Bioinformatic Analysis of Topoisomerase IIα Reveals Interdomain Interdependencies and Critical C-Terminal Domain Residues.

DNA Topoisomerase IIα (Top2A) is a nuclear enzyme that is a cancer drug target, and there is interest in identifying novel sites on the enzyme to inhibit cancer cells more selectively and to reduce off-target toxicity. The C-terminal domain (CTD) is one potential target, but it is an intrinsically disordered domain, which prevents structural analysis. Therefore, we set out to analyze the sequence of Top2A from 105 species using bioinformatic analysis, including the PSICalc algorithm, Shannon entropy analysis, and other approaches. Our results demonstrate that large (10th-order) interdependent clusters are found including non-proximal positions across the major domains of Top2A. Further, CTD-specific clusters of the third, fourth, and fifth order, including positions that had been previously analyzed via mutation and biochemical assays, were identified. Some of these clusters coincided with positions that, when mutated, either increased or decreased relaxation activity. Finally, sites of low Shannon entropy (i.e., low variation in amino acids at a given site) were identified and mapped as key positions in the CTD. Included in the low-entropy sites are phosphorylation sites and charged positions. Together, these results help to build a clearer picture of the critical positions in the CTD and provide potential sites/regions for further analysis.

Structural insights into the DNA topoisomerase II of the African swine fever virus.

Type II topoisomerases are ubiquitous enzymes that play a pivotal role in modulating the topological configuration of double-stranded DNA. These topoisomerases are required for DNA metabolism and have been extensively studied in both prokaryotic and eukaryotic organisms. However, our understanding of virus-encoded type II topoisomerases remains limited. One intriguing example is the African swine fever virus, which stands as the sole mammalian-infecting virus encoding a type II topoisomerase. In this work, we use several approaches including cryo-EM, X-ray crystallography, and biochemical assays to investigate the structure and function of the African swine fever virus type II topoisomerase, pP1192R. We determine the structures of pP1192R in different conformational states and confirm its enzymatic activity in vitro. Collectively, our results illustrate the basic mechanisms of viral type II topoisomerases, increasing our understanding of these enzymes and presenting a potential avenue for intervention strategies to mitigate the impact of the African swine fever virus.

Molecular choreography: Unveiling the dynamic landscape of type IIA DNA topoisomerases before T-segment passage through all-atom simulations.

Type IIA DNA topoisomerases are molecular nanomachines responsible for controlling topological states of DNA molecules. Here, we explore the dynamic landscape of yeast topoisomerase IIA during key stages of its catalytic cycle, focusing in particular on the events preceding the passage of the T-segment. To this end, we generated six configurations of fully catalytic yeast topo IIA, strategically inserted a T-segment into the N-gate in relevant configurations, and performed all-atom simulations. The essential motion of topo IIA protein dimer was characterized by rotational gyrating-like movement together with sliding motion within the DNA-gate. Both appear to be inherent properties of the enzyme and an inbuilt feature that allows passage of the T-segment through the cleaved G-segment. Coupled dynamics of the N-gate and DNA-gate residues may be particularly important for controlled and smooth passage of the T-segment and consequently the prevention of DNA double-strand breaks. QTK loop residue Lys367, which interacts with ATP and ADP molecules, is involved in regulating the size and stability of the N-gate. The unveiled features of the simulated configurations provide insights into the catalytic cycle of type IIA topoisomerases and elucidate the molecular choreography governing their ability to modulate the topological states of DNA topology.

Modeling allosteric mechanisms of eukaryotic type II topoisomerases.

Type II topoisomerases (TopoIIs) are ubiquitous enzymes that are involved in crucial nuclear processes such as genome organization, chromosome segregation, and other DNA metabolic processes. These enzymes function as large, homodimeric complexes that undergo a complex cycle of binding and hydrolysis of two ATP molecules in their ATPase domains, which regulates the capture and passage of one DNA double-helix through a second, cleaved DNA molecule. This process requires the transmission of information about the state of the bound nucleotide over vast ranges in the TopoII complex. How this information is transmitted at the molecular level to regulate TopoII functions and how protein substitutions disrupt these mechanisms remains largely unknown. Here, we employed extensive microsecond-scale molecular dynamics simulations of the yeast TopoII enzyme in multiple nucleotide-bound states and with amino acid substitutions near both the N and C termini of the complex. Simulation results indicate that the ATPase domains are remarkably flexible on the sub-microsecond timescale and that these dynamics are modulated by the identity of the bound nucleotides and both local and distant amino acid substitutions. Network analyses point toward specific allosteric networks that transmit information about the hydrolysis cycle throughout the complex, which include residues in both the protein and the bound DNA molecule. Amino acid substitutions weaken many of these pathways. Together, our results provide molecular level details on how the TopoII catalytic cycle is controlled through nucleotide binding and hydrolysis and how mutations may disrupt this process.

Natural product inspired diastereoselective synthesis of sugar-derived pyrano[3,2-c]quinolones and their in-silico studies.

Herein, we report the development of a diastereoselective and efficient route to construct sugar-derived pyrano[3,2-c]quinolones utilizing 1-C-formyl glycal and 4-hydroxy quinolone annulation. This methodology will open a route to synthesize nature inspired pyrano[3,2-c]quinolones. This is the first report for the stereoselective synthesis of sugar-derived pyrano[3,2-c]quinolones, where 100% stereoselectivity was observed. A total of sixteen compounds have been synthesized in excellent yields with 100% stereoselectivity. The molecular docking of the synthesized novel natural product analogues demonstrated their binding modes within the active site of type II topoisomerase. The results of the in-silico studies displayed more negative binding energies for the all the synthesized compounds in comparison to the natural product huajiosimuline A, indicating their affinity for the active pocket. Ten out of the sixteen novel synthesized compounds were found to have comparative or relatively more negative binding energy in comparison to the standard anti-cancer drug, doxorubicin. Additionally, the scalability and viability of this protocol was illustrated by the gram scale synthesis.

DNA fragility at topologically associated domain boundaries is promoted by alternative DNA secondary structure and topoisomerase II activity.

CCCTC-binding factor (CTCF) binding sites are hotspots of genome instability. Although many factors have been associated with CTCF binding site fragility, no study has integrated all fragility-related factors to understand the mechanism(s) of how they work together. Using an unbiased, genome-wide approach, we found that DNA double-strand breaks (DSBs) are enriched at strong, but not weak, CTCF binding sites in five human cell types. Energetically favorable alternative DNA secondary structures underlie strong CTCF binding sites. These structures coincided with the location of topoisomerase II (TOP2) cleavage complex, suggesting that DNA secondary structure acts as a recognition sequence for TOP2 binding and cleavage at CTCF binding sites. Furthermore, CTCF knockdown significantly increased DSBs at strong CTCF binding sites and at CTCF sites that are located at topologically associated domain (TAD) boundaries. TAD boundary-associated CTCF sites that lost CTCF upon knockdown displayed increased DSBs when compared to the gained sites, and those lost sites are overrepresented with G-quadruplexes, suggesting that the structures act as boundary insulators in the absence of CTCF, and contribute to increased DSBs. These results model how alternative DNA secondary structures facilitate recruitment of TOP2 to CTCF binding sites, providing mechanistic insight into DNA fragility at CTCF binding sites.

Virtual screening and multilevel precision-based prioritisation of natural inhibitors targeting the ATPase domain of human DNA topoisomerase II alpha.

Human DNA topoisomerase II alpha (hTopIIα) is a classic chemotherapeutic drug target. The existing hTopIIα poisons cause numerous side effects such as the development of cardiotoxicity, secondary malignancies, and multidrug resistance. The use of catalytic inhibitors targeting the ATP-binding cavity of the enzyme is considered a safer alternative due to the less deleterious mechanism of action. Hence, in this study, we carried out high throughput structure-based virtual screening of the NPASS natural product database against the ATPase domain of hTopIIα and identified the five best ligand hits. This was followed by comprehensive validation through molecular dynamics simulations, binding free energy calculation and ADMET analysis. On stringent multilevel prioritization, we identified promising natural product catalytic inhibitors that showed high binding affinity and stability within the ligand-binding cavity and may serve as ideal hits for anticancer drug development.Communicated by Ramaswamy H. Sarma.

Expression and functional analysis of various structural domains of tobacco topoisomerase II: To understand the mechanistic insights of plant type II topoisomerases.

In contrast to bacterial, yeast and animal systems, topoisomerases (topo) from plants have not been well studied. In this report, we generated four truncated topoisomerase II (Topo II) cDNA fragments encoding different functional domains of Nicotiana tabacum topo II (NtTopoII). Each of these recombinant polypeptides was expressed alone or in combination in temperature-sensitive topoisomerase II yeast mutants. Recombinant NtTopoII with truncated polypeptides fails to target the yeast nuclei and does not rescue the temperature-sensitive phenotype. In contrast complementation was achieved with the full-length NtTopoII, which localized to the yeast nucleus. These observations suggested the presence of a potent nuclear localization signal (NLS) in the extreme C-terminal 314 amino acid residues of NtTopoII that functioned effectively in the heterologous yeast system. Biochemical characterization of purified recombinant full-length and the partial NtTopoII polypeptides revealed that the ATP-binding and hydrolysis region of NtTopoIIwas located at 413 amino acid N-terminal region and this ATPase domain is functional both when it is expressed as a separate polypeptide or as part of the holoenzyme. The present findings also revealed that all NtTopoII truncated polypeptides were detrimental for in vitro supercoiled DNA relaxation and/or DNA nicking and ligation activity. Further, we discuss the possible disruption of coordinated macromolecular interface movements and the dimer interactions in truncated NtTopoII that are required for functional topoisomerase activity.

Evolution and Diversity of the TopoVI and TopoVI-like Subunits With Extensive Divergence of the TOPOVIBL subunit.

Type II DNA topoisomerases regulate topology by double-stranded DNA cleavage and ligation. The TopoVI family of DNA topoisomerase, first identified and biochemically characterized in Archaea, represents, with TopoVIII and mini-A, the type IIB family. TopoVI has several intriguing features in terms of function and evolution. TopoVI has been identified in some eukaryotes, and a global view is lacking to understand its evolutionary pattern. In addition, in eukaryotes, the two TopoVI subunits (TopoVIA and TopoVIB) have been duplicated and have evolved to give rise to Spo11 and TopoVIBL, forming TopoVI-like (TopoVIL), a complex essential for generating DNA breaks that initiate homologous recombination during meiosis. TopoVIL is essential for sexual reproduction. How the TopoVI subunits have evolved to ensure this meiotic function is unclear. Here, we investigated the phylogenetic conservation of TopoVI and TopoVIL. We demonstrate that BIN4 and RHL1, potentially interacting with TopoVIB, have co-evolved with TopoVI. Based on model structures, this observation supports the hypothesis for a role of TopoVI in decatenation of replicated chromatids and predicts that in eukaryotes the TopoVI catalytic complex includes BIN4 and RHL1. For TopoVIL, the phylogenetic analysis of Spo11, which is highly conserved among Eukarya, highlighted a eukaryal-specific N-terminal domain that may be important for its regulation. Conversely, TopoVIBL was poorly conserved, giving rise to ATP hydrolysis-mutated or -truncated protein variants, or was undetected in some species. This remarkable plasticity of TopoVIBL provides important information for the activity and function of TopoVIL during meiosis.

Topoisomerase 2ß and DNA topology during B cell development.

Topoisomerase 2ß (TOP2B) introduces transient double strand breaks in the DNA helix to remove supercoiling structures and unwind entangled DNA strains. Advances in genomic technologies have enabled the discovery of novel functions for TOP2B in processes such as releasing of the paused RNA polymerase II and maintaining the genome organization through DNA loop domains. Thus, TOP2B can regulate transcription directly by acting on transcription elongation and indirectly by controlling interactions between enhancer and promoter regions through genome folding. The identification of TOP2B mutations in humans unexpectedly revealed a unique role of TOP2B in B-cell progenitors. Here we discuss the functions of TOP2B and the mechanisms leading to the B-cell development defect in patients with TOP2B deficiency.

Catalytic Mechanism of ATP Hydrolysis in the ATPase Domain of Human DNA Topoisomerase IIα.

Human DNA topoisomerase IIα is a biological nanomachine that regulates the topological changes of the DNA molecule and is considered a prime target for anticancer drugs. Despite intensive research, many atomic details about its mechanism of action remain unknown. We investigated the ATPase domain, a segment of the human DNA topoisomerase IIα, using all-atom molecular simulations, multiscale quantum mechanics/molecular mechanics (QM/MM) calculations, and a point mutation study. The results suggested that the binding of ATP affects the overall dynamics of the ATPase dimer. Reaction modeling revealed that ATP hydrolysis favors the dissociative substrate-assisted reaction mechanism with the catalytic Glu87 serving to properly position and polarize the lytic water molecule. The point mutation study complemented our computational results, demonstrating that Lys378, part of the important QTK loop, acts as a stabilizing residue. The work aims to pave the way to a deeper understanding of these important molecular motors and to advance the development of new therapeutics.

A comprehensive structural analysis of the ATPase domain of human DNA topoisomerase II beta bound to AMPPNP, ADP, and the bisdioxopiperazine, ICRF193.

Human topoisomerase II beta (TOP2B) modulates DNA topology using energy from ATP hydrolysis. To investigate the conformational changes that occur during ATP hydrolysis, we determined the X-ray crystallographic structures of the human TOP2B ATPase domain bound to AMPPNP or ADP at 1.9 Å and 2.6 Å resolution, respectively. The GHKL domains of both structures are similar, whereas the QTK loop within the transducer domain can move for product release. As TOP2B is the clinical target of bisdioxopiperazines, we also determined the structure of a TOP2B:ADP:ICRF193 complex to 2.3 Å resolution and identified key drug-binding residues. Biochemical characterization revealed the N-terminal strap reduces the rate of ATP hydrolysis. Mutagenesis demonstrated residue E103 as essential for ATP hydrolysis in TOP2B. Our data provide fundamental insights into the tertiary structure of the human TOP2B ATPase domain and a potential regulatory mechanism for ATP hydrolysis.

Discovery of novel rigid analogs of 2-naphthol with potent anticancer activity through multi-target topoisomerase I & II and tyrosine kinase receptor EGFR & VEGFR-2 inhibition mechanism.

A novel oxygen-containing heterocyclic linked 1H-benzo[f]chromene moieties (4a-g) and (6a-g) with anti-proliferative activity against cancer cell lines was designed, synthesized, and established on the basis of spectral data. All the prepared compounds were evaluated in vitro for their anti-proliferative activity against MCF-7, HCT-116, HepG-2 cell lines and normal cell lines HFL-1, WI-38. Compounds 4a, 4b, and 6e exhibited good activity against MCF-7, HCT-116, and HepG-2 cell lines, comparable to that of Vinblastine and Doxorubicin, and weak active against normal cell lines. Moreover, the potential mechanisms of the cytotoxic activity of the promising compounds 4a, 4b, and 6e on the more sensitive cell line MCF-7 were studied. We found that compounds 4a, 4b, and 6e induce cell cycle arrest at G2/M phases for MCF-7 treated cells compared to untreated cells, which causes apoptosis and inhibits both the topoisomerase I and II enzymes. In addition, compounds 4a and 4b exhibited comparable inhibitory activity on tyrosine kinase receptors EGFR and VEGFR-2 kinases to that of the reference protein kinases inhibitor Sorafenib. The in silico molecular docking of the most active compounds into the active sites of EGFR kinase and Topo I & II enzymes provides us with a reasonable clarification of the interpreted biological data.

Topoisomerase VI is a chirally-selective, preferential DNA decatenase.

DNA topoisomerase VI (topo VI) is a type IIB DNA topoisomerase found predominantly in archaea and some bacteria, but also in plants and algae. Since its discovery, topo VI has been proposed to be a DNA decatenase; however, robust evidence and a mechanism for its preferential decatenation activity was lacking. Using single-molecule magnetic tweezers measurements and supporting ensemble biochemistry, we demonstrate that Methanosarcina mazei topo VI preferentially unlinks, or decatenates DNA crossings, in comparison to relaxing supercoils, through a preference for certain DNA crossing geometries. In addition, topo VI demonstrates a significant increase in ATPase activity, DNA binding and rate of strand passage, with increasing DNA writhe, providing further evidence that topo VI is a DNA crossing sensor. Our study strongly suggests that topo VI has evolved an intrinsic preference for the unknotting and decatenation of interlinked chromosomes by sensing and preferentially unlinking DNA crossings with geometries close to 90°.

Identification of novel topoisomerase II alpha inhibitors by virtual screening, molecular docking, and bioassay.

Breast cancer is one of the most common tumors, and its treatment still leaves room for improvement. Topoisomerase II alpha is a potential target for the treatment of human diseases such as breast cancer. In this article, we attempted to discover a novel anticancer drug. We have used the topoisomerase II alpha protein-Homo sapiens (Human) to hierarchically screen the Maybridge database. Based on their docking score, the top hit compounds have been assayed for inhibition in a topoisomerase II pBR322 DNA relaxation assay in vitro. Candidate compound 6 (CP6) was found to have the best inhibitory effect for topoisomerase II among the 20 tested compounds. In addition, CP6 had potent cytotoxicity against eight tested tumor cell lines. At the same time, CP6 was shown to have potential anti-multidrug resistance capabilities. This study identifies CP6, which can contribute to the development of new topoisomerase II inhibitors as anticancer agents.

Molecular dynamics simulations of sulfone derivatives in complex with DNA topoisomerase IIα ATPase domain.

Human topoisomerase II alpha (TopoIIα) is a crucial enzyme involved in maintaining genomic integrity during the process of DNA replication and mitotic division. It is a vital therapeutic target for designing novel anticancer agents in targeted cancer therapy. Sulfones, members of organosulfur compounds, have been reported to possess various biological activities such as antimicrobial, anti-inflammatory, anti-HIV, anticancer, and antimalarial properties. In the present study, a series of sulfones was selected to evaluate their inhibitory activity against TopoIIα using computational approaches. Molecular docking results revealed that several sulfone analogs bind efficiently to the ATPase domain of TopoIIα. Among them, sulfones 18a, 60a, *4 b, *8 b, *3c, and 8c exhibit higher binding affinity than the known TopoII inhibitor, salvicine. Molecular dynamics simulations and free energy calculations based on MM/PB(GB)SA method demonstrated that sulfone *8 b strongly interacts with amino acid residues in the ATP-binding pocket (E87, N91, D94, I125, I141, F142, S149, G161, and A167), driven mainly by an electrostatic attraction and a strong H-bond formation at G161 residue. Altogether, the obtained results predicted that sulfones could have a high potential to be a lead molecule for targeting TopoIIα.Communicated by Ramaswamy H. Sarma.

AK-I-190, a New Catalytic Inhibitor of Topoisomerase II with Anti-Proliferative and Pro-Apoptotic Activity on Androgen-Negative Prostate Cancer Cells.

Castration-resistant prostate cancer (CRPC) is a clinical challenge in treatment because of its aggressive nature and resistance to androgen deprivation therapy. Topoisomerase II catalytic inhibitors have been suggested as a strategy to overcome these issues. We previously reported AK-I-190 as a novel topoisomerase II inhibitor. In this study, the mechanism of AK-I-190 was clarified using various types of spectroscopic and biological evaluations. AK-I-190 showed potent topoisomerase II inhibitory activity through intercalating into DNA without stabilizing the DNA-enzyme cleavage complex, resulting in significantly less DNA toxicity than etoposide, a clinically used topoisomerase II poison. AK-I-190 induced G1 arrest and effectively inhibited cell proliferation and colony formation in combination with paclitaxel in an androgen receptor-negative CRPC cell line. Our results confirmed that topoisomerase II catalytic inhibition inhibited proliferation and induced apoptosis of AR-independently growing prostate cancer cells. These findings indicate the clinical relevance of topoisomerase II catalytic inhibitors in androgen receptor-negative prostate cancer.

Transcription-Replication Collisions-A Series of Unfortunate Events.

Transcription-replication interactions occur when DNA replication encounters genomic regions undergoing transcription. Both replication and transcription are essential for life and use the same DNA template making conflicts unavoidable. R-loops, DNA supercoiling, DNA secondary structure, and chromatin-binding proteins are all potential obstacles for processive replication or transcription and pose an even more potent threat to genome integrity when these processes co-occur. It is critical to maintaining high fidelity and processivity of transcription and replication while navigating through a complex chromatin environment, highlighting the importance of defining cellular pathways regulating transcription-replication interaction formation, evasion, and resolution. Here we discuss how transcription influences replication fork stability, and the safeguards that have evolved to navigate transcription-replication interactions and maintain genome integrity in mammalian cells.

Basic residues at the C-gate of DNA gyrase are involved in DNA supercoiling.

DNA gyrase is a type II topoisomerase that is responsible for maintaining the topological state of bacterial and some archaeal genomes. It uses an ATP-dependent two-gate strand-passage mechanism that is shared among all type II topoisomerases. During this process, DNA gyrase creates a transient break in the DNA, the G-segment, to form a cleavage complex. This allows a second DNA duplex, known as the T-segment, to pass through the broken G-segment. After the broken strand is religated, the T-segment is able to exit out of the enzyme through a gate called the C-gate. Although many steps of the type II topoisomerase mechanism have been studied extensively, many questions remain about how the T-segment ultimately exits out of the C-gate. A recent cryo-EM structure of Streptococcus pneumoniae GyrA shows a putative T-segment in close proximity to the C-gate, suggesting that residues in this region may be important for coordinating DNA exit from the enzyme. Here, we show through site-directed mutagenesis and biochemical characterization that three conserved basic residues in the C-gate of DNA gyrase are important for DNA supercoiling activity, but not for ATPase or cleavage activity. Together with the structural information previously published, our data suggest a model in which these residues cluster to form a positively charged region that facilitates T-segment passage into the cavity formed between the DNA gate and C-gate.

Facile One-Pot Multicomponent Synthesis of Pyrazolo-Thiazole Substituted Pyridines with Potential Anti-Proliferative Activity: Synthesis, In Vitro and In Silico Studies.

Pyrazolothiazole-substituted pyridine conjugates are an important class of heterocyclic compounds with an extensive variety of potential applications in the medicinal and pharmacological arenas. Therefore, herein, we describe an efficient and facile approach for the synthesis of novel pyrazolo-thiazolo-pyridine conjugate 4, via multicomponent condensation. The latter compound was utilized as a base for the synthesis of two series of 15 novel pyrazolothiazole-based pyridine conjugates (5-16). The newly synthesized compounds were fully characterized using several spectroscopic methods (IR, NMR and MS) and elemental analyses. The anti-proliferative impact of the new synthesized compounds 5-13 and 16 was in vitro appraised towards three human cancer cell lines: human cervix (HeLa), human lung (NCI-H460) and human prostate (PC-3). Our outcomes regarding the anti-proliferative activities disclosed that all the tested compounds exhibited cytotoxic potential towards all the tested cell lines with IC50 = 17.50-61.05 µM, especially the naphthyridine derivative 7, which exhibited the most cytotoxic potential towards the tested cell lines (IC50 = 14.62-17.50 µM) compared with the etoposide (IC50 = 13.34-17.15 µM). Moreover, an in silico docking simulation study was performed on the newly prepared compounds within topoisomerase II (3QX3), to suggest the binding mode of these compounds as anticancer candidates. The in silico docking results indicate that compound 7 was a promising lead anticancer compound which possesses high binding affinity toward topoisomerase II (3QX3) protein.