Inhibitor Of DNase Research Articles

Enhancing Salivary DNA Preservation via DNase I Inactivation: Role of Temperature and EDTA

Background Deoxyribonuclease I (DNase I) catalyzes the cleavage of double-stranded DNA into its end products. This enzyme requires cations for its activity. In addition, DNase activity is influenced by indirect DNase inhibitors (i.e. EDTA) and temperature. EDTA is an indirect DNase I inhibitor. Therefore, DNase inactivation is crucial for DNA preservation, especially for salivary samples, and the downstream application of genetic techniques using non-blood samples. Saliva is a complex mixture contaminated with oral microbes and contains DNase which is harmful for DNA preservation. This study aims to preserve salivary DNA through temperature and EDTA treatment. Methods This is an experimental study. Salivary DNA was extracted using the spin-column method. DNA degradation assays were carried out using either 5 µL of salivary supernatant or 2.5 mg/mL of DNase I which were incubated at -20°C, 2-8°C, room temperature, 40°C, and 50°C; and added with 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, and 2 mM EDTA for 60 minutes. After incubation, the results were visualized using agarose gel electrophoresis. Results The average concentration of salivary DNA was 61.70 µg/mL (30.50-109.05) and the purity was 1.893 µg/mL (1.800-2.005). Incubation at -20°C, 2-8°C, and room temperature degraded salivary DNA but incubation at 40 and 50°C did not completely degrade DNA. Addition 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, and 2 mM EDTA to 5 µL of salivary supernatant or 2.5 mg/mL DNase I preserved salivary DNA. Conclusions Temperature of 40 and 50°C, and 0.125 mM EDTA can inactivate DNase and preserve salivary DNA.

3’-Methyl-4-thio-1H-tetrahydropyranspiro-5’-hydantoin platinum complex as a novel deoxyribonuclease I inhibitor

Deoxyribonuclease I (DNase I) is one of the main nucleases involved in deoxyribonucleic acid (DNA) degradation during apoptosis. It catalyzes the hydrolytic cleavage of DNA, producing 5‘-oligonucleotides. The inhibition of DNase I may serve as an important mechanism for protecting DNA against premature degradation during cell damage. Fourteen hydantoin-containing compounds, including two newly synthesized and seven previously synthesized metal complexes, along with five previously synthesized hydantoin ligands, were evaluated in vitro for their inhibitory properties against bovine pancreatic DNase I. As a result, the 3’-methyl-4-thio-1H-tetrahydropyranspiro-5’-hydantoin platinum complex (8) inhibited the enzyme with an IC50 value of 110.20 ± 24.20 µM, a potency 3-fold greater than that of the reference crystal violet (IC50 = 378.27 ± 47.75 µM). To understand the binding mode and mechanism of inhibition of compound 8 with DNase I, molecular docking calculations were performed. The analysis revealed that compound 8 interacts with the most important catalytic residues of DNase I. To the best of our knowledge, this is the first report of a platinum complex inhibiting DNase I.

NETworking for Health and in Disease: Neutrophil Extracellular Traps in Pediatric Surgical Care.

This comprehensive review examines the role of Neutrophil Extracellular Traps (NETs) in pediatric surgery. Focusing on NET formation, functions, and implications, this study highlights their dual impact in infection control and contribution to tissue damage after surgery. It covers the role of NET formation in a range of pediatric conditions including immunothrombosis, formation of peritoneal adhesions, appendicitis, burns, gallstones, tumors, and necrotizing enterocolitis (NEC). The results underscore the significance of NETs in fighting infections and their association with complications like sepsis and delayed wound healing. The breakdown products of NETs as a diagnostic tool of the clinical course of acute appendicitis will also be discussed. Understanding NET formation in the pathophysiology can potentially help to find new therapeutic approaches such as the application of DNase and elastase inhibitors to change the clinical course of various diseases in pediatric surgery such as improvement of wound healing, adhesion formation, NEC, and many more.

Screening and identification of emodin as an EBV DNase inhibitor to prevent its biological functions

BackgroundThe Epstein-Barr virus (EBV) is a prevalent oncovirus associated with a variety of human illnesses. BGLF5, an EBV DNase with alkaline nuclease (AN) activity, plays important roles in the viral life cycle and progression of human malignancies and has been suggested as a possible diagnostic marker and target for cancer therapy. Methods used conventionally for the detection of AN activity, radioactivity-based nuclease activity assay and DNA digestion detection by gel electrophoresis, are not suitable for screening AN inhibitors; the former approach is unsafe, and the latter is complicated. In the present study, a fluorescence-based nuclease activity assay was used to screen several natural compounds and identify an EBV DNase inhibitor.ResultsFluorescence-based nuclease activity assays, in which the DNA substrate is labelled with PicoGreen dye, are cheaper, safer, and easier to perform. Herein, the results of the fluorescence-based nuclease activity assay were consistent with the results of the two conventional methods. In addition, the PicoGreen-labelling method was applied for the biochemical characterisation of viral nucleases. Using this approach, we explored EBV DNase inhibitors. After several rounds of screening, emodin, an anthraquinone derivative, was found to possess significant anti-EBV DNase activity. We verified the efficacy of emodin using the conventional DNA-cleavage assay. Furthermore, using comet assay and micronucleus formation detection, we confirmed that emodin can inhibit DNase-induced DNA damage and genomic instability. Additionally, emodin treatment inhibited EBV production.ConclusionsUsing a PicoGreen-mediated nuclease activity assay, we successfully demonstrated that emodin has the potential to inhibit EBV DNase nuclease activity. Emodin also inhibits EBV DNase-related biological functions, suggesting that it is a potential inhibitor of EBV DNase.

Thiazole derivatives as dual inhibitors of deoxyribonuclease I and 5-lipoxygenase: A promising scaffold for the development of neuroprotective drugs

A library of 43 thiazole derivatives, including 31 previously and 12 newly synthesized in the present study, was evaluated in vitro for their inhibitory properties against bovine pancreatic DNase I. Nine compounds (including three newly synthesized) inhibited the enzyme showing improved inhibitory properties compared to that of the reference crystal violet (IC50 = 346.39 μM). Two compounds (5 and 29) stood out as the most potent DNase I inhibitors, with IC50 values below 100 μM. The 5-LO inhibitory properties of the investigated derivatives were also analyzed due to the importance of this enzyme in the development of neurodegenerative diseases. Compounds (12 and 29) proved to be the most prominent new 5-LO inhibitors, with IC50 values of 60 nM and 56 nM, respectively, in cell-free assay. Four compounds, including one previously (41) and three newly (12, 29 and 30) synthesized, have the ability to inhibit DNase I with IC50 values below 200 μM and 5-LO with IC50 values below 150 nM in cell-free assay. Molecular docking and molecular dynamics simulations were used to clarify DNase I and 5-LO inhibitory properties of the most potent representatives at the molecular level. The newly synthesized compound 29 (4-((4-(3-bromo-4-morpholinophenyl)thiazol-2-yl)amino)phenol) represents the most promising dual DNase I and 5-LO inhibitor, as it inhibited 5-LO in the nanomolar and DNase I in the double-digit micromolar concentration ranges. The results obtained in the present study, together with our recently published results for 4-(4-chlorophenyl)thiazol-2-amines, represent a good basis for the development of new neuroprotective therapeutics based on dual inhibition of DNase I and 5-LO.

Low endogenous deoxyribonuclease activity in the colon as a factor contributing to the pathogenesis of ulcerative colitis

In inflammatory bowel disease (IBD), tissue damage and cell death occur, which are accompanied by the release of DNA into the extracellular space. Deoxyribonucleases (DNases) are enzymes that cleave phosphodiester bonds in DNA. Exogenous DNases have anti-inflammatory effects in animal models. It is not known whether endogenous DNase activity can affect the pathogenesis of IBD. Herein we hypothesize that lower DNase activity found in the distal colon compared with proximal parts of the gastrointestinal tract could be partially responsible for the limited localization of ulcerative colitis to distal parts of the gastrointestinal tract and contribute to the ineffective removal of pro-inflammatory extracellular DNA. Hypothesis testing was performed on homogenates of individual parts of the gastrointestinal tract, in which endogenous DNase activity and its change after addition of DNase as a therapeutic were measured. A more accurate determination of DNase activity in individual gastrointestinal regions and uncovering the unknown DNase inhibitors in the colon could help to optimize the preparation of targeted therapeutics for IBD. Localized exogenous delivery of DNases to cleave extracellular DNA and eliminate inflammation could thus represent an effective supplementary therapy.

Febrile-Range Hyperthermia Can Prevent Toxic Effects of Neutrophil Extracellular Traps on Mesenchymal Stem Cells.

Fracture healing is characterized by an inflammatory phase directly after fracture which has a strong impact on the healing outcome. Neutrophils are strong contributors here and can release neutrophil extracellular traps (NETs). NETs are found after trauma, originally thought to capture pathogens. However, they can lead to tissue damage and impede wound healing processes. Their role in fracture healing remains unclear. In this study, the effect of isolated NETs on the function of bone-forming mesenchymal stem cells (SCP-1 cells) was examined. NETs were isolated from stimulated healthy neutrophils and viability, migration, and differentiation of SCP-1 cells were analyzed after the addition of NETs. NETs severely impaired the viability of SCP-1 cells, induced necrosis and already nontoxic concentrations reduced migration significantly. Short-term incubation with NETs had a persistent negative effect on osteogenic differentiation, as measured by AP activity and matrix formation. The addition of DNase or protease inhibitors failed to reverse the negative effect of NETs, whereas a short febrile-range temperature treatment successfully reduced the toxicity and membrane destruction. Thus, the possible modification of the negative effects of NETs in fracture hematomas could be an interesting new target to improve bone healing, particularly in patients with chronic diseases such as diabetes.

Systemic Optimization of Gene Electrotransfer Protocol Using Hard-to-Transfect UT-7 Cell Line as a Model.

Non-adherent cells are difficult to transfect with chemical-mediated delivery methods. Electroporation is an attractive strategy to transfer the molecules of interest into suspension cells. Care must be taken with the viability of the transfected cells since parameters, which increase cell membrane permeability, subsequently increase transfection efficiency, leading to higher cell death indices. We intended to evaluate the distribution of hard-to-transfect UT-7 cells among different subpopulations: transfected/viable, untransfected/viable, transfected/dead, and untransfected/dead populations, for a better understanding of the relation between gene electrotransfer efficacy and cell death. The following electroporation parameters were tested: pulse strength, duration, plasmid DNA concentration, and ZnSO4 as DNase inhibitor. BTX T820 square-wave generator was used, and 48 h after electroporation, cells were observed for viability and fluorescence analysis. Increasing pulse strength correlated directly with an increased ratio of pEGFP-positive cells and inversely with cell viability. The best results, representing 21% pEGFP positive/viable cells, were obtained after EP with 1 HV 1400 V/cm pulse of 250 µs duration using 200 µg/mL plasmid concentration. Results demonstrated that plasmid concentration played the most significant role in pEGFP electrotransfer into UT-7 cells. These results can represent a relevant improvement of gene electrotransfer to obtain genetically modified suspension cells for further downstream experiments.

Molecular Basis for Luteolin as a Natural TatD DNase Inhibitor in Trueperella pyogenes

TatD960 and TatD825 are DNases that contribute to biofilm formation and virulence in Trueperella pyogenes (T. pyogenes). Luteolin is a natural flavonoid commonly found in plants that exhibits antimicrobial capacity. Our study aims to investigate the effects of luteolin on TatD DNases as a natural inhibitor. In this research, the expression of tatD genes and TatD proteins in T. pyogenes treated with luteolin was detected, and then the effect of luteolin on the hydrolysis of DNA by TatD DNases was analyzed using agarose gel electrophoresis. Moreover, the interactions between luteolin and TatD DNases were tested using surface plasmon resonance (SPR) assays and molecular docking analysis. After 1/2 MIC luteolin treatment, the transcription of tatD genes and expression of TatD proteins appeared to be reduced in 80–90% of T. pyogenes (n = 20). The gel assay revealed that luteolin can inhibit the activity of TatD DNases. The SPR assay showed that the KD values of luteolin to TatD960 and TatD825 were 6.268 × 10−6 M and 5.654 × 10−6 M, respectively. We found through molecular docking that hydrogen bonding is predominant in the interaction of luteolin and TatD DNases. Our data indicate that luteolin inhibited the ability of TatD DNases by decreasing their binding to DNA. The current study provides an insight into the development of luteolin as a DNase inhibitor in preventing biofilm formation and virulence in T. pyogenes.

Deoxyribonuclease (DNAse) and Its Inhibitor

Deoxyribonuclease (DNAse) is an enzyme that plays an important role in cell apoptosis and has the function of hydrolyzing DNA. There are two types of DNAse, namely i) DNAse I which works well at neutral pH (6.5-8.0) and requires bivalent ions such as magnesium (Mg2+) and calcium (Ca2+) for activation, and ii) DNAse II which works by good at acidic pH and does not require bivalent ions. DNAse inhibitors are required to control the activity of DNAse. Both DNAse and its inhibitors have a role in the pathogenesis of various diseases, such as in the autoimmune disease systemic lupus erythematosus. DNase inhibitors can be obtained from natural, conventional, or chemical sources.

Nucleases are upregulated in potato tubers afflicted with zebra chip disease.

The defense response of potato tubers afflicted with zebra chip disease involves oxidatively mediated upregulation of nucleases that likely modulate localized programmed cell death to restrict the phloem-mobile, CLso bacterial pathogen to the vasculature. Zebra chip (ZC) is a bacterial disease of potato (Solanum tuberosum L.) caused by Candidatus Liberibacter solanacearum (CLso). Tubers from infected plants develop characteristic brown discoloration of the vasculature, a result of localized programmed cell death (PCD). We examined the potential contribution of nucleases in the response of tubers to CLso infection. Specific activities of the major isozymes of dsDNase, ssDNase, and RNase were substantially upregulated in tubers from CLso-infected plants, despite their significantly lower soluble protein content. However, ZC disease had no effect on nuclease isozyme profiles. Activities of the predominant nuclease isoforms from healthy and CLso-infected tubers had similar pH optima, thermotolerance, and responses to metallic co-factors. Nuclease activities were heat stable to 60°C and resistant to precipitation with 70% (v/v) isopropanol, which constitute effective techniques for partial purification. DNase and RNase isozyme activities were highest at pH 7.2-8.5 and 6.8-7.2, respectively, and profiles were similar for tubers from CLso-infected and non-infected plants. RNase activities were mostly insensitive to inhibition by EDTA, except at pH 8.5 and above. DNase activities were inhibited by EDTA but less sensitive to inhibition at high pH than the RNases. The EDTA-mediated inhibition of DNase (ds/ss) activities was restored with ZnSO4, but not Ca+2 or Mg+2. By contrast, ZnSO4 inhibited the activities of RNases. DTT and CuSO4 inhibited the activities of all three nucleases. These results suggest that activation of tuber nucleases is dependent on the oxidation of sulfhydryl groups to disulfide and/or oxidation of Zn to Zn+2. In light of previous published results that established extensive CLso-induced upregulation of oxidative stress metabolism in tubers, we propose a model to show how increased nuclease activity could result from a glutathione-mediated oxidation of nuclease sulfhydryl groups in diseased tubers. DNases and RNases are likely an integral part of the hypersensitive response and may modulate PCD to isolate the pathogen to the vascular tissues of tubers.

Abstract 10926: Importance of Mitochondrial DNA Damage in Cigarette Smoke Extract-Induced Activation of Innate Immune System

Objective: Smoking is a well-known risk factor for atherosclerosis, yet the mechanism by which smoking causes atherosclerosis remains unclear. It has been reported that inflammation and DNA damage are involved in the formation and development of atherosclerosis. We have previously reported that nuclear DNA damage was increased in peripheral mononuclear cells of smokers compared to those of non-smokers, and that DNA damage was accumulated in atherosclerotic lesions. In this study, to clarify the mechanism by which smoking induces inflammation in vascular endothelial cells, we investigated the effect of cigarette smoke extract (CSE) on both nuclear and mitochondrial DNA damage and the subsequent cellular response in human umbilical vein endothelial cells (HUVEC). Methods and Results: CSE increased double-strand breaks in HUVEC. Notably, this increase was detected relatively late (after 3 days of CSE exposure) compared to other genotoxic agents. CSE also increased oxidative DNA damage both in the nucleus and mitochondria and induced mitochondrial dysfunction. Mitochondrial dysfunction decreased cytosolic protein levels of ICAD, an inhibitor of caspase-activated DNase (CAD), which causes nuclear DNA fragmentation, and increased CAD in the nucleus, suggesting that this is one of the mechanisms of nuclear DNA double-strand breaks. In addition, continuous stimulation by CSE induced the accumulation of cytosolic DNA. Interestingly, real-time PCR analysis revealed that the accumulated DNA in the cytosol was derived not only from the nucleus but also from mitochondria. We further examined whether DNA accumulated in the cytosol activated cytosolic DNA-sensing pathways. The production of cGAMP, a second messenger in cGAS (a DNA-sensing receptor) signaling, was increased by CSE. We also found that the mRNA expression of IL-6 was increased by CSE, and that the increase was suppressed by si-cGAS. Conclusions: This study showed that continuous exposure to CSE induces not only nuclear but also mitochondrial DNA damage, which leads to cytosolic DNA accumulation, and evokes chronic inflammation via the cGAS-STING pathway.

Antileukemic Natural Product Induced Both Apoptotic and Pyroptotic Programmed Cell Death and Differentiation Effect.

Acute myeloid leukemia (AML) is one of the most common forms of leukemia. Despite advances in the management of such malignancies and the progress of novel therapies, unmet medical needs still exist in AML because of several factors, including poor response to chemotherapy and high relapse rates. Ardisianone, a plant-derived natural component with an alkyl benzoquinone structure, induced apoptosis in leukemic HL-60 cells. The determination of dozens of apoptosis-related proteins showed that ardisianone upregulated death receptors and downregulated the inhibitor of apoptosis protein (IAPs). Western blotting showed that ardisianone induced a dramatic increase in tumor necrosis factor receptor 2 (TNFR2) protein expression. Ardisianone also induced downstream signaling by activating caspase-8 and -3 and degradation in Bid, a caspase-8 substrate. Furthermore, ardisianone induced degradation in DNA fragmentation factor 45 kDa (DFF45), a subunit of inhibitors of caspase-activated DNase (ICAD). Q-VD-OPh (a broad-spectrum caspase inhibitor) significantly diminished ardisianone-induced apoptosis, confirming the involvement of caspase-dependent apoptosis. Moreover, ardisianone induced pyroptosis. Using transmission electron microscopic examination and Western blot analysis, key markers including gasdermin D, high mobility group box1 (HMGB1), and caspase-1 and -5 were detected. Notably, ardisianone induced the differentiation of the remaining survival cells, which were characterized by an increase in the expression of CD11b and CD68, two markers of macrophages and monocytes. Wright–Giemsa staining also showed the differentiation of cells into monocyte and macrophage morphology. In conclusion, the data suggested that ardisianone induced the apoptosis and pyroptosis of leukemic cells through downregulation of IAPs and activation of caspase pathways that caused gasdermin D cleavage and DNA double-stranded breaks and ultimately led to programmed cell death. Ardisianone also induced the differentiation of leukemic cells into monocyte-like and macrophage-like cells. The data suggested the potential of ardisianone for further antileukemic development.

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.

1,2,3,4-Tetrahydroisoquinoline Derivatives as a Novel Deoxyribonuclease I Inhibitors.

Herein we report an assessment of 24 1,2,3,4-tetrahydroisoquinoline derivatives for potential DNase I (deoxyribonuclease I) inhibitory properties in vitro. Four of them inhibited DNase I with IC50 values below 200 μM. The most potent was 1-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)propan-2-one (2) (IC50 =134.35±11.38 μM) exhibiting slightly better IC50 value compared to three other active compounds, 2-[2-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]-1-phenylethan-1-one (15) (IC50 =147.51±14.87 μM), 2-[2-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]cyclohexan-1-one (18) (IC50 =149.07±2.98 μM) and 2-[6,7-dimethoxy-2-(p-tolyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]cyclohexan-1-one (22) (IC50 =148.31±2.96 μM). Cytotoxicity assessment of the active DNase I inhibitors revealed a lack of toxic effects on the healthy cell lines MRC-5. Molecular docking and molecular dynamics simulations suggest that interactions with Glu 39, His 134, Asn 170, Tyr 211, Asp 251 and His 252 are an important factor for inhibitors affinity toward the DNase I. Observed interactions would be beneficial for the discovery of new active 1,2,3,4-tetrahydroisoquinoline-based inhibitors of DNase I, but might also encourage researchers to further explore and utilize potential therapeutic application of DNase I inhibitors, based on a versatile role of DNase I during apoptotic cell death.

Green and Red Brazilian Propolis: Antimicrobial Potential and Anti-Virulence against ATCC and Clinically Isolated Multidrug-Resistant Bacteria.

Brazilian green and red propolis stand out as commercial products for different medical applications. In this article, we report the antimicrobial activities of the hydroalcoholic extracts of green (EGP) and red (ERP) propolis, as well as guttiferone E plus xanthochymol (8) and oblongifolin B (9) from red propolis, against multidrug-resistant bacteria (MDRB). We undertook the minimal inhibitory (MIC) and bactericidal (MBC) concentrations, inhibition of biofilm formation (MICB50 ), catalase, coagulase, DNase, lipase, and hemolysin assays, along with molecular docking simulations. ERP was more effective by displaying MIC and MBC values <100 μg mL-1 . Compounds 8 and 9 displayed the lowest MIC values (0.98 to 31.25 μg mL-1 ) against all tested Gram-positive MDRB. They also inhibited the biofilm formation of S. aureus (ATCC 43300 and clinical isolate) and S. epidermidis (ATCC 14990 and clinical isolate), with MICB50 values between 1.56 and 6.25 μg mL-1 . The molecular docking results indicated that 8 and 9 might interact with the catalase's amino acids. Compounds 8 and 9 have great antimicrobial potential.

Synthesis and analysis of 4-oxothiazolidines as potential dual inhibitors of deoxyribonuclease I and xanthine oxidase

In this study, seven new 4-oxothiazolidine derivatives were synthesized and assayed, along 7 known derivatives, for inhibitory properties against deoxyribonuclease I (DNase I) and xanthine oxidase (XO) in vitro. Among tested compounds, (5Z)-Ethyl-2-(2-(cyanomethylene)-4-oxothiazolidin-5-yliden)acetate (6) exhibited inhibitory activity against both enzymes (DNase I IC50 = 67.94 ± 5.99 μM; XO IC50 = 98.98 ± 13.47 μM), therefore being the first reported dual inhibitor of DNase I and XO. Observed DNase I inhibition qualifies compound 6 as the most potent small organic DNase I inhibitor reported so far. Derivatives of 2-alkyliden-4-oxothiazolidinone (1) inhibited DNase I below 200 μM, while the other tested 4-oxothiazolidine derivatives remained inactive against both enzymes. The molecular docking and molecular dynamics simulations into the binding sites of DNase I and XO enzyme allowed us to clarify the binding modes of this 4-oxothiazolidine derivative, which might aid future development of dual DNase I and XO.

Deoxyribonuclease I Inhibitory Properties, Molecular Docking and Molecular Dynamics Simulations of 1-(Pyrrolidin-2-yl)propan-2-one Derivatives.

Deoxyribonuclease I (DNase I) inhibitory properties of two 1-(pyrrolidin-2-yl)propan-2-one derivatives were examined in vitro. Determined IC50 values of 1-[1-(4-methoxyphenyl)pyrrolidin-2-yl]propan-2-one (1) (192.13±16.95 μM) and 1-[1-(3,4,5-trimethoxyphenyl)pyrrolidin-2-yl]propan-2-one (2) (132.62±9.92 μM) exceed IC50 value of crystal violet, used as a positive control, 1.89- and 2.73-times, respectively. Compounds are predicted to be nontoxic and to have favorable pharmacokinetic profiles, with high gastrointestinal absorption and blood-brain barrier permeability. Molecular docking and molecular dynamics simulations suggest that interactions with Glu 39, Glu 78, Arg 111, Pro 137, Asp 251 and His 252 are an important factor for inhibitors affinity toward the DNase I. Determined inhibitory properties along with predicted ADMET profiles and observed interactions would be beneficial for the discovery of new active 1-(pyrrolidin-2-yl)propan-2-one-based inhibitors of DNase I.

TatD DNases of African trypanosomes confer resistance to host neutrophil extracellular traps.

African trypanosomatid parasites escape host acquired immune responses through periodic antigenic variation of their surface coat. In this study, we describe a mechanism by which the parasites counteract innate immune responses. Two TatD DNases were identified in each of Trypanosoma evansi and Trypanosoma brucei. These DNases are bivalent metal-dependent endonucleases localized in the cytoplasm and flagella of the parasites that can also be secreted by the parasites. These enzymes possess conserved functional domains and have efficient DNA hydrolysis activity. Host neutrophil extracellular traps (NETs) induced by the parasites could be hydrolyzed by native and recombinant TatD DNases. NET disruption was prevented, and the survival rate of parasites was decreased, in the presence of the DNase inhibitor aurintricarboxylic acid. These data suggest that trypanosomes can counteract host innate immune responses by active secretion of TatD DNases to degrade NETs.

Analysis of deoxyribonuclease activity by conjugation-free fluorescence polarisation in sub-nanolitre droplets.

We report the analysis of deoxyribonuclease (DNase) activity by conjugation-free fluorescence polarisation in a droplet-based microfluidic chip. DNase is a DNA cleaving enzyme and its activity is important in the maintenance of normal cellular functions. Alterations in DNase activity have been implicated as the cause of various cancers and autoimmune diseases. To date, various methods for the analysis of DNase activity have been reported. However, they are not cost effective due to the requirement of large sample volumes and the need for the conjugation of fluorescent dyes. In this study, we have used ethidium bromide (EtBr), a DNA intercalating reagent, as a fluorescent reporter without any prior conjugation or modification of DNA. Degradation of DNA by DNase 1 was monitored at a steady state by making changes in the fluorescence polarisation of EtBr in droplets with a volume of 330 picolitre at a 40 hertz frequency under visible light. Using this technique, we successfully determined the half-maximal inhibitory concentration (IC50) of ethylenediaminetetraacetic acid (EDTA) for the inhibition of DNase 1 activity to be 1.56 ± 0.91 mM.