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.

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.

Inhibition of nuclease activity by a splice-switching oligonucleotide targeting deoxyribonuclease 1 mRNA prevents apoptosis progression and prolong viability of normal human CD4+ T-lymphocytes.

The nuclease activity of deoxyribonuclease 1 (DNase I) is regulated by alternative splicing (AS) of its mRNA. The aim of this study was to define the ability of a splice-switching oligonucleotide (SSO) that base-paired with DNase I pre-mRNA to induce AS and inhibit nuclease activity in human T, B and NK lymphocytes. The SSO for DNase I could significantly downregulate the expression of full-length active DNase I and upregulate a truncated splice variant with a deleted exon 4. Such an induction of AS resulted in inhibition of nuclease activity and slowed apoptosis progression in anti-CD95/FAS stimulated lymphocytes. These results should facilitate further investigations of apoptosis regulation in lymphocytes and demonstrate that SSOs for DNase I are promising cytoprotective agents.

4-(4-Chlorophenyl)thiazol-2-amines as pioneers of potential neurodegenerative therapeutics with anti-inflammatory properties based on dual DNase I and 5-LO inhibition.

Eleven new 4-(4-chlorophenyl)thiazol-2-amines were synthesized and, together with nine known derivatives, evaluated in vitro for inhibitory properties towards bovine pancreatic DNase I. Three compounds (18-20) inhibited DNase I with IC50 values below 100 µM, with compound 19 being the most potent (IC50 = 79.79 µM). Crystal violet, used as a positive control in the absence of a "golden standard", exhibited almost 5-fold weaker DNase I inhibition. Pharma/E-State RQSAR models clarified critical structural fragments relevant for DNase I inhibition. Molecular docking and molecular dynamics simulation defined the 4-(4-chlorophenyl)thiazol-2-amines interactions with the most important catalytic residues of DNase I. Ligand-based pharmacophore modeling and virtual screening confirmed the chemical features of 4-(4-chlorophenyl)thiazol-2-amines required for DNase I inhibition and proved the absence of structurally similar molecules in available databases. Compounds 18-20 have been shown as very potent 5-LO inhibitors with nanomolar IC50 values obtained in cell-free assay, with compound 20 being the most potent (IC50 = 50 nM). Molecular docking and molecular dynamics simulations into the binding site of 5-LO enzyme allowed us to clarify the binding mode of these dual DNase I/5-LO inhibitors. It was shown that compounds 18-20 uniquely show interactions with histidine residues in the catalytic site of DNase I and 5-LO enzyme. In the absence of potent organic DNase I inhibitors, compounds 18-20 represent a good starting point for the development of novel Alzheimer's therapeutics based on dual 5-LO and DNase I inhibition, which also have anti-inflammatory properties.

Removing DNA Contamination from RNA Samples by Treatment with RNase-Free DNase I.

RNA samples prepared using monophasic lysis reagents may contain small amounts of contaminating genomic DNA, which must be removed if the RNA will be used in subsequent analyses such as reverse transcriptase-polymerase chain reaction (RT-PCR) or quantitative real-time RT-PCR. In addition, the presence of contaminating DNA can render the quantitative determination of RNA in a sample inaccurate. The most common and effective method for removing trace to moderate amounts of DNA contamination from RNA samples is digestion with DNase I, as described here.

Synthesis and DNase I inhibitory properties of new benzocyclobutane-2,5-diones.

Aim: Eight new benzocyclobutane-2,5-diones (1a-1h) were synthesized, and their inhibitory properties against bovine pancreatic DNase I were examined in vitro. Methods & results: Compounds 1a-1h were synthesized using photocycloaddition of duroquinone with various phenyl-substituted ethylenes in the presence of 18W compact fluorescent lamp (visible light). Two compounds, 1,3,4,6-tetramethyl-7-phenylbicyclo[4.2.0]oct-3-ene-2,5-dione (1a) and 1,3,4,6-tetramethyl-7-p-tolylbicyclo[4.2.0]oct-3-ene-2,5-dione (1b) inhibited DNase I in a noncompetitive manner with IC50 values below 150 µM and showed to be more potent DNase I inhibitors than crystal violet, used as a positive control. In order to analyze potential binding sites for the studied compounds with DNase I, molecular docking study was performed. Conclusion: The studied benzocyclobutane-2,5-diones offer a good starting point for a design of new DNase I inhibitors.

Rutin as Deoxyribonuclease I Inhibitor.

DNase I inhibitory potential of water extract of nine Hypericum species (H. umbellatum, H. barbatum, H. rumeliacum, H. rochelii, H. perforatum, H. tetrapterum, H. olympicum, H. hirsutum, H. linarioides) and the most important Hypericum secondary metabolites (hypericin, hyperforin, quercetin, and rutin) was investigated. All examined Hypericum extracts inhibited DNase I with IC50 below 800 µg/ml, whereby H. perforatum was the most potent (IC50 =391.26±68.40 µg/ml). Among the investigated Hypericum secondary metabolites, rutin inhibited bovine pancreatic DNase I in a non-competitive manner with IC50 value of 108.90±9.73 µm. DNase I inhibitory ability of rutin was further confirmed on DNase I in rat liver homogenate (IC50 =137.17±16.65 µm). Due to the involvement of DNase I in apoptotic processes the results of this study indicate the importance of frequent rutin and H. perforatum consumption in daily human nutrition. Rutin is a dietary component that can contribute to male infertility prevention by showing dual mechanism of sperm DNA protection, DNase I inhibition and antioxidant activity.

Synthesis and DNase I inhibitory properties of some 4-thiazolidinone derivatives.

Twelve new thiazolidinones were synthesized and, together with 41 previously synthesized thiazolidinones, evaluated for inhibitory activity against deoxyribonuclease I (DNase I) in vitro. Ten compounds inhibited commercial bovine pancreatic DNase I with an IC50 below 200 µM and showed to be more potent DNase I inhibitors than crystal violet (IC50 = 365.90 ± 47.33 µM), used as a positive control. Moreover, three compounds were active against DNase I in rat liver homogenate, having an IC50 below 200 µM. (3-Methyl-1,4-dioxothiazolidin-2-ylidene)-N-(2-phenylethyl)ethanamide (41) exhibited the most potent DNase I inhibition against both commercial and rat liver DNase I with IC50 values of 115.96 ± 11.70 and 151.36 ± 15.85 µM, respectively. Site Finder and molecular docking defined the thiazolidinones interactions with the most important catalytic residues of DNase I, including the H-acceptor interaction with residues His 134 and His 252 and/or H-donor interaction with residues Glu 39 and Asp 168. The three most active compounds against both commercial and rat liver DNase I (31, 38, and 41) exhibited favorable physico-chemical, pharmacokinetic, and toxicological properties. These observations could be utilized to guide the rational design and optimization of novel thiazolidinone inhibitors. Thiazolidinones as novel DNase I inhibitors could have potential therapeutic applications due to the significant involvement of DNase I in the pathophysiology of many disease conditions.

Plant DNases are potent therapeutic agents against Echis carinatus venom-induced tissue necrosis in mice.

Echis carinatus envenomation leads to severe tissue necrosis at the bitten site by releasing DNA from immune cells that blocks the blood flow. An earlier report has shown that exogenous DNase 1 offers protection against such severe local tissue necrosis. Tricosanthus tricuspidata is a medicinal plant and the paste prepared from its leaves has been used extensively for the treatment of snakebite-induced tissue necrosis. Most studies including reports from our laboratory focused on plant secondary metabolite as therapeutic molecules against snakebite envenomation. However, the involvement of hydrolytic enzymes including DNase in treating snake venom-induced tissue necrosis has not been addressed. Several folk medicinal plants used against snakebite treatment showed the presence of DNase activity and found to be rich in T. tricuspidata. Further, purified T. tricuspidata DNase showed a single sharp peak in reversed-phase high-performance liquid chromatography (RP-HPLC) with an apparent molecular mass of 17 kDa. T. tricuspidata DNase exhibited potent DNA degrading activity performed using agarose gel electrophoresis, spectrophotometric assay, and DNA zymography. In addition, purified DNase from T. tricuspidata was able to neutralize E. carinatus venom-induced mouse tail tissue necrosis and normalized elevated serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels 30 minutes post venom injection. T. tricuspidata DNase was also able to reverse E. carinatus venom-induced histopathological changes and collagen depletion in mice tail tissue. All these observed pharmacological actions of T. tricuspidata DNase were inhibited by sodium fluoride (NaF). This study provides scientific validation of the traditional use of T. tricuspidata leaf paste in the healing of snakebite-induced tissue necrosis and might be exploited to treat snake venom-induced local toxicity.

Synthesis and DNase I inhibitory properties of some 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines.

A series of six novel and six known thieno[2,3-d]pyrimidin-4-amines 2-13 were synthesized, and further were used as a starting material for preparation of a small series of eight novel thieno[2,3-d]pyrimidin-4-phthalimides 14-21. Eight compounds, five amine and three phthalimide derivatives, inhibited bovine pancreatic DNase I with an IC50 below 200 µM, being more effective than referent inhibitor crystal violet. Phthalimide derivatives 16, 18 and 19 exhibited higher DNase I inhibitory activity compared to their amine precursors 7, 10 and 11. Compound 19, as the most potent (IC50 = 106 ±â€¯16 µM), offers a good starting point for a design of new DNase I inhibitors. The Pharma RQSAR model showed a significant enhancement of thieno[2,3-d]pyrimidines activity using aryl substituents at R1 position. The E-State RQSAR model clarified the most important structural fragments relevant for DNase I inhibition. Molecular docking and Site Finder module defined the thieno[2,3-d]pyrimidines interactions with the most important catalytic residues of DNase I, including Glu 39, His 134, Asp 168 and His 252. We also found that steric effects and increase of molecular volume play a vital role in DNase I inhibition.

Benzimidazoles as novel deoxyribonuclease I inhibitors.

Inhibitory potential of 19 benzimidazoles against bovine pancreatic deoxyribonuclease I (DNase I) was investigated in vitro. Three compounds inhibited DNase I with IC50 below 100 µM and proved to be more potent DNase I inhibitors than crystal violet (IC50 = 351.82 ± 29.41 µM), used as a positive control. Compound 9 showed the most potent DNase I inhibition with an IC 50 value of 79.46 ± 11.75 µM. To further explore the relationship between inhibitory activity and 2D pharmacophore features, Pharma/E-State R-group quantitative structure-activity relationship (RQSAR) models were generated and validated using Schrödinger Suite. RQSAR models showed a significant enhancement of benzimidazoles activity using hydrogen-bond acceptor substituents at the R2, R3, and R4 positions, or aryl substituents at the R4 position. The Site Finder module and molecular docking defined the benzimidazoles interactions with the most important catalytic residues of DNase I, including H-acceptor interaction with residue His 134 and His 252 and/or H-donor interaction with residue Glu 39. We also found a positive correlation between IC50 inhibition values and relative binding free energies of the most active benzimidazoles. In addition, a molecular dynamics simulation was performed for DNase I-compound 9 docking complex in Desmond. Trajectory analysis showed that docking complex and intermolecular interactions were stable throughout the entire production part of simulations. Furthermore, the results of protein structure alignment module suggested the potential translational impact of benzimidazoles against human DNase I. Due to the significant involvement of DNase I in the pathophysiology of many disease conditions, benzimidazoles as DNase I inhibitors could have potential therapeutic applications.

Potential of Pyridine Amphiphiles as Staphylococcal Nuclease Inhibitor.

The present study explores the potential of pyridine-based synthetic amphiphiles C1 and C2 having 4-carbon and 12-carbon hydrophobic tails, respectively, as staphylococcal nuclease inhibitors. UV-visible titration with calf-thymus DNA (CT-DNA) revealed a hypochromic shift in the absorbance bands of C1 and C2, whereas fluorescence titration indicated a reduction in the emission intensity of the monomer bands of the amphiphiles. Interaction of deoxyribonuclease I (DNase 1) and micrococcal nuclease (MNase) with C1 or C2 led to a decrease in the emission intensity of tryptophan at λ=345 nm along with an increase in the monomer emission intensity of C1 and C2 at λ=375 nm for DNase I and excimer emission intensity at λ=470 nm for both DNase I and MNase. Scatchard's analysis indicated superior interaction of C2 with DNase I. Circular dichroism spectroscopy revealed major changes in the secondary structures of both DNase I and MNase upon interaction with the amphiphiles. A solution-based nuclease assay in conjunction with gel electrophoresis indicated amphiphile-mediated protection against nuclease-directed DNA cleavage. Interestingly, C2 could render inhibition of nuclease present in the culture supernatant of Staphylococcus aureus MTCC 96, which highlights the therapeutic prospect of the amphiphile against S. aureus.

Ascorbic acid as DNase I inhibitor in prevention of male infertility.

Apoptotic and/or ROS-induced DNA fragmentation in sperm cells may contribute to the development of male infertility. As the known dietary antioxidant, ascorbic acid prevents ROS production and protects sperm cells from DNA damage. Here, we found that ascorbic acid has the ability to inhibit DNase I, one of the main endonucleases involved in DNA fragmentation during apoptosis. Site Finder and Molecular docking defined the ascorbic acid interactions with the most important residues of DNase I, including H-donor interactions with Asp 168 and Asn 170, and H-acceptor interaction with Asn 170. As a furan derivative, ascorbic acid could be considered a pioneer of substrate-based DNase I inhibitors. The results indicate to another possible mechanism for prevention of male infertility by ascorbic acid.

Mechanism of graphene-induced cytotoxicity: Role of endonucleases.

Graphene, a crystalline allotrope or carbon, presents numerous useful properties; however, its toxicity is yet to be determined. One of the most dramatic and irreversible toxic abilities of carbon nanomaterials is the induction of DNA fragmentation produced by endogenous cellular endonucleases. This study demonstrated that pristine graphene exposed to cultured kidney tubular epithelial cells is capable of inducing DNA fragmentation measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which is usually associated with cell death. TUNEL (cell death) and endonuclease activity measured using a near infrared fluorescence probe was significantly higher in cells containing graphene aggregates detected by Raman spectroscopy. The elevation of TUNEL coincided with the increased abundance of heme oxygenase 1 (HO-1), heat shock protein 90 (HSP90), active caspase-3 and endonucleases (deoxyribonuclease I [DNase I] and endonuclease G [EndoG]), as measured by quantitative immunocytochemistry. Specific inhibitors for HO-1, HSP90, caspase-3, DNase I and EndoG almost completely blocked the DNA fragmentation induced by graphene exposure. Therefore, graphene induces cell death through oxidative injury, caspase-mediated and caspase-independent pathways; and endonucleases DNase I and EndoG are important for graphene toxicity. Inhibition of these pathways may ameliorate cell injury produced by graphene. Copyright © 2017 John Wiley & Sons, Ltd.

Anti-DNase I antibodies in systemic lupus erythematosus: diagnostic value and share in the enzyme inhibition.

Diagnostic accuracy of anti-DNase I antibodies measurement in a differentiation between SLE and other autoimmune rheumatic diseases was evaluated. The share of anti-DNase I and actin in the DNase I activity decrease in SLE was established. Serum samples were obtained from 54 patients with verified SLE, 52 control patients with other autoimmune rheumatic diseases, and 44 healthy persons. Anti-DNase I concentrations were measured by ELISA. Free and actin inhibited DNase I activities were evaluated in the fresh serum samples. The appraisal of antibodies and actin effects on DNase I activity was made using multiple regression. Anti-DNase I antibodies were positive in 35 SLE and 8 control patients, without significant difference between the mean antibody concentrations. Sensitivity of this test was 64.81 %, and specificity-84.62 %. Mean free DNase I activity in SLE was somewhat lower than in the control group as a result of augmented frequency of extremely low enzyme activities. On the contrary, after the exclusion of the latter cases we have revealed elevated mean free DNase I activity in the other SLE patients comparing to the similar control subgroup. Unlike the controls, low serum DNase I activity in SLE arose not only from actin and antibody action, but also, in half of the cases, from unidentified factor, related to active SLE. The accuracy of the anti-DNase I antibodies measurement is approximate to the present reference standard of SLE diagnostics. We first demonstrated that neither antibodies nor actin caused DNase I activity decrease in SLE.

Catechols and 3-hydroxypyridones as inhibitors of the DNA repair complex ERCC1-XPF.

Catechol-based inhibitors of ERCC1-XPF endonuclease activity were identified from a high-throughput screen. Exploration of the structure-activity relationships within this series yielded compound 13, which displayed an ERCC1-XPF IC50 of 0.6 µM, high selectivity against FEN-1 and DNase I and activity in nucleotide excision repair, cisplatin enhancement and γH2AX assays in A375 melanoma cells. Screening of fragments as potential alternatives to the catechol group revealed that 3-hydroxypyridones are able to inhibit ERCC1-XPF with high ligand efficiency, and elaboration of the hit gave compounds 36 and 37 which showed promising ERCC1-XPF IC50 values of <10 µM.

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.

Novel high-throughput deoxyribonuclease 1 assay.

Deoxyribonuclease I (DNase I), the most active and abundant apoptotic endonuclease in mammals, is known to mediate toxic, hypoxic, and radiation injuries to the cell. Neither inhibitors of DNase I nor high-throughput methods for screening of high-volume chemical libraries in search of DNase I inhibitors are, however, available. To overcome this problem, we developed a high-throughput DNase I assay. The assay is optimized for a 96-well plate format and based on the increase of fluorescence intensity when fluorophore-labeled oligonucleotide is degraded by the DNase. The assay is highly sensitive to DNase I compared to other endonucleases, reliable (Z' ≥ 0.5), and operationally simple, and it has low operator, intraassay, and interassay variability. The assay was used to screen a chemical library, and several potential DNase I inhibitors were identified. After comparison, 2 hit compounds were selected and shown to protect against cisplatin-induced kidney cell death in vitro. This assay will be suitable for identifying inhibitors of DNase I and, potentially, other endonucleases.

Large negatively charged organic host molecules as inhibitors of endonuclease enzymes.

Three large negatively charged organic host molecules; ß-cyclodextrin sulphate, para-sulphonato-calix[6]arene and para-sulphonato-calix[8]arene have been shown to be effective inhibitors of endonuclease in the low micromolar range, additionally para-sulphonato-calix[8]arene is a partial inhibitor of rhDNase I.