Heterocyclic bibenzimidazole derivatives as topoisomerase I inhibitors

A series of 2'-heterocyclic derivatives of 5-phenyl-2,5'-1H-bibenzimidazoles were evaluated for topoisomerase I poisoning activity and cytotoxicity. Topo I poisoning activity was associated with 2'-derivatives that possessed a hydrogen atom capable of hydrogen bond formation, suggesting that the interatomic distances between such hydrogen atoms and the heteroatoms on the adjacent benzimidazole influence activity.     

New benzoxanthone derivatives as topoisomerase inhibitors and DNA cross-linkers

We synthesized 12 benzoxanthone derivatives classified as three different groups based on the tetracyclic ring shapes and evaluated their pharmacological activities to find potential anticancer agents. In the cytotoxicity test, most compounds showed effective cancer cell growth inhibition against the HT29 and DU145 cell lines. Among the compounds tested, compound 19 was the most effective in the cancer cell lines tested. Compound 9 showed dual inhibitory activities against DNA relaxation by topoisomerases I and II. The% inhibition of compound 9 on topoisomerase I was comparable to that of camptothecin. Compound 9 efficiently blocked topoisomerase II function by almost threefold than etoposide at 20 μM. Compound 19 had selective topoisomerase II inhibitory activity at 100 μM. The DNA cross-linking test revealed that only compounds 8 and 19, which possess epoxy groups, cross-linked DNA duplex, while 14 did not. From the combined pharmacological results, we proposed that the target through which compound 19 inhibits cancer cell growth may be the DNA duplex itself and/or DNA–topoisomerase II complex.     

Induction of mammalian DNA topoisomerase I mediated DNA cleavage by antitumor indolocarbazole derivatives.

DNA topoisomerases have been shown to be important therapeutic targets in cancer chemotherapy. We found that KT6006 and KT6528, synthetic antitumor derivatives of indolocarbazole antibiotic K252a, were potent inducers of a cleavable complex with topoisomerase I. In DNA cleavage assay using purified calf thymus DNA topoisomerase I and supercoiled pBR322 DNA, KT6006 induced topoisomerase I mediated DNA cleavage in a dose-dependent manner at drug concentrations up to 50 microM, while DNA cleavage induced by KT6528 was saturated at 5 microM. The maximal amount of nicked DNA produced by KT6006 was more than 50% of substrate DNA, which was comparable to that of camptothecin. Heat treatment (65 degrees C) of the reaction mixture containing these compounds and topoisomerase I resulted in a substantial reduction in DNA cleavage, suggesting that topoisomerase I mediated DNA cleavage induced by KT6006 and KT6528 is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex". Both KT6006 and KT6528 did not induce topoisomerase II mediated DNA cleavage in vitro. KT6006 and KT6528 were found to induce nearly identical topoisomerase I mediated DNA cleavage patterns, which was distinctly different from that with camptothecin. In contrast to the similarity between KT6006 and KT6528 in their structures and the nature of their cleavable complex with topoisomerase I, these drugs have different properties with respect to their interaction with DNA: KT6006 is a very weak intercalator whereas KT6528 is a strong intercalator with potentials comparable to that of adriamycin. These results indicate that KT6006 and KT6528 represent a new distinct class of mammalian DNA topoisomerase I active antitumor drugs.     

Structural analysis of catechin derivatives as mammalian DNA polymerase inhibitors

The inhibitory activities against DNA polymerases (pols) of catechin derivatives (i.e., flavan-3-ols) such as (+)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin, (+)-catechin gallate, (-)-epicatechin gallate, (-)-gallocatechin gallate, and (-)-epigallocatechin gallate (EGCg) were investigated. Among the eight catechins, some catechins inhibited mammalian pols, with EGCg being the strongest inhibitor of pol alpha and lambda with IC(50) values of 5.1 and 3.8 microM, respectively. EGCg did not influence the activities of plant (cauliflower) pol alpha and beta or prokaryotic pols, and further had no effect on the activities of DNA metabolic enzymes such as calf terminal deoxynucleotidyl transferase, T7 RNA polymerase, and bovine deoxyribonuclease I. EGCg-induced inhibition of pol alpha and lambda was competitive with respect to the DNA template-primer and non-competitive with respect to the dNTP (2'-deoxyribonucleotide 5'-triphosphate) substrate. Tea catechins also suppressed TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation, and the tendency of the pol inhibitory activity was the same as that of anti-inflammation. EGCg at 250 microg was the strongest suppressor of inflammation (65.6% inhibition) among the compounds tested. The relationship between the structure of tea catechins and the inhibition of mammalian pols and inflammation was discussed.     

Novel quinoline derivatives as inhibitors of bacterial DNA gyrase and topoisomerase IV

A structurally novel set of inhibitors of bacterial type II topoisomerases with potent in vitro and in vivo antibacterial activity was developed. Dual-targeting ability, hERG inhibition, and pharmacokinetic properties were also assessed.     

DNA topoisomerase I inhibitors from Rinorea anguifera

An organic extract prepared from Rinorea anguifera was investigated in order to identify the natural principle(s) responsible for stabilization of a topoisomerase I-DNA covalent binary complex. Bioassay-guided fractionation resulted in the isolation of mauritianin and (+)-syringaresinol as new topoisomerase I inhibitors, and also of the known inhibitor camptothecin.     

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.     

Monoclonal antibodies neutralizing mammalian DNA topoisomerase I activity

We have isolated three different monoclonal antibodies specific for mammalian type-I DNA topoisomerase. The antibodies react with three closely adjacent epitopes located in a central section of the enzyme (between amino acid residues 344 and 483). Two of the antibodies inhibit an early step of the nicking/closing pathway. We provide evidence showing that the antibodies do not block the association of the enzyme with DNA. The antibodies are useful for immunocytochemical investigation and for further exploration of the biochemical function of mammalian type-I DNA topoisomerase.     

Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I

Camptothecin, a cytotoxic drug, is a strong inhibitor of nucleic acid synthesis in mammalian cells and a potent inducer of strand breaks in chromosomal DNA. Neither the equilibrium dialysis nor the unwinding measurement indicates any interaction between camptothecin and purified DNA. However, camptothecin induces extensive single strand DNA breaks in reactions containing purified mammalian DNA topoisomerase I. DNA breakage in vitro is immediate and reversible. Analyses of camptothecin-induced DNA breaks show that topoisomerase I is covalently linked to the 3' end of the broken DNA. In addition, camptothecin inhibits the catalytic activity of mammalian DNA topoisomerase I. We propose that camptothecin blocks the rejoining step of the breakage-reunion reaction of mammalian DNA topoisomerase I. This blockage results in the accumulation of a cleavable complex which resembles the transient intermediate proposed for eukaryotic DNA topoisomerase I. The inhibition of nucleic acid synthesis and the induction of DNA strand breaks observed in vivo may be related to the formation of this drug-induced cleavable complex.     

Natural inhibitors of DNA topoisomerase I with cytotoxicities

DNA Topoisomerase I can cause DNA breaks and play a key role during cell proliferation and differentiation. It is an important target for anticancer agents. While screening for anticancer compounds, seven natural compounds, 1-7, showed potent cytotoxicities against a panel of ten cancer cell lines. Moreover, an inhibition assay demonstrated that they are also DNA topoisomerase I inhibitors, in which inhibitors 1-5 are new ones.     

3-Arylisoquinolines as novel topoisomerase I inhibitors

Topoisomerase I (topo I) is an essential enzyme for vital cellular processes. Inhibition of topo I activities is lethal and leads to cell death, thus establishing topo I as a promising target for cancer treatment. Camptothecin, a natural alkaloid, inhibits topo I. Topotecan and irinotecan, synthetic derivatives of camptothecin, are the most potent anticancer drugs in clinical use. However, several limitations of camptothecins such as solubility, toxicity, stability, resistance and the required high drug dose have encouraged the development of non-camptothecin topo I inhibitors. Natural alkaloid benzo[c]phenanthridines and synthetic indenoisoquinolines have been extensively studied as alternatives to camptothecin. Interestingly, these non-camptothecin topo I inhibitors share a common 3-arylisoquinoline scaffold. This review will describe the development of novel indeno[1,2-c]isoquinolines, isoindolo[2,1-b]isoquinolines, 12-oxobenzo[c]phenanthridines and benz[b]oxepines with a 3-arylisoquinoline nucleus as topo I inhibitors.     

Quadruplex-duplex motifs as new topoisomerase I inhibitors

In this article, 13 short chains that can form G-quadruplex and quadruplex-duplex motif have been designed. Fourteen oligonucleotides, including 13 short chains as well as a reference short chain all have certain level of inhibition to topoisomerase I, whether or not they form G-quadruplex and quadruplex-duplex motif, and the G-quadruplex and quadruplex-duplex motif show better activity than single short chain. The result confirmed that after forming G-quadruplex and quadruplex-duplex motif these 14 oligonucleotides are competitive inhibition, that is, through the priority binding with the topoisomerase I and precluding from its binding with the normal substrate pBR322 and, therefore, inhibiting the next reaction.     

Nick-containing oligonucleotides as human topoisomerase I inhibitors

A series of oligonucleotides with various lengths that contain nick and topoisomerase I-binding sites were designed. The interactions between these oligonucleotides and human topoisomerase I were investigated and the most efficient one among them has displayed IC₅₀ value of 6.3 nM. Our studies have also demonstrated that the position of the nick as well as the length of the oligonucleotides were crucial factors for the inhibition of this nuclear enzyme.     

Flavonoids as DNA topoisomerase I poisons

The therapeutic anticancer potential of flavonoids shown by recent research needs a greater understanding of these compounds. They are antioxidants and antimutagenic agents that can inhibit tumor promotion and transformation and can modify the activity of a large number of mammalian enzyme systems, such as human DNA-topoisomerases. Poisons of topoisomerases generate toxic DNA damage by stabilization of the covalent DNA-topoisomerase cleavage complex and some of them have therapeutic efficacy in human cancer. The present investigation has assayed ten flavonoids, isolated in our laboratory, as topoisomerase I poisons obtaining myricetin and myricetin-3-galactoside as two new topoiosomerase I poisons. These two flavonoids, and the plant extract from which they were isolated, were assayed for cytotoxic activity against three human cancer cell lines using the SRB assay. Taking into account our previous research, structural requisites implicated in the topoisomerase poisoning are discussed.     

Iridoids as DNA topoisomerase I poisons

The discovery of new topoisomerase I inhibitors is necessary since most of the antitumor drugs are targeted against type II and only a very few can specifically affect type I. Topoisomerase poisons generate toxic DNA damage by stabilization of the covalent DNA-topoisomerase cleavage complex and some have therapeutic efficacy in human cancer. Two iridoids, aucubin and geniposide, have shown antitumoral activities, but their activity against topoisomerase enzymes has not been tested. Here it was found that both compounds are able to stabilize covalent attachments of the topoisomerase I subunits to DNA at sites of DNA strand breaks, generating cleavage complexes intermediates so being active as poisons of topoisomerase I, but not topoisomerase II. This result points to DNA damage induced by topoisomerase I poisoning as one of the possible mechanisms by which these two iridoids have shown antitumoral activity, increasing interest in their possible use in cancer chemoprevention and therapy.     

Acidic phospholipids directly inhibit DNA binding of mammalian DNA topoisomerase I

Inhibition of mammalian DNA topoisomerase I by phospholipids was investigated using purified enzyme. Acidic phospholipids inhibited the DNA relaxation activity of topoisomerase I whereas neutral phospholipid, phosphatidylethanolamine, did not. Accumulation of a protein-DNA cleavable complex, an intermediate which is known to accumulate upon inhibition by a specific inhibitor camptothecin, did not occur. The filter binding assay revealed that the DNA binding activity of the enzyme was inhibited by acidic phospholipids. Moreover, direct binding of phosphatidylglycerol to topoisomerase I was demonstrated. These results indicated that the inhibitory effect of acidic phospholipids on topoisomerase I was due to the loss of the DNA binding of the enzyme as a result of direct interaction between phospholipids and the enzyme.     

Iridoids as DNA topoisomerase I poisons

The discovery of new topoisomerase I inhibitors is necessary since most of the antitumor drugs are targeted against type II and only a very few can specifically affect type I. Topoisomerase poisons generate toxic DNA damage by stabilization of the covalent DNA-topoisomerase cleavage complex and some have therapeutic efficacy in human cancer. Two iridoids, aucubin and geniposide, have shown antitumoral activities, but their activity against topoisomerase enzymes has not been tested. Here it was found that both compounds are able to stabilize covalent attachments of the topoisomerase I subunits to DNA at sites of DNA strand breaks, generating cleavage complexes intermediates so being active as poisons of topoisomerase I, but not topoisomerase II. This result points to DNA damage induced by topoisomerase I poisoning as one of the possible mechanisms by which these two iridoids have shown antitumoral activity, increasing interest in their possible use in cancer chemoprevention and therapy.     

Anthracycline antibiotics and their derivatives,inhibitors of topoisomerase I

The relationship between the structure of new semisynthetic derivatives of doxorubicin, daunorubicin, and carminomycin and their ability to inhibit topoisomerase I were studied. The new derivatives inhibit the activity of topoisomerase I at low concentrations, induce the death of K-562 leukemia cells in culture, and produce an antitumor effect in experimental animals bearing P388 leukemia.     

Netropsin and bis-netropsin analogs as inhibitors of the catalytic activity of mammalian DNA topoisomerase II and topoisomerase cleavable complexes.

This study examined the ability of netropsin and related minor groove binders to interfere with the actions of DNA topoisomerases II and I. We evaluated a series of netropsin dimers linked with flexible aliphatic chains of different lengths. These agents are potentially able to occupy longer stretches of DNA than the parental drug as a result of bidentate binding. Both netropsin and its dimers were found: (i) to inhibit the catalytic activity of isolated topoisomerase II and (ii) to interfere with the stabilization of the cleavable complexes of topoisomerase II and I in nuclei. Dimers with linkers consisting of 0-4 and 6-9 methylene groups (n) were far more inhibitory than netropsin against isolated enzyme and in the nuclear system. The compound with n = 5 was less active than netropsin in both assays while the dimer with n = 10 inhibited only the isolated enzyme. The comparison of dimers with fixed linker length (n = 2) but varying number of N-methylpyrrole residues (from 1 to 3) revealed that the inhibitory properties were enhanced with increasing number of N-methylpyrrole units. For dimers with varying linker length, drug ability to inhibit catalytic activity of isolated topoisomerase II was positively correlated with calf thymus DNA association constants. In contrast, no such correlation existed in nuclei. However, the inhibitory effects in the nuclear system were correlated with the association constants for poly(dAdT). The results indicate that bidentate binding can significantly enhance anti-topoisomerase activity of netropsin related dimeric minor groove binders. However, other factors such as the length of the linker, the number of pyrrole moieties and the nature of the target (isolated enzyme/DNA versus chromatin in nuclei) also contribute to these activities.     

Synthesis of nitrated indenoisoquinolines as topoisomerase I inhibitors

Indenoisoquinolines and dihydroindenoisoquinolines have been synthesized possessing a nitro-substituted isoquinoline ring in an effort to explore the effects of electron-withdrawing substituents on biological activity. The in vitro anticancer activities of these molecules have been tested in the National Cancer Institute's screen of 55 cell lines. The compounds have also been tested for topoisomerase I (top1) inhibition. The results indicate that these substances are a potent class of top1 inhibitors with sub-micromolar cytotoxicity mean graph midpoints (MGM) and top1 inhibition equal to camptothecin.