Dihydroindenoisoquinolines function as prodrugs of indenoisoquinolines

Dihydroindenoisoquinolines are analogs of cytotoxic indenoisoquinoline topoisomerase I (Top1) inhibitors, exhibiting potent cytotoxicity but weak inhibitory activity toward Top1. Through COMPARE analysis, cytotoxicity studies in Top1-deficient cells, chemical synthesis and biological evaluation of methylated dihydroindenoisoquinoline 5, we demonstrated that dihydroindenoisoquinolines function as prodrugs of indenoisoquinolines in cancer cells.     

Synthesis and biological evaluation of new fluorinated and chlorinated indenoisoquinoline topoisomerase I poisons

Fluorine and chlorine are metabolically stable, but generally less active replacements for a nitro group at the 3-position of indenoisoquinoline topoisomerase IB (Top1) poisons. A number of strategies were employed in the present investigation to enhance the Top1 inhibitory potencies and cancer cell growth inhibitory activities of halogenated indenoisoquinolines. In several cases, the new compounds’ activities were found to rival or surpass those of similarly substituted 3-nitroindenoisoquinolines, and several unusually potent analogs were discovered through testing in human cancer cell cultures. A hydroxyethylaminopropyl side chain on the lactam nitrogen of two halogenated indenoisoquinoline Top1 inhibitors was found to also impart inhibitory activity against tyrosyl DNA phosphodiesterases 1 and 2 (TDP1 and TDP2), which are enzymes that participate in the repair of DNA damage induced by Top1 poisons.     

The structure–activity relationships of A-ring-substituted aromathecin topoisomerase I inhibitors strongly support a camptothecin-like binding mode

Aromathecins are inhibitors of human topoisomerase I (Top1). These compounds are composites of several heteroaromatic systems, namely the camptothecins and indenoisoquinolines, and they possess notable Top1 inhibition and cytotoxicity when substituted at position 14. The SAR of these compounds overlaps with indenoisoquinolines, suggesting that they may intercalate into the Top1-DNA complex similarly. Nonetheless, the proposed binding mode for aromathecins is purely hypothetical, as an X-ray structure is unavailable. In the present communication, we have synthesized eight novel series of A-ring-substituted (positions 1–3) aromathecins, through a simple, modular route, as part of a comprehensive SAR study. Certain groups (such as 2,3-ethylenedioxy) moderately improve Top1 inhibition, and, often, antiproliferative activity, whereas other groups (2,3-dimethoxy and 3-substituents) attenuate bioactivity. Strikingly, these trends are very similar to those previously observed for the A-ring of camptothecins, and this considerable SAR overlap lends further support (in the absence of crystallographic data) to the hypothesis that aromathecins bind in the Top1 cleavage complex as interfacial inhibitors in a ‘camptothecin-like’ pose.     

Identification of novel PARP inhibitors using a cell-based TDP1 inhibitory assay in a quantitative high-throughput screening platform

Anti-cancer topoisomerase I (Top1) inhibitors (camptothecin and its derivatives irinotecan and topotecan, and indenoisoquinolines) induce lethal DNA lesions by stabilizing Top1-DNA cleavage complex (Top1cc). These lesions are repaired by parallel repair pathways including the tyrosyl-DNA phosphodiesterase 1 (TDP1)-related pathway and homologous recombination. As TDP1-deficient cells in vertebrates are hypersensitive to Top1 inhibitors, small molecules inhibiting TDP1 should augment the cytotoxicity of Top1 inhibitors. We developed a cell-based high-throughput screening assay for the discovery of inhibitors for human TDP1 using a TDP1-deficient chicken DT40 cell line (TDP1−/−) complemented with human TDP1 (hTDP1). Any compounds showing a synergistic effect with the Top1 inhibitor camptothecin (CPT) in hTDP1 cells should either be a TDP1-related pathway inhibitor or an inhibitor of alternate repair pathways for Top1cc. We screened the 400,000-compound Small Molecule Library Repository (SMLR, NIH Molecular Libraries) against hTDP1 cells in the absence or presence of CPT. After confirmation in a secondary screen using both hTDP1 and TDP1−/− cells in the absence or presence of CPT, five compounds were confirmed as potential TDP1 pathway inhibitors. All five compounds showed synergistic effect with CPT in hTDP1 cells, but not in TDP1−/− cells, indicating that the compounds inhibited a TDP1-related repair pathway. Yet, in vitro gel-based assay revealed that the five compounds did not inhibit TDP1 catalytic activity directly. We tested the compounds for their ability to inhibit poly(ADP-ribose)polymerase (PARP) because PARP inhibitors are known to potentiate the cytotoxicity of CPT by inhibiting the recruitment of TDP1 to Top1cc. Accordingly, we found that the five compounds inhibit catalytic activity of PARP by ELISA and Western blotting. We identified the most potent compound (Cpd1) that offers characteristic close to veliparib, a leading clinical PARP inhibitor. Cpd1 may represent a new scaffold for the development of PARP inhibitors.     

Synthesis and biological evaluation of 14-(aminoalkyl-aminomethyl)aromathecins as topoisomerase I inhibitors: Investigating the hypothesis of shared structure–activity relationships

The aromathecin topoisomerase I (top1) inhibitors offer promising scaffolds for the development of novel cancer chemotherapeutics. They are ‘composites’ of the camptothecin and indenoisoquinoline top1 inhibitors. Interestingly, some structure–activity relationship (SAR) overlap between the aromathecins and the indenoisoquinolines has been observed. For both classes, placement of certain polar groups in similar regions of the heteroaromatic system improves top1 inhibitory and antiproliferative activities. A series of water-soluble aromathecins substituted at position 14 with diaminoalkanes of various lengths has been prepared. These compounds all possess similar antiproliferative potency, but a general trend is observed: aromathecins with longer diaminoalkane substituents (>6 carbons) possess lower anti-top1 activity than their smaller counterparts (2–4 carbons), presumably as a result of unfavorable hydrophobic interactions. This trend is also noted with the indenoisoquinolines, revealing additional SAR overlap that supports the hypothesis that there is a ‘universal’ top1 inhibitor SAR.     

Exploring DNA topoisomerase I ligand space in search of novel anticancer agents

DNA topoisomerase I (Top1) is over-expressed in tumour cells and is an important target in cancer chemotherapy. It relaxes DNA torsional strain generated during DNA processing by introducing transient single-strand breaks and allowing the broken strand to rotate around the intermediate Top1-DNA covalent complex. This complex can be trapped by a group of anticancer agents interacting with the DNA bases and the enzyme at the cleavage site, preventing further topoisomerase activity. Here we have identified novel Top1 inhibitors as potential anticancer agents by using a combination of structure- and ligand-based molecular modelling methods. Pharmacophore models have been developed based on the molecular characteristics of derivatives of the alkaloid camptothecin (CPT), which represent potent antitumour agents and the main group of Top1 inhibitors. The models generated were used for in silico screening of the National Cancer Institute (NCI, USA) compound database, leading to the identification of a set of structurally diverse molecules. The strategy is validated by the observation that amongst these molecules are several known Top1 inhibitors and agents cytotoxic against human tumour cell lines. The potential of the untested hits to inhibit Top1 activity was further evaluated by docking into the binding site of a Top1-DNA complex, resulting in a selection of 10 compounds for biological testing. Limited by the compound availability, 7 compounds have been tested in vitro for their Top1 inhibitory activity, 5 of which display mild to moderate Top1 inhibition. A further compound, found by similarity search to the active compounds, also shows mild activity. Although the tested compounds display only low in vitro antitumour activity, our approach has been successful in the identification of structurally novel Top1 inhibitors worthy of further investigation as potential anticancer agents.     

Mus81-mediated DNA cleavage resolves replication forks stalled by topoisomerase I-DNA complexes

Deoxyribonucleic acid (DNA) topoisomerases are essential for removing the supercoiling that normally builds up ahead of replication forks. The camptothecin (CPT) Top1 (topoisomerase I) inhibitors exert their anticancer activity by reversibly trapping Top1-DNA cleavage complexes (Top1cc's) and inducing replication-associated DNA double-strand breaks (DSBs). In this paper, we propose a new mechanism by which cells avoid Top1-induced replication-dependent DNA damage. We show that the structure-specific endonuclease Mus81-Eme1 is responsible for generating DSBs in response to Top1 inhibition and for allowing cell survival. We provide evidence that Mus81 cleaves replication forks rather than excises Top1cc's. DNA combing demonstrated that Mus81 also allows efficient replication fork progression after CPT treatment. We propose that Mus81 cleaves stalled replication forks, which allows dissipation of the excessive supercoiling resulting from Top1 inhibition, spontaneous reversal of Top1cc, and replication fork progression.     

TDP2 promotes repair of topoisomerase I-mediated DNA damage in the absence of TDP1

The abortive activity of topoisomerases can result in clastogenic and/or lethal DNA damage in which the topoisomerase is covalently linked to the 3'- or 5'-terminus of a DNA strand break. This type of DNA damage is implicated in chromosome translocations and neurological disease and underlies the clinical efficacy of an important class of anticancer topoisomerase 'poisons'. Tyrosyl DNA phosphodiesterase-1 protects cells from abortive topoisomerase I (Top1) activity by hydrolyzing the 3'-phosphotyrosyl bond that links Top1 to a DNA strand break and is currently the only known human enzyme that displays this activity in cells. Recently, we identified a second tyrosyl DNA phosphodiesterase (TDP2; aka TTRAP/EAPII) that possesses weak 3'-tyrosyl DNA phosphodiesterase (3'-TDP) activity, in vitro. Herein, we have examined whether TDP2 contributes to the repair of Top1-mediated DNA breaks by deleting Tdp1 and Tdp2 separately and together in murine and avian cells. We show that while deletion of Tdp1 in wild-type DT40 cells and mouse embryonic fibroblasts decreases DNA strand break repair rates and cellular survival in response to Top1-induced DNA damage, deletion of Tdp2 does not. However, deletion of both Tdp1 and Tdp2 reduces rates of DNA strand break repair and cell survival below that observed in Tdp1(-)(/)(-) cells, suggesting that Tdp2 contributes to cellular 3'-TDP activity in the absence of Tdp1. Consistent with this idea, over-expression of human TDP2 in Tdp1(-)(/)(-)/Tdp2(-)(/)(-)(/)(-) DT40 cells increases DNA strand break repair rates and cell survival above that observed in Tdp1(-)(/)(-) DT40 cells, suggesting that Tdp2 over-expression can partially complement the defect imposed by loss of Tdp1. Finally, mice lacking both Tdp1 and Tdp2 exhibit greater sensitivity to Top1 poisons than do mice lacking Tdp1 alone, further suggesting that Tdp2 contributes to the repair of Top1-mediated DNA damage in the absence of Tdp1. In contrast, we failed to detect a contribution for Tdp1 to repair Top2-mediated damage. Together, our data suggest that Tdp1 and Tdp2 fulfil overlapping roles following Top1-induced DNA damage, but not following Top2-induced DNA damage, in vivo.     

Enhanced CPT sensitivity of yeast cells and selective relaxation of Ga14 motif-containing DNA by novel Gal4-topoisomerase I fusion proteins

Human topoisomerase I-B (Top1) efficiently relaxes DNA supercoils during basic cellular processes, and can be transformed into a DNA-damaging agent by antitumour drugs, enzyme mutations and DNA lesions. Here, we describe Gal4-Top1 chimeric proteins (GalTop) with an N-terminal truncation of Top1, and mutations of the Gal4 Zn-cluster and/or Top1 domains that impair their respective DNA-binding activities. Expression levels of chimeras were similar in yeast cells, however, GalTop conferred an increased CPT sensitivity to RAD52- yeast cells as compared to a GalTop with mutations of the Gal4 domain, showing that a functional Gal4 domain can alter in vivo functions of Top1. In vitro enzyme activity was tested with a DNA relaxation assay using negatively supercoiled plasmids with 0 to 5 Gal4 consensus motifs. Only GalTop with a functional Gal4 domain could direct DNA relaxation activity of Top1 specifically to DNA molecules containing Gal4 motifs. By using a substrate competition assay, we could demonstrate that the Gal4-anchored Top1 remains functional and efficiently relax DNA substrates in cis. The enhanced CPT sensitivity of GalTop in yeast cells may then be due to alterations of the chromatin-binding activity of Top1. The GalTop chimeras may indeed mimic a normal mechanism by which Top1 is recruited to chromatin sites in living cells. Such hybrid Top1s may be helpful in further dissecting enzyme functions, and constitute a prototype of a site-specific DNA cutter endowed with high cell lethality.     

Active site mutations in DNA topoisomerase I distinguish the cytotoxic activities of camptothecin and the indolocarbazole, rebeccamycin.

DNA topoisomerase I (Top1p) catalyzes topological changes in DNA and is the cellular target of the antitumor agent camptothecin (CPT). Non-CPT drugs that target Top1p, such as indolocarbazoles, are under clinical development. However, whether the cytotoxicity of indolocarbazoles derives from Top1p poisoning remains unclear. To further investigate indolocarbazole mechanism, rebeccamycin R-3 activity was examined in vitro and in yeast. Using a series of Top1p mutants, where substitution of residues around the active site tyrosine has well-defined effects on enzyme catalysis, we show that catalytically active, CPT-resistant enzymes remain sensitive to R-3. This indolocarbazole did not inhibit yeast Top1p activity, yet was effective in stabilizing Top1p-DNA complexes. Similar results were obtained with human Top1p, when Ser or His were substituted for Asn-722. The mutations altered enzyme function and sensitivity to CPT, yet R-3 poisoning of Top1p was unaffected. Moreover, top1delta, rad52delta yeast cells expressing human Top1p, but not catalytically inactive Top1Y723Fp, were sensitive to R-3. These data support hTop1p as the cellular target of R-3 and indicate that distinct drug-enzyme interactions at the active site are required for efficient poisoning by R-3 or CPT. Furthermore, resistance to one poison may potentiate cell sensitivity to structurally distinct compounds that also target Top1p.     

Platinated DNA adducts enhance poisoning of DNA topoisomerase I by camptothecin

Camptothecins constitute a novel class of chemotherapeutics that selectively target DNA topoisomerase I (Top1) by reversibly stabilizing a covalent enzyme-DNA intermediate. This cytotoxic mechanism contrasts with that of platinum drugs, such as cisplatin, which induce inter- and intrastrand DNA adducts. In vitro combination studies using platinum drugs combined with Top1 poisons, such as topotecan, showed a schedule-dependent synergistic activity, with promising results in the clinic. However, whereas the molecular mechanism of these single agents may be relatively well understood, the mode of action of these chemotherapeutic agents in combination necessitates a more complete understanding. Indeed, we recently reported that a functional homologous recombination pathway is required for cisplatin and topotecan synergy yet represses the synergistic toxicity of 1-beta-D-arabinofuranosyl cytidine in combination with topotecan (van Waardenburg, R. C., de Jong, L. A., van Delft, F., van Eijndhoven, M. A., Bohlander, M., Bjornsti, M. A., Brouwer, J., and Schellens, J. H. (2004) Mol. Cancer Ther. 3, 393-402). Here we provide direct evidence for Pt-1,3-d(GTG) poisoning of Top1 in vitro and demonstrate that persistent Pt-DNA adducts correlate with increased covalent Top1-DNA complexes in vivo. This contrasts with a lack of persistent lesions induced by the alkylating agent bis[chloroethyl]nitrosourea, which exhibits only additive activity with topotecan in a range of cell lines. In human IGROV-1 ovarian cancer cells, the synergistic activity of cisplatin with topotecan requires processive DNA polymerization, whereas overexpression of Top1 enhances yeast cell sensitivity to cisplatin. These results indicate that the cytotoxic activity of cisplatin is due, in part, to poisoning of Top1, which is exacerbated in the presence of topotecan.     

A novel DNA topoisomerase I inhibitor with different mechanism from camptothecin induces G2/M phase cell cycle arrest to K562 cells

DNA topoisomerase I (Top1) is an essential nuclear enzyme and a validated target for anticancer agent screening. In a previous study, we found that indolizinoquinoline-5,12-dione derivatives show significant biological activity against several human cancer cell lines. To understand their mechanism of inhibition of cancer cell growth, one indolizinoquinoline-5,12-dione derivative, CY13II, was further studied as lead. Our present results indicate that CY13II shows more potent antiproliferative activity against K562 cells than camptothecin. Additionally, K562 cells were arrested in G2/M, and their growth rate decreased after treatment with CY13II at micromolar concentration. Biochemical Top1 assays indicate that CY13II exhibits a different inhibitory mechanism from camptothecin. Unlike camptothecin, CY13II specifically inhibits the catalytic cleavage activity of Top1 instead of forming the drug-enzyme-DNA covalent ternary complex.     

甜菜夜蛾拓扑异构酶I氨基酸突变对其DNA解旋活性的影响

【目的】为了探究甜菜夜蛾 Spodoptera exigua 拓扑异构酶I（topoisomerase I, Top I）氨基酸突变对其DNA解旋活性的影响。【方法】通过克隆甜菜夜蛾 Top I 基因,构建原核表达载体,采用完全重叠PCR定点突变技术,向甜菜夜蛾Top I 的V420, L530, A653和S729（根据人Top I 氨基酸序列编号）4个位点引入突变,将改造成功的重组 Top I 基因转化至大肠杆菌BL21 (DE3)中,诱导重组蛋白表达、纯化,测定Top I突变对其解旋活性的影响。【结果】完全重叠PCR能实现甜菜夜蛾 Top I 定点突变。重组蛋白在体外得到稳定的表达,表达产物经SDS-PAGE电泳分析在96.0 kDa处出现特异性条带。通过对重组蛋白分离纯化并测定对质粒pBR322解旋酶活性,发现引入V420I, L530P和A653T突变后Top I的比活力显著降低,而引入S729T突变后比活力与野生型蛋白无显著差异。【结论】本研究证明在甜菜夜蛾Top I中引入V420I, L530P和A653T突变后,其对底物pBR322的解旋活性显著降低,为后期探索甜菜夜蛾Top I的定点突变与其对喜树碱及其衍生物敏感性的关系奠定了基础。     

Binding and activation of DNA topoisomerase III by the Rmi1 subunit

Rmi1 is a conserved oligonucleotide and oligosaccharide binding-fold protein that is associated with RecQ DNA helicase complexes from humans (BLM-TOP3 alpha) and yeast (Sgs1-Top3). Although human RMI1 stimulates the dissolution activity of BLM-TOP3 alpha, its biochemical function is unknown. Here we examined the role of Rmi1 in the yeast complex. Consistent with the similarity of top3Delta and rmi1Delta phenotypes, we find that a stable Top3.Rmi1 complex can be isolated from yeast cells overexpressing these two subunits. Compared with Top3 alone, this complex displays increased superhelical relaxation activity. The isolated Rmi1 subunit also stimulates Top3 activity in reconstitution experiments. In both cases elevated temperatures are required for optimal relaxation unless the substrate contains a single-strand DNA (ssDNA) bubble. Interestingly, Rmi1 binds only weakly to ssDNA on its own, but it stimulates the ssDNA binding activity of Top3 5-fold. Top3 and Rmi1 also cooperate to bind the Sgs1 N terminus and promote its interaction with ssDNA. These results demonstrate that Top3-Rmi1 functions as a complex and suggest that Rmi1 stimulates Top3 by promoting its interaction with ssDNA.     

Mapping the DNA topoisomerase III binding domain of the Sgs1 DNA helicase

Several members of the RecQ family of DNA helicases are known to interact with DNA topoisomerase III (Top3). Here we show that the Saccharomyces cerevisiae Sgs1 and Top3 proteins physically interact in cell extracts and bind directly in vitro. Sgs1 and Top3 proteins coimmunoprecipitate from cell extracts under stringent conditions, indicating that Sgs1 and Top3 are present in a stable complex. The domain of Sgs1 which interacts with Top3 was identified by expressing Sgs1 truncations in yeast. The results indicate that the NH(2)-terminal 158 amino acids of Sgs1 are sufficient for the high affinity interaction between Sgs1 and Top3. In vitro assays using purified Top3 and NH(2)-terminal Sgs1 fragments demonstrate that at least part of the interaction is through direct protein-protein interactions with these 158 amino acids. Consistent with these physical data, we find that mutant phenotypes caused by a point mutation or small deletions in the Sgs1 NH(2) terminus can be suppressed by Top3 overexpression. We conclude that Sgs1 and Top3 form a tight complex in vivo and that the first 158 amino acids of Sgs1 are necessary and sufficient for this interaction. Thus, a primary role of the Sgs1 amino terminus is to mediate the Top3 interaction.