Dynophore-Based Approach in Virtual Screening: A Case of Human DNA Topoisomerase IIα.
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ABSTRACT:
SUBMITTER: Janezic M
PROVIDER: S-EPMC8703789 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:DNA topoisomerases are enzymes responsible for the relaxation of DNA torsional strain, as well as for the untangling of DNA duplexes after replication, and are important cancer drug targets. One class of topoisomerase inhibitors, "poisons", binds to the transient enzyme-DNA complex which occurs during the mechanism of action, and inhibits the religation of DNA. This ultimately leads to the accumulation of DNA double strand breaks and cell death. Different types of topoisomerases occur in human cells and several poisons of topoisomerase I and II are widely used clinically. However, their use is compromised by a variety of side effects. Recent studies confirm that the inhibition of the α-isoform of topoisomerase II is responsible for the cytotoxic effect, whereas the inhibition of the β-isoform leads to development of adverse drug reactions. Thus, the discovery of agents selective for topoisomerase IIα is an important strategy for the development of topoisomerase II poisons with improved clinical profiles. Here, we present a computer-aided drug design study leading to the identification of structurally novel topoisomerase IIα poisons. The study combines ligand- and structure-based drug design methods including pharmacophore models, homology modelling, docking, and virtual screening of the National Cancer Institute compound database. From the 8 compounds identified from the computational work, 6 were tested for their capacity to poison topoisomerase II in vitro: 4 showed selective inhibitory activity for the α- over the β-isoform and 3 of these exhibited cytotoxic activity. Thus, our study confirms the applicability of computer-aided methods for the discovery of novel topoisomerase II poisons, and presents compounds which could be investigated further as selective topoisomerase IIα inhibitors.
| S-EPMC4260913 | biostudies-literature
Project description:Human DNA topoisomerase II (Top2) is a promising target for cancer treatment. To overcome the limitations of known Top2 inhibitors, novel Top2 catalytic inhibitors with new scaffolds were identified by structure-based virtual screening. In particular, compound 8 showed good in vitro antiproliferative activity with a broad spectrum. Top2-mediated cleavage assay and molecular modeling rationalized the mode of action. The new Top2 inhibitors are considered as good starting points for further hit-to-lead optimization in anticancer drug discovery.
| S-EPMC6071756 | biostudies-literature
Project description:Human type II topoisomerases, molecular motors that alter the DNA topology, are a major target of modern chemotherapy. Groups of catalytic inhibitors represent a new approach to overcome the known limitations of topoisomerase II poisons such as cardiotoxicity and induction of secondary tumors. Here, we present a class of substituted 4,5'-bithiazoles as catalytic inhibitors targeting the human DNA topoisomerase IIα. Based on a structural comparison of the ATPase domains of human and bacterial type II topoisomerase, a focused chemical library of 4,5'-bithiazoles was assembled and screened to identify compounds that better fit the topology of the human topo IIα adenosine 5'-triphosphate (ATP) binding site. Selected compounds showed inhibition of human topo IIα comparable to that of the etoposide topo II drug, revealing a new class of inhibitors targeting this molecular motor. Further investigations showed that compounds act as catalytic inhibitors via competitive ATP inhibition. We also confirmed binding to the truncated ATPase domain of topo IIα and modeled the inhibitor molecular recognition with molecular simulations and dynophore models. The compounds also displayed promising cytotoxicity against HepG2 and MCF-7 cell lines comparable to that of etoposide. In a more detailed study with the HepG2 cell line, there was no induction of DNA double-strand breaks (DSBs), and the compounds were able to reduce cell proliferation and stop the cell cycle mainly in the G1 phase. This confirms the mechanism of action of these compounds, which differs from topo II poisons also at the cellular level. Substituted 4,5'-bithiazoles appear to be a promising class for further development toward efficient and potentially safer cancer therapies exploiting the alternative topo II inhibition paradigm.
| S-EPMC7469689 | biostudies-literature
Project description:The seeds of Nigella sativa (often referred to as black seed) have long been utilized as a medicinal herb in Middle Eastern, Northern African, and Indian cultures. Historically, black seed has been used to treat a variety of illnesses associated with inflammation. More recent studies have found that it induces apoptosis and displays anticancer activity in animal and cellular models. The major bioactive compound of black seed is thymoquinone, which shares structural features with 1,4-benzoquinone and other covalent topoisomerase II poisons. Because a number of anticancer drugs target type II topoisomerases, we determined the effects of thymoquinone and a series of related quinones on human topoisomerase IIα. Thymoquinone enhanced enzyme-mediated DNA cleavage ~5-fold, which is similar to the increase seen with the anticancer drug etoposide. In order to enhance cleavage, compounds had to have at least two positions available for acylation. Furthermore, activity was decreased by the inclusion of electron-donating groups or bulky substituents. As predicted for a covalent topoisomerase II poison, the activity of thymoquinone (and related compounds) was abrogated by the addition of a reducing agent. Also, thymoquinone inhibited topoisomerase IIα activity when incubated with the enzyme prior to the addition of DNA. Cleavage complexes formed in the presence of the compound were stable for at least 8 h. Lastly, black seed extract and black seed oil both increased levels of enzyme-mediated DNA cleavage, suggesting that thymoquinone is active even in more complex herbal formulations. These findings indicate that thymoquinone can be added to the growing list of dietary and medicinal natural products with activity against human type II topoisomerases.
| S-EPMC4033629 | biostudies-other
Project description:The human type IIA topoisomerases (Top2) are essential enzymes that regulate DNA topology and chromosome organization. The Topo IIα isoform is a prime target for antineoplastic compounds used in cancer therapy that form ternary cleavage complexes with the DNA. Despite extensive studies, structural information on this large dimeric assembly is limited to the catalytic domains, hindering the exploration of allosteric mechanism governing the enzyme activities and the contribution of its non-conserved C-terminal domain (CTD). Herein we present cryo-EM structures of the entire human Topo IIα nucleoprotein complex in different conformations solved at subnanometer resolutions (3.6-7.4 Å). Our data unveils the molecular determinants that fine tune the allosteric connections between the ATPase domain and the DNA binding/cleavage domain. Strikingly, the reconstruction of the DNA-binding/cleavage domain uncovers a linker leading to the CTD, which plays a critical role in modulating the enzyme's activities and opens perspective for the analysis of post-translational modifications.
| S-EPMC8137924 | biostudies-literature
Project description:Phenotypic screening of α-substituted thiocarbohydrazones revealed promising activity of 1,5-bis(salicylidene)thiocarbohydrazide against leukemia and breast cancer cells. Supplementary cell-based studies indicated an impairment of DNA replication via the ROS-independent pathway. The structural similarity of α-substituted thiocarbohydrazone to previously published thiosemicarbazone catalytic inhibitors targeting the ATP-binding site of human DNA topoisomerase IIα prompted us to investigate the inhibition activity on this target. Thiocarbohydrazone acted as a catalytic inhibitor and did not intercalate the DNA molecule, which validated their engagement with this cancer target. A comprehensive computational assessment of molecular recognition for a selected thiosemicarbazone and thiocarbohydrazone provided useful information for further optimization of this discovered lead compound for chemotherapeutic anticancer drug discovery.
| S-EPMC10054454 | biostudies-literature
Project description:Coordination between the N-terminal gate and the catalytic core of topoisomerase II allows the proper capture, cleavage, and transport of DNA during the catalytic cycle. Because the activities of these domains are tightly linked, it has been difficult to discern their individual contributions to enzyme-DNA interactions and drug mechanism. To further address the roles of these domains, we analyzed the activity of the catalytic core of human topoisomerase IIα. The catalytic core and the wild-type enzyme both maintained higher levels of cleavage with negatively (as compared to positively) supercoiled plasmid, indicating that the ability to distinguish supercoil handedness is embedded within the catalytic core. However, the catalytic core alone displayed little ability to cleave DNA substrates that did not intrinsically provide the enzyme with a transport segment (i.e., substrates that did not contain crossovers). Finally, in contrast to interfacial topoisomerase II poisons, covalent poisons did not enhance DNA cleavage mediated by the catalytic core. This distinction allowed us to further characterize the mechanism of etoposide quinone, a drug metabolite that functions primarily as a covalent poison. Etoposide quinone retained some ability to enhance DNA cleavage mediated by the catalytic core, indicating that it still can function as an interfacial poison. These results further define the distinct contributions of the N-terminal gate and the catalytic core to topoisomerase II function. The catalytic core senses the handedness of DNA supercoils during cleavage, while the N-terminal gate is critical for capturing the transport segment and for the activity of covalent poisons.
| S-EPMC4204876 | biostudies-other
Project description:Currently, DNA topoisomerase I (Topo I) inhibitors constitute a family of antitumor agents with demonstrated clinical effects on human malignancies. However, the clinical uses of these agents have been greatly limited due to their severe toxic effects. Therefore, it is urgent to find and develop novel low toxic Topo I inhibitors. In recent years, during our ongoing research on natural antitumor products, a collection of low cytotoxic or non-cytotoxic compounds with various structures were identified from marine invertebrates, plants, and their symbiotic microorganisms. In the present study, new Topo I inhibitors were discovered from low cytotoxic and non-cytotoxic natural products by virtual screening with docking simulations in combination with bioassay test. In total, eight potent Topo I inhibitors were found from 138 low cytotoxic or non-cytotoxic compounds from coral-derived fungi and plants. All of these Topo I inhibitors demonstrated activities against Topo I-mediated relaxation of supercoiled DNA at the concentrations of 5-100 µM. Notably, the flavonoids showed higher Topo I inhibitory activities than other compounds. These newly discovered Topo I inhibitors exhibited structurally diverse and could be considered as a good starting point for the development of new antitumor lead compounds.
| S-EPMC5532659 | biostudies-literature
Project description:Studies in animal models have indicated that dietary isothiocyanates (ITCs) exhibit cancer preventive activities through carcinogen detoxification-dependent and -independent mechanisms. The carcinogen detoxification-independent mechanism of cancer prevention by ITCs has been attributed at least in part to their ability to induce apoptosis of transformed (initiated) cells (e.g. through suppression of IκB kinase and nuclear factor κB as well as other proposed mechanisms). In the current studies we show that ITC-induced apoptosis of oncogene-transformed cells involves thiol modification of DNA topoisomerase II (Top2) based on the following observations. 1) siRNA-mediated knockdown of Top2α in both SV40-transformed MEFs and Ras-transformed human mammary epithelial MCF-10A cells resulted in reduced ITC sensitivity. 2) ITCs, like some anticancer drugs and cancer-preventive dietary components, were shown to induce reversible Top2α cleavage complexes in vitro. 3) ITC-induced Top2α cleavage complexes were abolished by co-incubation with excess glutathione. In addition, proteomic analysis revealed that several cysteine residues on human Top2α were covalently modified by benzyl-ITC, suggesting that ITC-induced Top2α cleavage complexes may involve cysteine modification. Interestingly, consistent with the thiol modification mechanism for Top2α cleavage complex induction, the thiol-reactive selenocysteine, but not the non-thiol-reactive selenomethionine, was shown to induce Top2α cleavage complexes. In the aggregate, our results suggest that thiol modification of Top2α may contribute to apoptosis induction in transformed cells by ITCs.
| S-EPMC3190902 | biostudies-literature
Project description:DNA topoisomerase IIα (170 kDa, TOP2α/170) induces transient DNA double-strand breaks in proliferating cells to resolve DNA topological entanglements during chromosome condensation, replication, and segregation. Therefore, TOP2α/170 is a prominent target for anticancer drugs whose clinical efficacy is often compromised due to chemoresistance. Although many resistance mechanisms have been defined, acquired resistance of human cancer cell lines to TOP2α interfacial inhibitors/poisons is frequently associated with a reduction of Top2α/170 expression levels. Recent studies by our laboratory, in conjunction with earlier findings by other investigators, support the hypothesis that a major mechanism of acquired resistance to TOP2α-targeted drugs is due to alternative RNA processing/splicing. Specifically, several TOP2α mRNA splice variants have been reported which retain introns and are translated into truncated TOP2α isoforms lacking nuclear localization sequences and subsequent dysregulated nuclear-cytoplasmic disposition. In addition, intron retention can lead to truncated isoforms that lack both nuclear localization sequences and the active site tyrosine (Tyr805) necessary for forming enzyme-DNA covalent complexes and inducing DNA damage in the presence of TOP2α-targeted drugs. Ultimately, these truncated TOP2α isoforms result in decreased drug activity against TOP2α in the nucleus and manifest drug resistance. Therefore, the complete characterization of the mechanism(s) regulating the alternative RNA processing of TOP2α pre-mRNA may result in new strategies to circumvent acquired drug resistance. Additionally, novel TOP2α splice variants and truncated TOP2α isoforms may be useful as biomarkers for drug resistance, prognosis, and/or direct future TOP2α-targeted therapies.
| S-EPMC7304410 | biostudies-literature