Dimethylmyricacene: An In Vitro and In Silico Study of a Semisynthetic Non-Camptothecin Derivative Compound, Targeting Human DNA Topoisomerase 1B.

Human topoisomerase 1B regulates the topological state of supercoiled DNA enabling all fundamental cell processes. This enzyme, which is the unique molecular target of the natural anticancer compound camptothecin, acts by nicking one DNA strand and forming a transient protein-DNA covalent complex. The interaction of human topoisomerase 1B and dimethylmyricacene, a compound prepared semisynthetically from myricanol extracted from Myrica cerifera root bark, was investigated using enzymatic activity assays and molecular docking procedures. Dimethylmyricacene was shown to inhibit both the cleavage and the religation steps of the enzymatic reaction, and cell viability of A-253, FaDu, MCF-7, HeLa and HCT-116 tumor cell lines.

From Antarctica to cancer research: a novel human DNA topoisomerase 1B inhibitor from Antarctic sponge Dendrilla antarctica.

Nature has been always a great source of possible lead compounds to develop new drugs against several diseases. Here we report the identification of a natural compound, membranoid G, derived from the Antarctic sponge Dendrilla antarctica displaying an in vitro inhibitory activity against human DNA topoisomerase 1B. The experiments indicate that membranoid G, when pre-incubated with the enzyme, strongly and irreversibly inhibits the relaxation of supercoiled DNA. This compound completely inhibits the cleavage step of the enzyme catalytic mechanism by preventing protein binding to the DNA. Membranoid G displays also a cytotoxic effect on tumour cell lines, suggesting its use as a possible lead compound to develop new anticancer drugs.

Cow Milk Extracellular Vesicle Effects on an In Vitro Model of Intestinal Inflammation.

Extracellular vesicles (EVs) are lipid bilayer nano-dimensional spherical structures and act mainly as signaling mediators between cells, in particular modulating immunity and inflammation. Milk-derived EVs (mEVs) can have immunomodulatory and anti-inflammatory effects, and milk is one of the most promising food sources of EVs. In this context, this study aimed to evaluate bovine mEVs anti-inflammatory and immunomodulating effects on an in vitro co-culture (Caco-2 and THP-1) model of intestinal inflammation through gene expression evaluation with RT-qPCR and cytokine release through ELISA. After establishing a pro-inflammatory environment due to IFN-γ and LPS stimuli, CXCL8, IL1B, TNFA, IL12A, IL23A, TGFB1, NOS2, and MMP9 were significantly up-regulated in inflamed Caco-2 compared to the basal co-culture. Moreover, IL-17, IL-1ß, IL-6, TNF-α release was increased in supernatants of THP-1. The mEV administration partially restored initial conditions with an effective anti-inflammatory activity. Indeed, a decrease in gene expression and protein production of most of the tested cytokines was detected, together with a significant gene expression decrease in MMP9 and the up-regulation of MUC2 and TJP1. These results showed a fundamental capability of mEVs to modulate inflammation and their potential beneficial effect on the intestinal mucosa.

In Vitro and In Silico Characterization of an Antimalarial Compound with Antitumor Activity Targeting Human DNA Topoisomerase IB.

Human DNA topoisomerase IB controls the topological state of supercoiled DNA through a complex catalytic cycle that consists of cleavage and religation reactions, allowing the progression of fundamental DNA metabolism. The catalytic steps of human DNA topoisomerase IB were analyzed in the presence of a drug, obtained by the open-access drug bank Medicines for Malaria Venture. The experiments indicate that the compound strongly and irreversibly inhibits the cleavage step of the enzyme reaction and reduces the cell viability of three different cancer cell lines. Molecular docking and molecular dynamics simulations suggest that the drug binds to the human DNA topoisomerase IB-DNA complex sitting inside the catalytic site of the enzyme, providing a molecular explanation for the cleavage-inhibition effect. For all these reasons, the aforementioned drug could be a possible lead compound for the development of an efficient anti-tumor molecule targeting human DNA topoisomerase IB.

Natural Compounds as Therapeutic Agents: The Case of Human Topoisomerase IB.

Natural products are widely used as source for drugs development. An interesting example is represented by natural drugs developed against human topoisomerase IB, a ubiquitous enzyme involved in many cellular processes where several topological problems occur due the formation of supercoiled DNA. Human topoisomerase IB, involved in the solution of such problems relaxing the DNA cleaving and religating a single DNA strand, represents an important target in anticancer therapy. Several natural compounds inhibiting or poisoning this enzyme are under investigation as possible new drugs. This review summarizes the natural products that target human topoisomerase IB that may be used as the lead compounds to develop new anticancer drugs. Moreover, the natural compounds and their derivatives that are in clinical trial are also commented on.

Swapping of The N-Terminal Domain of Human Topoisomerase 1B with the Corresponding Plasmodium Falciparum Counterpart Strongly Impairs Enzyme Activity.

BACKGROUND: DNA topoisomerases 1B are a class of ubiquitous enzyme that solves the topological problems associated with biological processes such as replication, transcription and recombination. Numerous sequence alignment of topoisomerase 1B from different species shows that the lengths of different domains as well as their amino acids sequences are quite different. In the present study a hybrid enzyme, generated by swapping the N-terminal of Plasmodium falciparum into the corresponding domain of the human, has been characterized. METHODS: The chimeric enzyme was generated using different sets of PCR. The in vitro characterization was carried out using different DNA substrate including radio-labelled oligonucleotides. RESULTS: The chimeric enzyme displayed slower relaxation activity, cleavage and re-ligation kinetics strongly perturbed when compared to the human enzyme. CONCLUSION: These results indicate that the N-terminal domain has a crucial role in modulating topoisomerase activity in different species.

Topoisomerase IB: a relaxing enzyme for stressed DNA.

DNA topoisomerase I enzymes relieve the torsional strain in DNA; they are essential for fundamental molecular processes such as DNA replication, transcription, recombination, and chromosome condensation; and act by cleaving and then religating DNA strands. Over the past few decades, scientists have focused on the DNA topoisomerases biological functions and established a unique role of Type I DNA topoisomerases in regulating gene expression and DNA chromosome condensation. Moreover, the human enzyme is being investigated as a target for cancer chemotherapy. The active site tyrosine is responsible for initiating two transesterification reactions to cleave and then religate the DNA backbone, allowing the release of superhelical tension. The different steps of the catalytic mechanism are affected by various inhibitors; some of them prevent the interaction between the enzyme and the DNA while others act as poisons, leading to TopI-DNA lesions, breakage of DNA, and eventually cellular death. In this review, our goal is to provide an overview of mechanism of human topoisomerase IB action together with the different types of inhibitors and their effect on the enzyme functionality.

The human DNA topoisomerase I mutant Gly717Asp: Higher religation rate is not always associated with camptothecin resistance.

DNA topoisomerases are key enzyme responsible for modulating the topological state of the DNA by breaking and rejoining of DNA strand. Characterization of a Gly717Asp mutation in the human topoisomerase was performed using several catalytic assays. The mutant enzyme was shown to have comparable cleavage and fast religation rate as compared to the wild-type protein. Addition of the anticancer drug camptothecin significantly reduced the religation step. The simulative approaches and analysis of the cleavage/religation equilibrium indicate that the mutation is able to modify the architecture of the drug binding site, increasing the persistence of the drug for the enzyme-DNA covalent complex. Taken together these results indicate that the structure modification of the drug binding site is the key reason for the increasing CPT persistence and furthermore provide the possibility for new anti-cancer drug discovery.

Real-time analysis of cleavage and religation activity of human topoisomerase 1 based on ternary fluorescence resonance energy transfer DNA substrate.

Human topoisomerase 1B is a ubiquitous and essential enzyme involved in relaxing the topological state of supercoiled DNA to allow the progression of fundamental DNA metabolism. Its enzymatic catalytic cycle consists of cleavage and religation reaction. A ternary fluorescence resonance energy transfer biosensor based on a suicide DNA substrate conjugated with three fluorophores has been developed to monitor both cleavage and religation Topoisomerase I catalytic function. The presence of fluorophores does not alter the specificity of the enzyme catalysis on the DNA substrate. The enzyme-mediated reaction can be tracked in real-time by simple fluorescence measurement, avoiding the use of risky radioactive substrate labeling and time-consuming denaturing gel electrophoresis. The method is applied to monitor the perturbation brought by single mutation on the cleavage or religation reaction and to screen the effect of the camptothecin anticancer drug monitoring the energy transfer decrease during religation reaction. Pathological mutations usually affect only the cleavage or the religation reaction and the proposed approach represent a fast protocol for assessing chemotherapeutic drug efficacy and analyzing mutant's properties.

Real-Time Label-Free Direct Electronic Monitoring of Topoisomerase Enzyme Binding Kinetics on Graphene.

Monolayer graphene field-effect sensors operating in liquid have been widely deployed for detecting a range of analyte species often under equilibrium conditions. Here we report on the real-time detection of the binding kinetics of the essential human enzyme, topoisomerase I interacting with substrate molecules (DNA probes) that are immobilized electrochemically on to monolayer graphene strips. By monitoring the field-effect characteristics of the graphene biosensor in real-time during the enzyme-substrate interactions, we are able to decipher the surface binding constant for the cleavage reaction step of topoisomerase I activity in a label-free manner. Moreover, an appropriate design of the capture probes allows us to distinctly follow the cleavage step of topoisomerase I functioning in real-time down to picomolar concentrations. The presented results are promising for future rapid screening of drugs that are being evaluated for regulating enzyme activity.