Neutrophil Extracellular Traps-DNase Balance and Autoimmunity.

Neutrophil extracellular traps (NETs) are macromolecular structures programmed to trap circulating bacteria and viruses. The accumulation of NETs in the circulation correlates with the formation of anti-double-stranded (ds) DNA antibodies and is considered a causative factor for systemic lupus erythematosus (SLE). The digestion of DNA by DNase1 and DNases1L3 is the rate- limiting factor for NET accumulation. Mutations occurring in one of these two DNase genes determine anti-DNA formation and are associated with severe Lupus-like syndromes and lupus nephritis (LN). A second mechanism that may lead to DNase functional impairment is the presence of circulating DNase inhibitors in patients with low DNase activity, or the generation of anti-DNase antibodies. This phenomenon has been described in a relevant number of patients with SLE and may represent an important mechanism determining autoimmunity flares. On the basis of the reviewed studies, it is tempting to suppose that the blockade or selective depletion of anti-DNase autoantibodies could represent a potential novel therapeutic approach to prevent or halt SLE and LN. In general, strategies aimed at reducing NET formation might have a similar impact on the progression of SLE and LN.

Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance.

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.

Afatinib Exerts Immunomodulatory Effects by Targeting the Pyrimidine Biosynthesis Enzyme CAD.

Current clinical trials of combined EGFR-tyrosine kinase inhibitors (TKI) and immune checkpoint blockade (ICB) therapies show no additional effect. This raises questions regarding whether EGFR-TKIs attenuate ICB-enhanced CD8+ T lymphocyte function. Here we show that the EGFR-TKI afatinib suppresses CD8+ T lymphocyte proliferation, and we identify CAD, a key enzyme of de novo pyrimidine biosynthesis, to be a novel afatinib target. Afatinib reduced tumor-infiltrating lymphocyte numbers in Lewis lung carcinoma (LLC)-bearing mice. Early afatinib treatment inhibited CD8+ T lymphocyte proliferation in patients with non-small cell lung cancer, but their proliferation unexpectedly rebounded following long-term treatment. This suggests a transient immunomodulatory effect of afatinib on CD8+ T lymphocytes. Sequential treatment of afatinib with anti-PD1 immunotherapy substantially enhanced therapeutic efficacy in MC38 and LLC-bearing mice, while simultaneous combination therapy showed only marginal improvement over each single treatment. These results suggest that afatinib can suppress CD8+ T lymphocyte proliferation by targeting CAD, proposing a timing window for combined therapy that may prevent the dampening of ICB efficacy by EGFR-TKIs. SIGNIFICANCE: This study elucidates a mechanism of afatinib-mediated immunosuppression and provides new insights into treatment timing for combined targeted therapy and immunotherapy. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3270/F1.large.jpg.

Selections and screenings of DNA-encoded chemical libraries against enzyme and cellular targets.

The use of DNA-encoded libraries (DELs) has increased greatly over the last decade, and today a majority of pharmaceutical companies employ the technology. The technology may be applied to most soluble and purified targets. However, standard DEL technology has limitations; some targets are challenging to purify, and it is not possible to directly screen for cellular or biochemical activity. Numerous creative methods have been reported to overcome these limitations and expand DEL target scope. Reported proof-of-concept experiments include DEL selections of cell surfaces, and inside of living cells. Additional alternatives include the construction and biochemical screening of one-bead-one-compound (OBOC) DELs using picoliter aqueous droplets or microfabricated wells as containers. In these cases, the small-molecule moiety of the library member is liberated from its DNA barcode, and able to interact freely with the desired target. Lastly, patent literature suggests the ability to conduct cellular functional screens using OBOC DELs.

Design, synthesis and biological evaluation of water soluble and non-aggregated silicon phthalocyanines, naphthalocyanines against A549, SNU-398, SK-MEL128, DU-145, BT-20 and HFC cell lines as potential anticancer agents.

Cancer has become an important public problem in worldwide since cancer incidence and mortality are growing rapidly. In this study, water soluble and non-aggregated silicon (IV) phthalocyanines and naphthalocyanines containing (3,5-bis{3-[3-(diethylamino)phenoxy]propoxy}phenyl)methoxy groups have been synthesized and characterized to investigate their anticancer potential. Their DNA binding/nuclease, topoisomerases inhibition were investigated using UV-Vis absorption, thermal denaturation and agarose gel electrophoresis. The in vitro cytotoxic properties of the compounds on human lung (A549), breast (BT-20), liver (SNU-398), prostate (DU-145), melanoma (SK-Mel 128) carcinoma and human fibroblast (HFC) normal cell lines were evaluated by using MTT assay. In order to determine the mechanism of cancer cell growth suppression, cell cycle analysis was carried out using flow cytometer on A549 cell line. The Kb values of SiPc1a and SiNc2a were 6.85 ± (0.35) × 106 and 1.72 ± (0.16) × 104 M-1 and Tm values of ct-DNA were calculated as 82.02 °C and 78.07 °C, respectively in the presence of both compounds. The ΔTm values of SiPc1a and SiNc2a were calculated as 6.45 and 2.50 °C, respectively. The nuclease effects of SiPc1a and SiNc2a with supercoiled plasmid pBR322 DNA demonstrated that both compounds did not trigger any DNA nuclease effects at the lowest concentrations without irradiation whereas both compounds in the presence of activating agent (H2O2) showed significant plasmid DNA nuclease actions under irradiation (22.5 J/cm2). SiPc1a and SiNc2a inhibited to topoisomerase I on increasing concentrations whilst they had lower inhibition action toward topoisomerase II that of topoisomerase I. The in vitro cytotoxicity studies displayed that SiPc1a had the highest cytotoxic effects among the tested compounds against A549, SNU-398, SK-MEL128, DU-145, BT-20 and HFC cell lines with CC50 values ranged from 0.49 to 2.99 µM. Furthermore, SiPc1a inhibited cell proliferation by cell cycle arrest in G0/G1 phase. All of these results suggested that SiPc1a is a promising candidate as an anticancer agent.

A combined experimental and theoretical investigation of ruthenium(II)-hydrazone complex with DNA: Spectroscopic, nuclease activity, topoisomerase inhibition and molecular docking.

A new Dichlorotetra(4-hydroxy-N'-(pyridin-4-ylmethylene)benzohydrazone)Ru(II) complex 1 has been synthesized and characterized using spectroscopic techniques. The structure of complex 1 has been optimized through ORCA computational programme package using B3LYP functionals. The complex binds efficiently with calf thymus DNA (CT-DNA) as monitored by UV-visible titrations (Kb = 4.8 × 105), ethidium bromide displacement studies (Ksv = 1.39) as well as Circular Dichroism (CD) titrations. The complex intercalates with DNA base pairs. It shows cleavage of supercoiled (SC) DNA into nicked circular (NC) DNA efficiently via oxidative pathway. Complex 1 also inhibits Topoisomerase I (Topo I) relaxation activity at concentration < 20 µM. The molecular docking studies support that Topo I inhibition occur via blocking religation of G11 hydroxyl group.

Graphene oxide severely inhibits DNase activity.

Graphene oxide (GO) is an important type of 2D nanomaterial and widely used in biomedicine, sensors, photocatalysis and electronic materials. With the extensive exposure of GO, its biological effect is debatable. In this study, we found a novel biological effect of GO, ie, suppression of deoxyribonuclease (DNase). GO inhibited DNA degradation when DNA or the DNA/RNA mixture was exposed to DNase. Moreover, GO suppressed nuclear fragmentation when the nuclei were treated with DNase. Interestingly, GO neither interacted with DNA nor influenced the interaction between DNase and DNA. Further investigation revealed that GO had a strong activity of adsorbing l-phenylalanine and l-histidine, key amino acid residues in the active site of DNase. These results suggest that GO could suppress the activity of DNase by interaction with the active site of DNase, and have an impact on DNase-related cellular processes (eg, apoptosis), implying its potential application in treating diseases associated with disorderly DNase function.

In silico prediction of active site and in vitro DNase and RNase activities of Helicoverpa-inducible pathogenesis related-4 protein from Cicer arietinum.

Plants are endowed with an innate immune system, which enables them to protect themselves from pest and pathogen. The participation of pathogenesis-related (PR) proteins is one of the most crucial events of inducible plant defense response. Herein, we report the characterization of CaHaPR-4, a Helicoverpa-inducible class II PR-4 protein from chickpea. Bioinformatic analysis of CaHaPR-4 protein indicated the presence of a signal peptide, barwin domain but it lacks the chitin-binding site/hevein domain. The recombinant CaHaPR-4 is bestowed with RNase and bivalent ion-dependent DNase activity. Further, the RNA and DNA binding sites were identified and confirmed by analyzing interactions between mutated CaHaPR-4 with the altered active site and ribonuclease inhibitor, 5'ADP and DNase inhibitor, 2­nitro­5­thiocyanobenzoic acid (NTCB) using 3D modeling and docking studies. Moreover, CaHaPR-4 shows antifungal activity as well as growth inhibiting properties against neonatal podborer larvae. To the best of our knowledge, this is the first report of a PR-4 showing RNase, DNase, antifungal and most importantly insect growth inhibiting properties against Helicoverpa armigera simultaneously.

Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein.

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor.IMPORTANCE The CRISPR-Cas immune system provides bacteria with resistance to invasion by potentially harmful viruses, plasmids, and other foreign mobile genetic elements. This study presents the first structural and mechanistic insight into a phage-encoded protein that inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa The interaction of this anti-CRISPR protein with the CRISPR-associated helicase/nuclease proteins Cas3 shuts down the CRISPR-Cas system and protects phages carrying this gene from destruction. This interaction also allows the repurposing of the endogenous type I-E CRISPR system into a programmable transcriptional repressor, providing a new biotechnological tool for genetic studies of bacteria encoding this type I-E CRISPR-Cas system.

EGCG, a tea polyphenol, as a potential mitigator of hematopoietic radiation injury in mice.

Agents capable of providing protection, mitigation or therapy against radiation injuries have long been of interest of radiation biologists owing to the ever expanding application of radiation in our day to day life despite the well reported ill effects of exposure. The current study investigates radiomitigating potential of EGCG (epigallocatechin gallate), a tea polyphenol with known DNMT inhibitory property, in C57 Bl/6 mice model. Treatment with 0.1833mg/kg body weight EGCG, 1.5h post-irradiation to lethally whole body irradiated mice rendered 45% survival for 30days and also helped restoring the body weight of the animals. An early recovery of various hematological parameters was observed in EGCG treated animals compared to radiation alone group. Significant recovery in the number of bone marrow colony forming cells was observed in EGCG treated irradiated animals. EGCG reduced cytogenetic damage to bone marrow cells in radiation exposed mice significantly as studied by micronucleus assay without any significant affect on cell cycle distribution of the bone marrow cells. ELISA assay with bone marrow cell lysates showed EGCG as an inhibitor of HDAC activity and DNase accessibility assay showed EGCG treatment increased the accessibility of chromatin to the enzyme. The results suggest EGCG provides mitigation against radiation injury to the hemopoietic system of mice and also inhibits HDAC enzyme activity. However, further studies are required to understand its mechanism of action.

Functional Analysis of a Type-I Ribosome Inactivating Protein Balsamin from Momordica balsamina with Anti-Microbial and DNase Activity.

Ribosome inactivating proteins (RIPs) have received considerable attention in biomedical research because of their unique activities towards tumor and virus-infected cells. We extracted balsamin, a type-I RIP, from Momordica balsamina. In the present study, a detailed investigation on DNase activity, antioxidant capacity and antibacterial activity was conducted using purified balsamin. DNase-like activity of balsamin towards plasmid DNA was pH, incubation time and temperature dependent. Moreover, the presence of Mg(2+) (10-50 mM) influenced the DNA cleavage activity. Balsamin also demonstrated reducing power and a capacity to scavenge free radicals in a dose dependent manner. Furthermore, the protein exhibited antibacterial activity against Staphylococcus aureus, Salmonella enterica, Staphylococcus epidermidis and Escherichia coli, which suggests potential utility of balsamin as a nutraceutical.

Protein synthesis directly from PCR: progress and applications of cell-free protein synthesis with linear DNA.

A rapid, versatile method of protein expression and screening can greatly facilitate the future development of therapeutic biologics, proteomic drug targets and biocatalysts. An attractive candidate is cell-free protein synthesis (CFPS), a cell-lysate-based in vitro expression system, which can utilize linear DNA as expression templates, bypassing time-consuming cloning steps of plasmid-based methods. Traditionally, such linear DNA expression templates (LET) have been vulnerable to degradation by nucleases present in the cell lysate, leading to lower yields. This challenge has been significantly addressed in the recent past, propelling LET-based CFPS as a useful tool for studying, screening and engineering proteins in a high-throughput manner. Currently, LET-based CFPS has promise in fields such as functional proteomics, protein microarrays, and the optimization of complex biological systems.

Modular Nuclease-Responsive DNA Three-Way Junction-Based Dynamic Assembly of a DNA Device and Its Sensing Application.

Here, we explored a modular strategy for rational design of nuclease-responsive three-way junctions (TWJs) and fabricated a dynamic DNA device in a "plug-and-play" fashion. First, inactivated TWJs were designed, which contained three functional domains: the inaccessible toehold and branch migration domains, the specific sites of nucleases, and the auxiliary complementary sequence. The actions of different nucleases on their specific sites in TWJs caused the close proximity of the same toehold and branch migration domains, resulting in the activation of the TWJs and the formation of a universal trigger for the subsequent dynamic assembly. Second, two hairpins (H1 and H2) were introduced, which could coexist in a metastable state, initially to act as the components for the dynamic assembly. Once the trigger initiated the opening of H1 via TWJs-driven strand displacement, the cascade hybridization of hairpins immediately switched on, resulting in the formation of the concatemers of H1/H2 complex appending numerous integrated G-quadruplexes, which were used to obtain label-free signal readout. The inherent modularity of this design allowed us to fabricate a flexible DNA dynamic device and detect multiple nucleases through altering the recognition pattern slightly. Taking uracil-DNA glycosylase and CpG methyltransferase M.SssI as models, we successfully realized the butt joint between the uracil-DNA glycosylase and M.SssI recognition events and the dynamic assembly process. Furthermore, we achieved ultrasensitive assay of nuclease activity and the inhibitor screening. The DNA device proposed here will offer an adaptive and flexible tool for clinical diagnosis and anticancer drug discovery.

Photoinduced conformational changes in DNA by poly(vinyl alcohol) carrying a malachite green moiety for protecting DNA against attack by nuclease.

Light is a highly advantageous means of specific cell targeting. Though targeted gene delivery is an important characteristic of an ideal delivery vehicle, there has been little effort to develop a photoresponsive vector. Among nonviral vectors, cationic substances interact effectively with negatively charged DNA. With this property in mind, we designed copolymers of poly(vinyl alcohol) carrying a malachite green moiety (PVAMG) with different molecular weights. Though PVAMG has no affinity for DNA in the absence of light, it undergoes photoionization in the presence of light to afford cationic DNA binding sites. The DNA-PVAMG complex was investigated with respect to DNA conformational changes and its protective nature, which are important properties for nonviral vectors. PVAMG irradiation promoted DNA conformational transitions from coils to partial globules to compacted globules. The complex had a protective effect against DNase I after PVAMG irradiation, while DNA was degraded under dark conditions. The effect on DNA transition and the protective nature were sensitive to the molecular weight of PVAMG. The data regarding binding constants and binding mode provided insight into the structure of the DNA-PVAMG complex. To withstand DNase I attacks, complexation results in the compaction of DNA, which is further covered with PVAMG.

EDTA-mediated inhibition of DNases protects circulating cell-free DNA from ex vivo degradation in blood samples.

OBJETIVES: The extracellular DNA occurring in plasma-EDTA and serum is a biomarker of growing interest, especially in prenatal diagnosis and oncology. The objectives of the present study were to compare the DNase activity in these specimens and to investigate its ex-vivo impact over the circulating cell-free DNA yield (ccfDNA), using the circulating cell-free fetal DNA (ccffDNA) as a tool. DESIGN AND METHODS: EDTA-plasma and serum from women bearing male fetus were submitted to an endogenous DNase activity assay based on qPCR hydrolysis probe degradation, they were treated with DNAse I to investigate the action of an exogenous nuclease and also submitted to different temperature conditions to investigate the temperature-dependent degradation of the ccffDNA. In all instances, all male ccffDNA were quantified by qPCR targeting the Y chromosome-specific sequence DYS-14. Moreover, a serial dilution of EDTA was added to nonanticoagulated plasma and serum before the endogenous DNAse activity assay, to investigate the EDTA-mediated inhibition of the blood's DNase. RESULTS: The endogenous nuclease activity was 14.9-fold higher in serum compared to EDTA-plasma. The DNAse I treatment did not alter the ccffDNA yields in EDTA-plasma, but completely degraded it in serum. The addition of increasing doses of EDTA to nonanticoagulated plasma and serum resulted in a stepwise inhibition of their nucleases activity. Finally, we observed a much more pronounced temperature-mediated decrease on the ccffDNA amount in serum compared to EDTA-plasma. CONCLUSION: The exogenous and endogenous DNases are more active in serum, the anticoagulant EDTA indirectly inhibits blood DNases, and consequently ccfDNA is protected from the blood's DNase preanalytical impact in EDTA-plasma.

Partial characterization of Acanthamoeba castellanii (T4 genotype) DNase activity.

The deoxyribonuclease (DNase) activities of Acanthamoeba castellanii belonging to the T4 genotype were investigated. Using zymographic assays, the DNase activities had approximate molecular masses of 25 and 35 kDa. A. castellanii DNases exhibited activity at wide-ranging temperature of up to 60 °C and at pH ranging from 4 to 9. The DNases activities were unaffected by proteinase-K treatment, divalent cations such as Ca(++), Cu(++), Mg(++), and Zn(++), or divalent cation chelating agent ethylenediaminetetraacetic acid (EDTA) or sodium dodecyl sulfate (SDS). The non-reliance on divalent cations and homology data suggests that A. castellanii DNases belong to the class of eukaryotic lysosomal DNase II but exhibit robust properties. The DNases activity in A. castellanii interfered with the genomic DNA extraction. Extraction methods involving EDTA, SDS, and proteinase-K resulted in low yield of genomic DNA. On the other hand, these methods resulted in high yield of genomic DNA from human cells suggesting the robust nature of A. castellanii DNases that are unaffected by reagents normally used in blocking eukaryotic DNases. In contrast, the use of chaotropic agent such as guanidine thiocyanate improved the yield of genomic DNA from A. castellanii cells significantly. Further purification and characterization of Acanthamoeba DNases is needed to study their non-classic distinct properties and to determine their role in the biology, cellular differentiation, cell cycle progression, and arrest of Acanthamoeba.

Deoxyribonuclease inhibitors.

Deoxyribonucleases (DNases) are a class of enzymes able to catalyze DNA hydrolysis. DNases play important roles in cell function, while DNase inhibitors control or modify their activities. This review focuses on DNase inhibitors. Some DNase inhibitors have been isolated from various natural sources, such as humans, animals (beef, calf, rabbit and rat), plants (Nicotiana tabacum), and microorganisms (some Streptomyces and Adenovirus species, Micromonospora echinospora and Escherichia coli), while others have been obtained by chemical synthesis. They differ in chemical structure (various proteins, nucleotides, anthracycline and aminoglycoside antibiotics, synthetic organic and inorganic compounds) and mechanism of action (forming complexes with DNases or DNA). Some of the inhibitors are specific toward only one type of DNase, while others are active towards two or more. Physico-chemical properties of DNase inhibitors are calculated using the Molinspiration tool and most of them meet all criteria for good solubility and permeability. DNase inhibitors may be used as pharmaceuticals for preventing, monitoring and treating various diseases.

Nucleases in homologous recombination as targets for cancer therapy.

Genomic DNA is constantly challenged from endogenous as well as exogenous sources. The DNA damage response (DDR) mechanism has evolved to combat these challenges and ensure genomic integrity. In this review, we will focus on repair of DNA double-strand breaks (DSB) by homologous recombination and the role of several nucleases and other recombination factors as suitable targets for cancer therapy. Their inactivation as well as overexpression have been shown to sensitize cancer cells by increasing toxicity to DNA-damaging agents and radiation or to be responsible for resistance of cancer cells. These factors can also be used in targeted cancer therapy by taking advantage of specific genetic abnormalities of cancer cells that are not present in normal cells and that result in cancer cell lethality.

[The effect of middle ear effusion on enzymatic digestion of DNA in middle ear effusions of chronic otitis media with effusion].

OBJECTIVE: To determine whether or not the bacterial DNA which was detected by PCR comes from viable bacteria. METHOD: Observing the affection of middle ear effusion (MEE) on DNA viscosity and enzymatic digestion of DNA. RESULT: The middle ear effusion and DNA are stable and DNase 1 rapidly digests DNA. The effusion does not seem to degrade DNA. The middle ear effusion signficantly inhibits DNase 1. CONCLUSION: Middle ear effusion provides an inhibition of the enzymatic digestion of purified DNA. Thus any DNA found in effusion by PCR techniques could well be fossilized remains and chronic otitis media with effusion may not be the bacterial infection.

Poly(thymine)-templated fluorescent copper nanoparticles for ultrasensitive label-free nuclease assay and its inhibitors screening.

Noble-metal fluorescent nanoparticles have attracted considerable interest on account of their excellent properties and potential applicable importance in many fields. Particularly, we recently found that poly(thymine) (poly T) could template the formation of fluorescent copper nanoparticles (CuNPs), offering admirable potential as novel functional biochemical probes. However, exploration of poly T-templated CuNPs for application is still at a very early stage. We report herein for the first example to develop a novel ultrasensitive label-free method for the nuclease (S1 nuclease as a model system) assay, and its inhibitors screening using the poly T-templated fluorescent CuNPs. In this assay, the signal reporter of poly T of 30 mer (T30) kept the original long state in the absence of nuclease, which could effectively template the formation of fluorescent CuNPs. In the presence of nuclease, poly T was digested to mono- or oligonucleotide fragments with decrease of fluorescence. The proposed method was low-cost and simple in its operation without requirement for complex labeling of probe DNA or sophisticated synthesis of the fluorescent compound. The assay process was very rapid with only 5 min for the formation of fluorescent CuNPs. The capabilities for target detection from complex fluids and screening of nuclease inhibitors were verified. A high sensitivity exhibited with a detectable minimum concentration of 5 × 10(-7) units µL(-1) S1 nuclease, which was about 1-4 orders of magnitude more sensitive than the developed approaches.