Synthesis and in vitro cytotoxicity evaluation of ß-carboline-combretastatin carboxamides as apoptosis inducing agents: DNA intercalation and topoisomerase-II inhibition.

To explore a new set of cytotoxic agents, ß-carboline-combretastatin carboxamide conjugates were designed, synthesized and evaluated for their in vitro cytotoxicity potential, DNA binding affinity and Topoisomerase-II (topo-II) inhibition activity. Among the designed hybrids, 10v and 10af have shown significant cytotoxic effect against A549 (lung cancer) cell line having IC50 value 1.01 µM and 1.17 µM respectively. Further, it was speculated that treatment with compound 10v may induce apoptosis among A549 cells, which was supported by Hoechst staining, DCFDA, Annexin V-FITC and morphological assays. Flow cytometric analysis revealed that the hybrid 10v arrests A549 cells in G2/M phase of cell cycle in a dose dependent manner. Amongst the active hybrids, most potent hybrid 10v was tested for DNA topo-II inhibition activity. Moreover, to further support the biological activity and to infer the mode of interaction between ligands and DNA, spectroscopy and molecular docking studies were carried out. The docking and spectroscopy results showed that the ligands exhibited an intercalative mode of binding with DNA and could efficiently bind to DNA and form topo-II ternary complex. Based on these experiments, the hybrids 10v and 10af were identified as proficient new scaffolds which need to be developed as hit molecules for therapeutic interest.

Selective lead compounds against kinetoplastid tubulin.

Kinetoplastid parasites are responsible for the potentially fatal diseases leishmaniasis, African sleeping sickness and Chagas disease. The current treatments for these diseases are far from ideal and new compounds are needed as antiparasitic drug candidates. Tubulin is the accepted target for treatments against cancer and helminths, suggesting that kinetoplastid tubulin is also a suitable target for antiprotozoal compounds. Selective lead compounds against kinetoplastid tubulin have been identified that could represent a starting point for the development of new drug candidates against these parasites.

The HSP90 and DNA topoisomerase VI inhibitor radicicol also inhibits human type II DNA topoisomerase.

Radicicol derivatives are currently investigated as promising antitumoral drugs because they inhibit the activity of the molecular chaperone heat shock protein (HSP90), causing the destabilization and eventual degradation of HSP90 client proteins that are often associated with tumor cells. These drugs interact with the ATP-binding site of HSP90 which is characterized by a structural element known as the Bergerat fold, also present in type II DNA topoisomerases (Topo II). We have previously shown that radicicol inhibits archaeal DNA topoisomerase VI, the prototype of Topo II of the B family (present in archaea, some bacteria and all the plants sequenced so far). We show here that radicicol also inhibits the human Topo II, a member of the A family (comprising the eukaryotic Topo II, bacterial gyrase, Topo IV and viral Topo II), which is a major target for antitumoral drugs. In addition, radicicol prevents in vitro induction of DNA cleavage by human Topo II in the presence of the antitumoral drug etoposide. The finding that radicicol can inhibit at least two different antitumoral drug targets in human, and interferes with drugs currently used in cancer treatment, could have implications in cancer therapy.

Modulation of radiation response by inhibiting topoisomerase II catalytic activity.

Due to the essential role played by DNA topoisomerases (topos) in cell survival, the use of topoisomerase inhibitors as chemotherapeutic drugs in combination with radiation has become a common strategy for the treatment of cancer. Catalytic inhibitors of these enzymes would be promising to improve the effectiveness of radiation and therefore, it appears reasonable to incorporate them in combined modality trials. In this work, we have investigated the capacity of both ICRF-193 and Aclarubicin (ACLA), two catalytic inhibitors of topoisomerase II (Topo II), to modulate radiation response in Chinese hamster V79 cell line and its radiosensitive mutant irs2. We also have explored potential mechanisms underlying these interactions. Experiments were performed in the presence and absence of either ICRF-193 or ACLA, and topo II activity was measured using an assay based upon decatenation of kinetoplast DNA (kDNA). For the combined experiments cells were incubated for 3 h in the presence of various inhibitor concentrations and irradiated 30 min prior to the end of treatments and cell survival was determined by clonogenic assay. DNA-damaging activity was measured by single-cell gel electrophoresis. Our results demonstrate that combinations of catalytic inhibitors of topo II and radiation produce an increase in cell killing induced by ionising radiation. The mechanism of radiation enhancement may involve a direct or indirect participation of topo II in the repair of radiation-induced DNA damage.

Stilbenoids of Kobresia nepalensis (Cyperaceae) exhibiting DNA topoisomerase II inhibition.

Resveratrol oligomers, nepalensinol A, B and C, were isolated from the stem of Kobresia nepalensis (Cyperaceae). The structures were established on the basis of chemical properties and spectroscopic evidence including 2D NMR spectroscopic analysis. Nepalensinol A, B and C showed a potent inhibitory effect on topoisomerase II -- stronger than etoposide (VP-16), a topoisomerase II inhibitor used as an anti-cancer drug. Nepalensinol B, in particular, exhibited the most potent activity with an IC(50) of 0.02 microg/ml.

Interaction of strong DNA-intercalating bioreductive compounds with topoisomerases I and II.

Nitro(imidazole/triazole)-linked acridines (NLAs) have been previously developed in our laboratory as DNA-intercalating bioreductive drugs. Such compounds demonstrate toxicity through the formation of bulky monoadducts with cellular macromolecules upon activation and reductive metabolism under hypoxic conditions. However, NLAs also demonstrate considerable aerobic toxicity. Based on the ability of NLAs to bind strongly to DNA through intercalation, we investigated whether their relatively high aerobic cytotoxicity and their relatively low hypoxic selectivity in vitro are associated with topoisomerases I and II (Topo I and II) inhibition. DNA Topo I or II-mediated activity studies have been performed using supercoiled or kinetoplast DNA plasmids. Calf thymus or human Topo I and human Topo II purified enzymes were used. All NLA derivatives strongly inhibited relaxation of supercoiled DNA catalyzed by either Topo I or II, in a concentration-dependent manner, without stabilization of a cleavable complex. Aerobic toxicity correlated well with the inhibition of Topo II-mediated decatenation of kinetoplast DNA, whereas the intracellular concentrations of NLAs were 27-152-fold greater than those needed for 50% inhibition of Topo-II mediated decatenation of DNA. These results suggest that topoisomerase inhibition accounts for NLAs aerobic toxicity.

Increased temperature and 2-methyl-2,4-pentanediol change the DNA structure of both curved and uncurved adenine/thymine-rich sequences.

DNA curvature is affected by elevated temperature and dehydrating agents such as 2-methyl-2,4-pentanediol (MPD) (used in crystallization). This effect of MPD has been ascribed to a specific distortion of the structure of adenine tracts (A-tracts), probably through a deformation of the characteristic narrow minor groove. Uranyl photoprobing indicates that a narrowed minor groove is present in all A/T regions containing four or more A/T base pairs. Consequently, this technique may be employed to study conformational changes in other A/T-rich sequences than pure A-tracts. In this study we use uranyl photoprobing to demonstrate that the effect of elevated temperature and MPD is analogous on both "normal" and curve-inducing A/T-rich sequences. The results therefore indicate that under these conditions the minor groove is widened in all A/T sequences and not only in pure A-tracts as previously suggested. Thus, the rather subtle structural difference of AT regions and A-tracts in nonbent DNA versus A-tracts in bent DNA may be quantitative rather than qualitative; i.e., the structure is more persistent and/or rigid in bent DNA.

Inhibition of human DNA topoisomerase II by hydroquinone and p-benzoquinone, reactive metabolites of benzene.

Chronic exposure of humans to benzene (BZ) causes acute myeloid leukemia (AML). Both BZ and therapy-related secondary AML are characterized by chromosomal translocations that may occur by inappropriate recombinational events. DNA topoisomerase II (topo II) is an essential sulfhydryl (SH)-dependent endonuclease required for replication, recombination, chromosome segregation, and chromosome structure. Topo II cleaves DNA at purine(R)/pyrimidine(Y) repeat sequences that have been shown to be highly recombinogenic in vivo. Certain antineoplastic drugs stabilize topo II-DNA cleavage complexes at RY repeat sequences, which leads to translocations of the type observed in leukemia. Hydroquinone (HQ) is metabolized to p-benzoquinone (BQ) in a peroxidase-mediated reaction in myeloid progenitor cells. BQ interacts wit SH groups of SH-dependent enzymes. Consequently, the aims of this research were to determine whether HQ and BQ are topo II inhibitors. The ability of the compounds to inhibit the activity of topo III was tested using an assay system that depends on the conversion, by homogeneous human topo II, of catenated kinetoplast DNA into open and/or nicked open circular DNA that can be separated from the catenated DNA by electrophoresis in a 1% agarose-ethidium bromide gel. We provide preliminary data that indicate that both HQ and BQ cause a time and concentration (microM)-dependent inhibition of topo II activity. These compounds, which potentially can form adducts with DNA, have no effect on the migration of the supercoiled and open circular forms in the electrophoretic gradient, and BQ-adducted KDNA can be decatenated by topo II. Using a pRYG plasmid DNA with a single RY repeat as a cleavage site, it was determined that BQ does not stimulate the production of linear DNA indicative of an inhibition of topo II religation of strand breaks by stabilization of the covalent topo III-DNA cleavage complex. Rather, BQ most probably inhibits the SH-dependent topo II by binding to an essential SH group. The inhibition of topo II by BQ has implications for the formation of deleterious translocations that may be involved in BZ-induced initiation of leukemogenesis.

A cisplatin-resistant murine leukemia cell line exhibits increased topoisomerase II activity.

cis-Dichlorodiammineplatinum(II) (CDDP) resistance in L1210/10 murine leukemia cells is multifactorial and involves decreased drug uptake, increased glutathione content, and enhanced DNA repair activity. We show here that 0.35 M NaCl nuclear extracts from L1210/10 cells possess an approximately 3-fold increase in DNA topoisomerase II activity, compared with parental L1210 cells, as measured by decatenation of kinetoplast DNA. No difference in topoisomerase I activity is observed between the two cell lines. Immunoblot analysis of topoisomerase II protein in resistant and sensitive cells suggests that the observed differences in topoisomerase II activity cannot be explained by differences in the level of protein expressed. L1210/10 cells are 2.5-fold more sensitive than L1210 cells to the cytotoxic effects of the topoisomerase II inhibitor 4'-(9-acridylamino)methane-sulfon-m-anisidide. Sequential treatment with 4'-(9-acridyl-amino)methanesulfon-m-anisidide and CDDP leads to an additive cytotoxic effect of the two drugs in sensitive L1210 cells, as determined by colony formation in semi-solid medium. In contrast, the same treatment leads to a supra-additive effect in L1210/10 cells, which strongly suggests a role for topoisomerase II in the CDDP resistance of this cell line.

Inhibition of topoisomerases in African trypanosomes.

African trypanosomiasis continues to pose a challenge for the development of new chemotherapy. Type II topoisomerases, essential enzymes in nucleic acid metabolism, have proven highly suitable as targets for antibacterial and antitumor therapy. Well-characterized topoisomerase II inhibitors affect the cognate nuclear and mitochondrial enzymes in Trypanosoma equiperdum. Inhibition is accompanied by extensive fragmentation and structural alteration in nuclear and mitochondrial DNA. Some clinically important antitrypanosomal drugs bind to DNA (i.e., pentamidine, isometamidium, diminazene). These agents inhibit the mitochondrial, but not nuclear, topoisomerase II of trypanosomes. These studies suggest that type II topoisomerase inhibitors may prove to be effective and safe new antitrypanosomal drugs.