

The SUMO pathway is required for selective degradation of DNA topoisomerase IIbeta induced by a catalytic inhibitor ICRF-193(1)

DNA topoisomerase I and II have been shown to be modified with a ubiquitin-like protein SUMO in response to their specific inhibitors called 'poisons'. These drugs also damage DNA by stabilizing the enzyme-DNA cleavable complex and induce a degradation of the enzymes through the 26S proteasome system. A plausible link between sumoylation and degradation has not yet been elucidated. We demonstrate here that topoisomerase IIbeta, but not its isoform IIalpha, is selectively degraded through proteasome by exposure to the catalytic inhibitor ICRF-193 which does not damage DNA. The beta isoform immunoprecipitated from ICRF-treated cells was modified by multiple modifiers, SUMO-2/3, SUMO-1, and polyubiquitin. When the SUMO conjugating enzyme Ubc9 was conditionally knocked out, the ICRF-induced degradation of topoisomerase IIbeta did not occur, suggesting that the SUMO modification pathway is essential for the degradation.     

Some fused heterocyclic compounds as eukaryotic topoisomerase II inhibitors

Our previously synthesized 37 compounds, which are 2,5,6-substituted benzoxazole, benzimidazole, benzothiazole, and oxazolo(4,5-b)pyridine derivatives, were tested for their eukaryotic DNA topoisomerase II inhibitory activity in cell free system and 28 were found to inhibit the topoisomerase II at an initial concentration of 100 microg/ml. After further testing at a lower range of concentrations, 12 derivatives, which were considered as positive topoisomerase inhibitors, exhibited IC50 values between 11.4 and 46.8 microM. Etoposide was used as the standard reference drug to compare the inhibitor activity. Among these compounds, 2-phenoxymethylbenzothiazole (3f), 6-nitro-2-(2-methoxyphenyl)benzoxazole (1a), 5-methylcarboxylate-2-phenylthiomethylbenzimidazole (3c), and 6-methyl-2-(2-nitrophenyl)benzoxazole (1c) were found to be more active than the reference drug etoposide. Present results point out that, besides the very well-known bi- and ter-benzimidazoles, compounds with single bicycle fused ring systems in their structure such as benzimidazole, benzoxazole, benzothiazole, and/or oxazolopyridine derivatives also exhibit significant topoisomerase II inhibitory activity.     

DNA topoisomerase II as the target for the anticancer drug TOP-53: mechanistic basis for drug action

TOP-53 is a promising anticancer agent that displays high activity against non-small cell lung cancer in animal tumor models [Utsugi, T., et al. (1996) Cancer Res. 56, 2809-2814]. Compared to its parent compound, etoposide, TOP-53 is considerably more toxic to non-small cell lung cancer cells, is more active at generating chromosomal breaks, and displays improved cellular uptake and pharmacokinetics in animal lung tissues. Despite the preclinical success of TOP-53, several questions remain regarding its cytotoxic mechanism. Therefore, this study characterized the basis for drug action. Results indicate that topoisomerase II is the primary cytotoxic target for TOP-53. Furthermore, the drug kills cells by acting as a topoisomerase II poison. TOP-53 exhibits a DNA cleavage site specificity that is identical to that of etoposide. Like its parent compound, the drug increases the number of enzyme-mediated DNA breaks by interfering with the DNA religation activity of the enzyme. TOP-53 is considerably more efficient than etoposide at enhancing topoisomerase II-mediated DNA cleavage and exhibits high activity against human topoisomerase IIalpha and IIbeta in vitro and in cultured cells. Therefore, at least in part, the enhanced cytotoxic activity of TOP-53 can be attributed to an enhanced activity against topoisomerase II. Finally, TOP-53 displays nearly wild-type activity against a mutant yeast type II enzyme that is highly resistant to etoposide. This finding suggests that TOP-53 can retain activity against systems that have developed resistance to etoposide, and indicates that substituents on the etoposide C-ring are important for topoisomerase II-drug interactions.     

Cytotoxicity and DNA topoisomerase inhibitory activity of benz[f]indole-4,9-dione analogs

A series of benz[f]indole-4,9-diones, based on the antitumor activity of 1,4-naphthoquinone, were synthesized and evaluated for their cytotoxic activity in cultured human cancer cell lines A549 (lung cancer), Col2 (colon cancer), and SNU-638 (stomach cancer), and also for the inhibition of human DNA topoisomerases I and II activity in vitro. Several compounds including 2-amino-3-ethoxycarbonyl-N-methyl-benz[f]indole-4,9-dione showed a potential cytotoxic activity judged by IC50<20.0 microg/ml in the panel of cancer cell lines. Especially, 2-hydroxy-3-ethoxycarbonyl-N-(3,4-dimethylphenyl)-benz[f]indole-4,9-dione had potential selective cytotoxicity against lung cancer cells (IC50=0.4 microg/ml)) compared to colon (IC50>20.0 microg/ml) and stomach (IC50>20.0 microg/ml) cancer cells. To further investigate the cytotoxic mechanism, the effects of test compounds on DNA topoisomerase I and II activities were used. In a topoisomerase I-mediated relaxation assay using human placenta DNA topoisomerase I and supercoiled pHOTI plasmid DNA, 2-amino-3-ethoxycarbonyl-N-(4-fluorophenyl)-benz[f]indole-4,9-dione had the most potent inhibitory activity among the compounds tested. However, most of the compounds showed only weak inhibition of the DNA topoisomerase II-mediated KDNA (Kinetoplast DNA) decatenation assay, except for 2-amino-3-ethoxycarbonyl-N-(4-methylphenyl)-benz[f]indole-4,9-dione and 2-amino-3-ethoxycarbonyl-N-(2-bromoehtyl)-benz[f]indole-4,9-dione with a moderate inhibitory activity. These results suggest that several active compounds had relatively selective inhibitory activity against toposiomearse I compared to toposiomerase II. No obvious correlation was observed between the cytotoxicity of the individual compound and the inhibitory activity of DNA relaxation and decatenation by topoisomerase I and II, respectively, in vitro.     

N-acetyl-p-benzoquinone imine, the toxic metabolite of acetaminophen, is a topoisomerase II poison

Although acetaminophen is the most widely used analgesic in the world, it is also a leading cause of toxic drug overdoses. Beyond normal therapeutic doses, the drug is hepatotoxic and genotoxic. All of the harmful effects of acetaminophen have been attributed to the production of its toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Since many of the cytotoxic/genotoxic events triggered by NAPQI are consistent with the actions of topoisomerase II-targeted drugs, the effects of this metabolite on human topoisomerase IIalpha were examined. NAPQI was a strong topoisomerase II poison and increased levels of enzyme-mediated DNA cleavage >5-fold at 100 microM. The compound induced scission at a number of DNA sites that were similar to those observed in the presence of the topoisomerase II-targeted anticancer drug etoposide; however, the relative site utilization differed. NAPQI strongly impaired the ability of topoisomerase IIalpha to reseal cleaved DNA molecules, suggesting that inhibition of DNA religation is the primary mechanism underlying cleavage enhancement. In addition to its effects in purified systems, NAPQI appeared to increase levels of DNA scission mediated by human topoisomerase IIalpha in cultured CEM leukemia cells. In contrast, acetaminophen did not significantly affect the DNA cleavage activity of the human enzyme in vitro or in cultured CEM cells. Furthermore, the analgesic did not interfere with the actions of etoposide against the type II enzyme. These results suggest that at least some of the cytotoxic/genotoxic effects caused by acetaminophen overdose may be mediated by the actions of NAPQI as a topoisomerase II poison.     

Synthesis of 6-chloroisoquinoline-5,8-diones and pyrido[3,4-b]phenazine-5,12-diones and evaluation of their cytotoxicity and DNA topoisomerase II inhibitory activity

The substituted chloroisoquinolinediones and pyrido[3,4-b]phenazinediones were synthesized, and the cytotoxic activity and topoisomerase II inhibitory activity of the prepared compounds were evaluated. Chloroisoquinolinediones have been prepared by the reported method employing 6,7-dichloroisoquinoline-5,8-dione. The cyclization to pyrido[3,4-b]phenazinediones was achieved by adding the aqueous sodium azide solution to the dimethylformamide solution of corresponding chloroisoquinoline-5,8-dione. The cytotoxicity of the synthesized compounds was evaluated by a SRB (Sulforhodamine B) assay against various cancer cell lines such as A549 (human lung cancer cell line), SNU-638 (human stomach cancer cell), Col2 (human colon cancer cell line), HT1080 (human fibrosarcoma cell line), and HL-60 (human leukemia cell line). Almost all the synthesized pyrido[3,4-b]phenazinediones showed greater cytotoxic potential than ellipticine (IC(50)=1.82-5.97 microM). In general, the cytotoxicity of the pyrido[3,4-b]phenazinediones was higher than that of the corresponding chloroisoquinolinediones. The caco-2 cell permeability of selected compounds was 0.62 x 10(-6)-35.3 x 10(-6)cm/s. The difference in cytotoxic activity among tested compounds was correlated with the difference in permeability to some degree. To further investigate the cytotoxic mechanism, the topoisomerase II inhibitory activity of the synthesized compounds was estimated by a plasmid cleavage assay. Most of compounds showed the topoisomerase II inhibitory activity (28-100%) at 200 microM. IC(50) values for the most active compound 6a were 0.082 microM. However, the compounds were inactive for DNA relaxation by topoisomerase I at 200 microM.