Relaxation Of Supercoiled DNA Research Articles

Dicobalt(ii) helices kill colon cancer cells via enantiomer-specific mechanisms; DNA damage or microtubule disruption.

Highly diastereoselective self-assembly reactions give both enantiomers (Λ and Δ) of anti-parallel triple-stranded bimetallic Co(ii) and Co(iii) cationic helices, without the need for resolution; the first such reaction for Co. The complexes are water soluble and stable, even in the case of Co(ii). Studies in a range of cancer and healthy cell lines indicate high activity and selectivity, and substantial differences between enantiomers. The oxidation state has little effect, and correspondingly, Co(iii) compounds are reduced to Co(ii) e.g. by glutathione. In HCT116 colon cancer cells the Λ enantiomer induces dose-dependent G2-M arrest in the cell cycle and disrupts microtubule architectures. This Co(ii) Λ enantiomer is ca. five times more potent than the isostructural Fe(ii) compound. Since the measured cellular uptakes are similar this implies a higher affinity of the Co system for the intracellular target(s); while the two systems are isostructural they have substantially different charge distributions as shown by calculated hydrophobicity maps. In contrast to the Λ enantiomer, Δ-Co(ii) induces G1 arrest in HCT116 cells, efficiently inhibits the topoisomerase I-catalyzed relaxation of supercoiled plasmid DNA, and, unlike the isostructural Fe(ii) system, causes DNA damage. It thus seems very likely that redox chemistry plays a role in the latter.

Characterization of Rheum palmatum mitochondrial genome and comparative analysis among Caryophyllales species

Objective: This work aimed to report the first complete mitochondrial genome (mitogenome) of Rheum palmatum, summarize the features of Caryophyllales mitogenomes, and to reveal the potential of utilizing the mitogenomes of R. palmatum and other Caryophyllales species for inferring phylogenetic relationships and species identification. Methods: Both Illumina short reads and PacBio HiFi reads were utilized to obtain a complete mitogenome of R. palmatum. A variety of bioinformatics tools were employed to characterize the R. palmatum mitogenome, compare the reported mitogenomes in Caryophyllales and conduct phylogenetic analysis. Results: The mitogenome of R. palmatum was assembled into a single master circle of 302,993 bp, encoding 35 known protein-coding genes, 18 transfer RNA genes, and three ribosome RNA genes. A total of 249 long repeats and 49 simple sequence repeats were identified in this mitogenome. The sizes of mitogenomes in Caryophyllales varied from 253 kb to 11.3 Mb. Among them, 23 mitogenomes were circular molecules, one was linear, and one consisted of relaxed circles, linear molecules, and supercoiled DNA. Out of the total mitogenomes, 11 were single-chromosome structure, whereas the remaining 14 were multi-chromosomal organizations. The phylogenetic analysis is consistent with both the Engler system (1964) and the Angiosperm Phylogeny Group III system. Conclusions: We obtained the first mitogenome of R. palmatum, which consists of a master circle. Mitogenomes in Caryophyllales have variable genome sizes and structures even within the same species. Circular molecules are still the dominant pattern in Caryophyllales. Single-chromosome mitogenomes account for nearly a half of all the mitogenomes in Caryophyllales, in contrast to previous studies. It is feasible to utilize mitochondrial genomes for inferring phylogenetic relationships and conducting species identification.

Antibacterial Activity and Mechanism of Madecassic Acid against Staphylococcus aureus.

Antibacterial resistance has become one of the most serious problems threating global health. To overcome this urgent problem, many scientists have paid great attention to developing new antibacterial drugs from natural products. Hence, for exploring new antibacterial drugs from Chinese medicine, a series of experiments were carried out for verifying and elucidating the antibacterial activity and mechanisms of madecassic acid (MA), which is an active triterpenoid compound isolated from the traditional Chinese medicine, Centella asiatica. The antibacterial activity was investigated through measuring the diameter of the inhibition zone, the minimum inhibitory concentration (MIC), the growth curve, and the effect on the bacterial biofilm, respectively. Meanwhile, the antibacterial mechanism was also discussed from the aspects of cell wall integrity variation, cell membrane permeability, and the activities of related enzymes in the respiratory metabolic pathway before and after the intervention by MA. The results showed that MA had an inhibitory effect on eight kinds of pathogenic bacteria, and the MIC values for Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Bacillus megaterium were 31.25, 62.5, 250, 125, 62.5, and 62.5 µg/mL, respectively. For instance, 31.25 µg/mL MA could inhibit the growth of Staphylococcus aureus within 28 h. The antibacterial mechanism experiments confirmed that MA could destroy the integrity of the cell membrane and cell wall of Staphylococcus aureus, causing the leakage of macromolecular substances, inhibiting the synthesis of soluble proteins, reducing the activities of succinate dehydrogenase and malate dehydrogenase, and interacting with DNA, leading to the relaxation and ring opening of supercoiled DNA. Besides, the activities of DNA topoisomerase I and II were both inhibited by MA, which led to the cell growth of Staphylococcus aureus being repressed. This study provides a theoretical basis and reference for the application of MA in the control and inhibition of food-borne Staphylococcus aureus.

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.

G-quadruplex-containing oligodeoxynucleotides as DNA topoisomerase I inhibitors

DNA topoisomerase I was found to be highly abundant in fast-proliferating tumor cells and is a potential target for anticancer therapy. A series of G-quadruplex-containing oligodeoxynucleotides (ODNs) were designed and used as inhibitors of DNA topoisomerase I. It was demonstrated that ODNs with G-quadruplexes can efficiently inhibit the supercoiled DNA relaxation reaction catalyzed by DNA topoisomerase I. Compared with the other conformations, the parallel propeller-type G-quadruplex was the most efficient DNA topoisomerase I inhibitor. Further studies revealed that integrating G-quadruplexes with duplexes to form quadruplex-duplex hybrids could significantly improve the inhibition efficiency. In addition, a circular ODN that consists of a G-quadruplex motif and DNA topoisomerase I binding site was synthesized and used as a DNA topoisomerase I inhibitor. The results showed that the particularly designed circular ODN displayed high inhibitory efficiency on the activity of DNA topoisomerase I with an IC50 value of 54.8 nM. Moreover, the circular ODN exhibited excellent thermal stability and nuclease resistance. Considering the low cytotoxicity of DNA-based biopharmaceuticals, the design strategy and results reported in this study may shed new light on nucleic acid-based DNA topoisomerase I inhibitor construction for potential anticancer drugs.

AFM imaging of the transcriptionally active chromatin in mammalian cells' nuclei

BackgroundNuclear rigidity is traditionally associated with lamina and densely packed heterochromatin. Actively transcribed DNA is thought to be less densely packed. Currently, approaches for direct measurements of the transcriptionally active chromatin rigidity are quite limited. MethodsIsolated nuclei were subjected to mechanical stress at 60 g and analyzed by Atomic Force Microscopy (AFM). ResultsNuclei of the normal fibroblast cells were completely flattened under mechanical stress, whereas nuclei of the cancerous HeLa were extremely resistant. In the deformed HeLa nuclei, AFM revealed a highly-branched landscape assembled of ~400 nm closed-packed globules and their structure was changing in response to external influence. Normal and cancerous cells' isolated nuclei were strikingly different by DNA resistance to applied mechanical stress. Paradoxically, more transcriptionally active and less optically dense chromatin of the nuclei of the cancerous cells demonstrated higher physical rigidity. A high concentration of the transcription inhibitor actinomycin D led to complete flattening of HeLa nuclei, that might be related to the relaxation of supercoiled DNA tending to deformation. At a low concentration of actinomycin D, we observed the intermediary formation of stochastically distributed nanoloops and nanofilaments with different shapes but constant width ~ 180 nm. We related this phenomenon with partial DNA relaxation, while non-relaxed DNA still remained rigid. ConclusionsThe resistance to deformation of nuclear chromatin correlates with fundamental biological processes in the cell nucleus, such as transcription, as assessed by AFM. General significanceA new outlook to studying internal nuclei structure is proposed.

The Effects of DNA Topoisomerase 1 Alteration in Herpes Simplex Virus Type 1 Infections

Herpes simplex virus type 1 (HSV‐1) is a universal human pathogen that infects nearly 70% of the population. In addition to producing its own viral factors, HSV‐1 utilizes numerous host proteins to facilitate its infection cycle. One host protein of particular interest, found to specifically interact with the HSV‐1 genome throughout replication, is human topoisomerase 1 (Top1). In human cells, Top1 is an enzyme required for normal growth and development. Cellular Top1 ensures proper transcription and DNA replication by creating single stranded nicks in DNA to relieve topological stress and allow for relaxation of supercoiled DNA. Based on its role in cellular processes, we believe that Top1 aids in the regulation of viral transcription and/or replication. We therefore hypothesize that altering Top1 expression in HSV‐1 infected cells will decrease viral yield by not allowing for an efficient infection cycle. To alter Top1 expression, the commercial inhibitor, camptothecin (CPT) was used. Our studies revealed the novel observation that Top1 inhibition via CPT does reduce viral yield. Currently, an inducible Top1 shRNA knockdown cell line is being created as another method of alteration. In the future, we will determine if viral transcription or DNA replication are altered after Top1 inhibition or knockdown. These studies will further pinpoint the role of Top1 in HSV‐1 infection.

Trans-Dichloro(triphenylarsino)(N,N-dialkylamino)platinum(II) Complexes: In Search of New Scaffolds to Circumvent Cisplatin Resistance.

The high incidence of the resistance phenomenon represents one of the most important limitations to the clinical usefulness of cisplatin as an anticancer drug. Notwithstanding the considerable efforts to solve this problem, the circumvention of cisplatin resistance remains a challenge in the treatment of cancer. In this work, the synthesis and characterization of two trans-dichloro(triphenylarsino)(N,N-dialkylamino)platinum(II) complexes (1 and 2) were described. The trypan blue exclusion assay demonstrated an interesting antiproliferative effect for complex 1 in ovarian carcinoma-resistant cells, A2780cis. Quantitative analysis performed by ICP-AES demonstrated a scarce ability to platinate DNA, and a significant intracellular accumulation. The investigation of the mechanism of action highlighted the ability of 1 to inhibit the relaxation of supercoiled plasmid DNA mediated by topoisomerase II and to stabilize the cleavable complex. Cytofluorimetric analyses indicated the activation of the apoptotic pathway and the mitochondrial membrane depolarization. Therefore, topoisomerase II and mitochondria could represent possible intracellular targets. The biological properties of 1 and 2 were compared to those of the related trans-dichloro(triphenylphosphino)(N,N-dialkylamino)platinum(II) complexes in order to draw structure–activity relationships useful to face the resistance phenotype.

Copper(II) and silver(I)-1,10-phenanthroline-5,6-dione complexes interact with double-stranded DNA: further evidence of their apparent multi-modal activity towards Pseudomonas aeruginosa

Tackling microbial resistance requires continuous efforts for the development of new molecules with novel mechanisms of action and potent antimicrobial activity. Our group has previously identified metal-based compounds, [Ag(1,10-phenanthroline-5,6-dione)2]ClO4 (Ag-phendione) and [Cu(1,10-phenanthroline-5,6-dione)3](ClO4)2.4H2O (Cu-phendione), with efficient antimicrobial action against multidrug-resistant species. Herein, we investigated the ability of Ag-phendione and Cu-phendione to bind with double-stranded DNA using a combination of in silico and in vitro approaches. Molecular docking revealed that both phendione derivatives can interact with the DNA by hydrogen bonding, hydrophobic and electrostatic interactions. Cu-phendione exhibited the highest binding affinity to either major (− 7.9 kcal/mol) or minor (− 7.2 kcal/mol) DNA grooves. In vitro competitive quenching assays involving duplex DNA with Hoechst 33258 or ethidium bromide demonstrated that Ag-phendione and Cu-phendione preferentially bind DNA in the minor grooves. The competitive ethidium bromide displacement technique revealed Cu-phendione has a higher binding affinity to DNA (Kapp = 2.55 × 106 M−1) than Ag-phendione (Kapp = 2.79 × 105 M−1) and phendione (Kapp = 1.33 × 105 M−1). Cu-phendione induced topoisomerase I-mediated DNA relaxation of supercoiled plasmid DNA. Moreover, Cu-phendione was able to induce oxidative DNA injuries with the addition of free radical scavengers inhibiting DNA damage. Ag-phendione and Cu-phendione avidly displaced propidium iodide bound to DNA in permeabilized Pseudomonas aeruginosa cells in a dose-dependent manner as judged by flow cytometry. The treatment of P. aeruginosa with bactericidal concentrations of Cu-phendione (15 µM) induced DNA fragmentation as visualized by either agarose gel or TUNEL assays. Altogether, these results highlight a possible novel DNA-targeted mechanism by which phendione-containing complexes, in part, elicit toxicity toward the multidrug-resistant pathogen P. aeruginosa.Graphical abstract

Topoisomerase Assays.

Topoisomerases are enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of DNA topoisomerases: type I enzymes, which make single-stranded cuts in DNA, and type II enzymes, which cut and decatenate double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. Provided in this article are protocols to assess activities of topoisomerases and their inhibitors. Included are an assay for topoisomerase I activity based on relaxation of supercoiled DNA; an assay for topoisomerase II based on the decatenation of double-stranded DNA; and approaches for enriching and quantifying DNA-protein covalent complexes formed as obligatory intermediates in the reactions of type I and II topoisomerases with DNA; and assays for measuring DNA cleavage in vitro. Topoisomerases are not the only proteins that form covalent adducts with DNA in living cells, and the approaches described here are likely to find use in characterizing other protein-DNA adducts and exploring their utility as targets for therapy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Assay of topoisomerase I activity Basic Protocol 2: Assay of topoisomerase II activity Basic Protocol 3: In vivo determination of topoisomerase covalent complexes using the in vivo complex of enzyme (ICE) assay Support Protocol 1: Preparation of mouse tissue for determination of topoisomerase covalent complexes using the ICE assay Support Protocol 2: Using recombinant topoisomerase standard for absolute quantification of cellular TOP2CC Basic Protocol 4: Quantification of topoisomerase-DNA covalent complexes by RADAR/ELISA: The rapid approach to DNA adduct recovery (RADAR) combined with the enzyme-linked immunosorbent assay (ELISA) Basic Protocol 5: Analysis of protein-DNA covalent complexes by RADAR/Western Support Protocol 3: Adduct-Seq to characterize adducted DNA Support Protocol 4: Nuclear fractionation and RNase treatment to reduce sample complexity Basic Protocol 6: Determination of DNA cleavage by purified topoisomerase I Basic Protocol 7: Determination of inhibitor effects on DNA cleavage by topoisomerase II using a plasmid linearization assay Alternate Protocol: Gel electrophoresis determination of topoisomerase II cleavage.

DNA-Topology Simplification by Topoisomerases.

The topological properties of DNA molecules, supercoiling, knotting, and catenation, are intimately connected with essential biological processes, such as gene expression, replication, recombination, and chromosome segregation. Non-trivial DNA topologies present challenges to the molecular machines that process and maintain genomic information, for example, by creating unwanted DNA entanglements. At the same time, topological distortion can facilitate DNA-sequence recognition through localized duplex unwinding and longer-range loop-mediated interactions between the DNA sequences. Topoisomerases are a special class of essential enzymes that homeostatically manage DNA topology through the passage of DNA strands. The activities of these enzymes are generally investigated using circular DNA as a model system, in which case it is possible to directly assay the formation and relaxation of DNA supercoils and the formation/resolution of knots and catenanes. Some topoisomerases use ATP as an energy cofactor, whereas others act in an ATP-independent manner. The free energy of ATP hydrolysis can be used to drive negative and positive supercoiling or to specifically relax DNA topologies to levels below those that are expected at thermodynamic equilibrium. The latter activity, which is known as topology simplification, is thus far exclusively associated with type-II topoisomerases and it can be understood through insight into the detailed non-equilibrium behavior of type-II enzymes. We use a non-equilibrium topological-network approach, which stands in contrast to the equilibrium models that are conventionally used in the DNA-topology field, to gain insights into the rates that govern individual transitions between topological states. We anticipate that our quantitative approach will stimulate experimental work and the theoretical/computational modeling of topoisomerases and similar enzyme systems.

Probing the Impact of Serine and Threonine Patches in the C‐terminus on the Catalytic of Human Topoisomerase IIα

Topoisomerase IIα is an essential nuclear enzyme involved in resolving knots and tangles in DNA during replication and cell division. The enzyme is a homodimer with a complex, multidomain structure. While the N-terminal and core regions of the protein are well-studied, the C-terminal domain is poorly understood but appears to be involved in enzyme regulation. In addition, the C-terminus varies widely among isoforms of topoisomerase II and appears to be a major region of post-translational modification including several patches of Ser and Thr residues. Many of these Ser and Thr residues have not been studied for biochemical effects. Therefore, we generated a series of mutant versions of human topoisomerase IIα where we selectively changed patches of Ser and Thr residues in the C-terminal domain to Ala and Gly residues. We designed, purified, and examined the activity of eleven mutant enzymes for changes in catalytic activity. The amino acid changes were made in the region of positions 1272 to 1525 with 1-7 residues changed per mutant. Several mutants displayed varying increased levels of DNA cleavage without displaying any change in plasmid DNA relaxation or DNA binding. For example, mutations in the regions 1272-1279, 1324-1343, 1351-1365, and 1374-1377 all had larger increases in DNA cleavage in the presence of etoposide when compared with wild-type topoisomerase IIα. One mutant displayed decreased DNA cleavage below wild-type levels. This mutant had changes between residues 1469-1476 and displayed a decreased ability to both relax supercoiled DNA and to bind to plasmid DNA. Importantly, several residues in this region (Ser1469, Thr1470, Ser1471, and Ser1474) are known to have a role in mitosis indicating these are key regulators of enzyme function. Together, these results indicate that the C-terminal domain of topoisomerase IIα influences catalytic activity and likely interacts with substrate DNA at various positions. Additional studies are warranted to explore these results in more detail.

DNA Binding Interactions and DNA Topoisomerase I Inhibition Activities of Crude Extracts from <i>Annona Squamosa</i> (L.) and <i>Annona Muricata</i> (L.) Fruits

Some plants metabolites serve as antiprotozoal and antitumour by binding to nuclear enzyme; DNA Topo I affecting DNA function and cell survival. This study was aimed at screening DNA binding interactions and DNA Topo I inhibitory activity of crude extracts from fruits of Annona muricata (L) and Annona squamosa (L) which can form the basis of developing efficacious, safe and low cost antiprotozoal and antitumor agents. Aqueous, Methanolic, Ethyl acetate and Hexane extracts from fruits of two hypothesized antiprotozoal and antitumour plants; Annona muricata (L) and Annona squamosa (L) were screened for DNA-binding interaction and DNA Topo I inhibition. For DNA-methyl green test, 50 µL crude extracts were incubated with 200 μL DNA-methyl green in darkness at 25°C for 24 hours. Absorbance decrease at 650 nm using UV-vis spectrophotometer was calculated as a percentage of untreated DNA-methyl green value whereas with IC50 calculated by regression analysis. For DNA Topo I inhibitory activity, crude extracts were incubated in 10 µg/mL with 0.5 µg of supercoiled pBR322 DNA and 1U of DNA Topo I at 37°C for 2 hours, reaction terminated using stop buffer containing 3% SDS, 60 mM EDTA, 50% glycerol, 0.25% bromophenol blue. Products were determined by electrophoresis on 1% agarose gel in Tris-acetate-EDTA (TAE) running buffer at 65 V/cm for 2 hours. 24 extracts were studied, percentage decrease in absorbance were between 18.14±2.67 - 38.06±1.47 (Aqueous), 17.14±2.67 - 41.01±1.09% (Methanolic), 9.05±1.67 - 20.50±2.01% (Ethyl acetate) and 4.04±1.12 - 10.09±1.39% (Hexane)., IC50 values were between 50 μg/mL – 100 μg/mL (6), 100 μg/mL – 150 μg/mL (8), 150 μg/mL – 200 μg/mL (7) and <200 μg/mL (3). The activity against DNA Topo I mediated relaxation of supercoiled pBR322 DNA at 5 µM, 25 µM and 100 µM observed in 8 hits with percentage decrease in absorbance between 17.14±2.67 - 40.01±1.09% with IC50 between 62.97±3.37 μg/mL - 131.37±10.77 μg/mL. The extracts of A. muricata and A. squamosa showed DNA Topo I inhibitory activities by inhibiting the relaxation of supercoiled DNA pBR322. However, further studies need to be conducted on the purified fractions of aqueous and methanolic extracts.

Novel 2′-alkoxymethyl substituted klavuzon derivatives as inhibitors of Topo I and CRM1

In this work, 2′-alkoxymethyl substituted klavuzon derivatives were prepared starting from 2-methyl-1-naphthoic acid in eight steps. Anticancer potencies of the synthesized compounds were evaluated by performing MTT cell viability test over cancerous and healthy pancreatic cell lines, along with CRM1 inhibitory properties in HeLa cells by immunostaining and Topo I inhibition properties by supercoiled DNA relaxation assay. Their cytotoxic activities were also presented in hepatocellular carcinoma cells (HuH-7) derived 3D spheroids. Among the tested klavuzon derivatives, isobutoxymethyl substituted klavuzon showed the highest selectivity of cytotoxic activity against pancreatic cancer cell line. They showed potent Topo I inhibition while their CRM1 inhibitory properties somehow diminished compared to 4′-alkylsubstituted klavuzons. The most cytotoxic 2′-methoxymethyl derivative inhibited the growth of the spheroids derived from HuH-7 cell lines and PI staining exhibited time and concentration dependent cell death in 3D spheroids.

3,6-Disubstituted 1,2,4-Triazolo[3,4-b]Thiadiazoles with Anticancer Activity Targeting Topoisomerase II Alpha.

BackgroundTopoisomerase IIα (topIIα) maintains the topology of DNA in order to ensure the proper functioning of numerous DNA processes. Inhibition of topIIα leads to the killing of cancer cells thus constituting such inhibitors as useful tools in cancer therapeutics. Triazolo[3,4-b]thiadiazole derivatives are known for their wide range of pharmacological activities while previous studies have documented their in vitro anticancer activity. The purpose of the current study was to investigate if these chemical compounds can act as topIIα inhibitors in cell-free and cell-based systems.Materials and MethodsThe MTT assay was performed in DLD-1, HT-29, and LoVo cancer cells so as to evaluate the antiproliferative activity of KA25, KA26, and KA39 triazolo[3,4-b]thiadiazole derivatives. The KA39 compound was tested as a potential topIIα inhibitor using the plasmid-based topoisomerase II drug screening kit. The inhibitory effect of the three derivatives on topIIα phosphorylation was studied in HT-29 and LoVo cancer cells according to Human Phospho-TOP2A/Topoisomerase II Alpha Cell-Based Phosphorylation ELISA Kit. Moreover, flow cytometry was utilized in order to explore apoptotic induction and cell cycle growth arrest, upon treatment with KA39, in DLD-1 and HT-29 cells, respectively. In silico studies were also carried out for further investigation.ResultsAll three triazolo[3,4-b]thiadiazole derivatives showed an in vitro antiproliferative effect with the KA39 compound being the most potent one. Our results indicated that KA39 induced both early and late apoptosis as well as cell cycle growth arrest in S phase. In addition, the compound blocked the relaxation of supercoiled DNA while it also inhibited topIIα phosphorylation (upon treatment; P<0.001).ConclusionAmong the three triazolo[3,4-b]thiadiazole derivatives, KA39 was shown to be the most potent anticancer agent and catalytic inhibitor of topIIα phosphorylation as well.

Synthesis, Biological Evaluation and Molecular Docking Study of Cyclic Diarylheptanoids as Potential Anticancer Therapeutics.

Cancer is one of the leading causes of mortality globally. To cope with cancer, it is necessary to develop anticancer drugs. Bioactive natural products, i.e. diarylheptanoids, have gained significant attention of researchers owing to their intriguing structures and potent biological activities. In this article, considering the development of anticancer drugs with enhanced selectivity towards cancerous cells, a series of Cyclic Diarylheptanoids (CDHs) are designed, synthesized and evaluated their biological activity. To establish an easy route for the synthesis of diarylheptanoids, and evaluate their antiproliferative, and topoisomerase-I & -IIα inhibitory activities, for developing potential anticancer drugs among CDHs. Diarylheptanoids were synthesized from reported linear diarylheptanoids using the classical Ullmann reaction. Antibacterial activity was evaluated by the filter paper disc diffusion method. Cell viability was assessed by measuring mitochondrial dehydrogenase activity with a Cell Counting Kit (CCK-8). Topoisomerases I and II (topo-I and -IIα) inhibitory activity was measured by the assessment of relaxation of supercoiled pBR322 plasmid DNA. IFD protocol of Schrodinger Maestro v11.1 was used to characterize the binding pattern of studied compounds with the ATPase domain of the human topo-IIα. The synthesized CDHs were evaluated for their biological activities (antibacterial, antiproliferative, and topoisomerase-I & -IIα inhibitory activities, respectively). Leading to obtain a series of anticancer agents with the least inhibitory activities against different microbes, improving their selectivity for cancer cells. In brief, most of the synthesized CDHs had excellent antiproliferative activity against T47D (human breast cancer cell line). Pterocarine possessed the strongest activity (2i; IC50 = 0.63µM) against T47D. The cyclic diarylheptanoid 2b induced 30% inhibition of topoisomerase-IIα activity at 100μM compared with the reference of etoposide, which induced 72% inhibition. Among the tested compounds, galeon (2h) displayed very low activity against four bacterial strains. Compounds 2b, 2h, and 2i formed hydrogen bonds with Thr215, Asn91, Asn120, Ala167, Lys168 and Ile141 residues, which are important for binding of ligand compound to the ATPase binding site of topoisomerase IIα by acting as ATP competitive molecule validated by docking study. In silico Absorption, Distribution, Metabolism and Excretion (ADME) analysis revealed the predicted ADME parameters of the studied compounds which showed recommended values. A series of CDHs were synthesized and evaluated for their antibacterial, antiproliferative, and topo-I & -IIα inhibitory activities. SARs study, molecular docking study and in silico ADME analysis were conducted. Five compounds exhibited excellent and selective antiproliferative activity against the human breast cancer cell line (T47D). Among them, a compound 2h showed topo-IIα activity by 30% at 100µM, which represented a moderate intensity of inhibition compared with etoposide. Three of them formed hydrogen bonds with Thr215, Asn91, Asn120, and Ala167 residues, which are considered as crucial residues for binding to the ATPase domain of topoisomerase IIα. According to in silico drug-likeness property analysis, three compounds are expected to show superiority over etoposide in case of absorption, distribution, metabolism and excretion.

(S)-10-Hydroxycamptothecin Inhibits Esophageal Squamous Cell Carcinoma Growth In Vitro and In Vivo Via Decreasing Topoisomerase I Enzyme Activity.

Topoisomerase (TOP) I plays a major role in the process of supercoiled DNA relaxation, thereby facilitating DNA replication and cell cycle progression. The expression and enzymatic activity of TOP I is positively correlated with tumor progression. Although the anticancer activity of (S)-10-Hydroxycamptothecin (HCPT), a TOP I specific inhibitor, has been reported in various cancers, the effect of HCPT on esophageal cancer is yet to be examined. In this study, we investigate the potential of HCPT to inhibit the growth of ESCC cells in vitro and verify its anti-tumor activity in vivo by using a patient-derived xenograft (PDX) tumor model in mice. Our study revealed the overexpression of TOP I in ESCC cells and treatment with HCPT inhibited TOP I enzymatic activity at 24 h and decreased expression at 48 h and 72 h. HCPT also induced DNA damage by increasing the expression of H2A.XS139. HCPT significantly decreased the proliferation and anchorage-independent growth of ESCC cells (KYSE410, KYSE510, KYSE30, and KYSE450). Mechanistically, HCPT inhibited the G2/M phase cell cycle transition, decreased the expression of cyclin B1, and elevated p21 expression. In addition, HCPT stimulated ESCC cells apoptosis, which was associated with elevated expression of cleaved PARP, cleaved caspase-3, cleaved caspase-7, Bax, Bim, and inhibition of Bcl-2 expression. HCPT dramatically suppressed PDX tumor growth and decreased the expression of Ki-67 and TOP I and increased the level of cleaved caspase-3 and H2A.XS139 expression. Taken together, our data suggested that HCPT inhibited ESCC growth, arrested cell cycle progression, and induced apoptosis both in vitro and in vivo via decreasing the expression and activity of TOP I enzyme.

Clarifying the Mechanism of Copper(II) α-(N)-Heterocyclic Thiosemicarbazone Complexes on DNA Topoisomerase IIα and IIβ.

Topoisomerase II is a nuclear enzyme involved in the maintenance of DNA and is an effective anticancer drug target. However, several clinical topoisomerase II-targeted agents display significant off-target toxicities and adverse events. Thus, it is important to continue characterizing compounds with activity against topoisomerase II. We previously analyzed α-(N)-heterocyclic thiosemicarbazone copper(II) complexes against human topoisomerase IIα (TOP2A), but humans also express topoisomerase IIβ (TOP2B), which has distinct functional roles. Therefore, we examined two α-(N)-heterocyclic thiosemicarbazone copper [Cu(II)] complexes for activity against TOP2B in a purified system. The Cu(II) complexes, Cu(APY-ETSC)Cl and Cu(BZP-ETSC)Cl, were examined using plasmid DNA cleavage, supercoiled DNA relaxation, enzyme inactivation, protein cross-linking, DNA ligation, and ATP hydrolysis assays with TOP2B to determine whether these compounds act similarly against both enzymes. Both of the Cu(II) thiosemicarbazone (Cu-TSC) complexes we tested disrupted the function of TOP2B in a way similar to the effect on TOP2A. In particular, TOP2B DNA cleavage activity is increased in the presence of these compounds, while the relaxation and ATPase activities are inhibited. Further, both Cu-TSCs stabilize the N-terminal DNA clamp of TOP2A and TOP2B and rapidly inactivate TOP2B when the compounds are present before DNA. Our data provide evidence that the Cu-TSC complexes we tested utilize a similar mechanism against both isoforms of the enzyme. This mechanism may involve interaction with the ATPase domain of TOP2A and TOP2B outside of the ATP binding pocket. Additionally, these data support a model of TOP2 function where the ATPase domain communicates with the DNA cleavage/ligation domain.

Complete labelling of pneumococcal DNA-binding proteins with seleno-L-methionine

Streptococcus pneumoniae is a pathogenic and opportunistic Gram-positive bacterium that is the leading cause of community-acquired respiratory diseases, varying from mild- to deathly- infections. The appearance of antibiotic-resistant isolates has prompted the search for novel strategies and targets to tackle the bacterial resistances. One of the most promising approaches is the structure-based knowledge of possible targets in conjunction with rational design and docking of inhibitors of the chosen targets. A useful technique that helps to solve protein structures is to label them with an amino acid derivative like seleno-methionine that facilitates tracing of some of the amino acid residues. We have chosen two pneumococcal DNA-binding proteins, namely the relaxase domain of MobM protein from plasmid pMV158, and the RelB-RelE antitoxin-toxin protein complex. Through several changes that improve substantially a previous protocol (Budisa et al., 1995), we have used seleno-L-methionine to incorporate selenium into the amino acid sequence of the selected proteins. We have achieved 100% labelling of the proteins and could demonstrate that the labelled proteins retained full activity as judged from the relaxation of supercoiled plasmid DNA and from gel-retardation assays.

Crystal structure of the 65-kilodalton amino-terminal fragment of DNA topoisomerase I from the gram-positive model organism Streptococcus mutans

Herein we report the first structure of topoisomerase I determined from the gram-positive bacterium, S. mutans. Bacterial topoisomerase I is an ATP-independent type 1A topoisomerase that uses the inherent torsional strain within hyper-negatively supercoiled DNA as an energy source for its critical function of DNA relaxation. Interest in the enzyme has gained momentum as it has proven to be essential in various bacterial organisms. In order to aid in further biochemical characterization, the apo 65-kDa amino-terminal fragment of DNA topoisomerase I from the gram-positive model organism Streptococcus mutans was crystalized and a three-dimensional structure was determined to 2.06 Å resolution via x-ray crystallography. The overall structure illustrates the four classic major domains that create the traditional topoisomerase I “lock” formation comprised of a sizable toroidal aperture atop what is considered to be a highly dynamic body. A catalytic tyrosine residue resides at the interface between two domains and is known to form a 5’ phosphotyrosine DNA-enzyme intermediate during transient single-stranded cleavage required for enzymatic relaxation of hyper negative DNA supercoils. Surrounding the catalytic tyrosine residue is the remainder of the highly conserved active site. Within 5 Å from the catalytic center, only one dissimilar residue is observed between topoisomerase I from S. mutans and the gram-negative model organism E. coli. Immediately adjacent to the conserved active site, however, S. mutans topoisomerase I displays a somewhat unique nine residue loop extension not present in any bacterial topoisomerase I structures previously determined other than that of an extremophile.