A Systems Biology Approach to Understanding the Mechanisms of Action of an Alternative Anticancer Compound in Comparison to Cisplatin

PubMed Central

Wright, Elise P.; Padula, Matthew P.; Higgins, Vincent J.; Aldrich-Wright, Janice R.; Coorssen, Jens R.

2014-01-01

Many clinically available anticancer compounds are designed to target DNA. This commonality of action often yields overlapping cellular response mechanisms and can thus detract from drug efficacy. New compounds are required to overcome resistance mechanisms that effectively neutralise compounds like cisplatin and those with similar chemical structures. Studies have shown that 56MESS is a novel compound which, unlike cisplatin, does not covalently bind to DNA, but is more toxic to many cell lines and active against cisplatin-resistant cells. Furthermore, a transcriptional study of 56MESS in yeast has implicated iron and copper metabolism as well as the general yeast stress response following challenge with 56MESS. Beyond this, the cytotoxicity of 56MESS remains largely uncharacterised. Here, yeast was used as a model system to facilitate a systems-level comparison between 56MESS and cisplatin. Preliminary experiments indicated that higher concentrations than seen in similar studies be used. Although a DNA interaction with 56MESS had been theorized, this work indicated that an effect on protein synthesis/ degradation was also implicated in the mechanism(s) of action of this novel anticancer compound. In contrast to cisplatin, the different mechanisms of action that are indicated for 56MESS suggest that this compound could overcome cisplatin resistance either as a stand-alone treatment or a synergistic component of therapeutics. PMID:28250393

Identification of Disubstituted Sulfonamide Compounds as Specific Inhibitors of Hepatitis B Virus Covalently Closed Circular DNA Formation

PubMed Central

Cai, Dawei; Mills, Courtney; Yu, Wenquan; Yan, Ran; Aldrich, Carol E.; Saputelli, Jeffry R.; Mason, William S.; Xu, Xiaodong; Guo, Ju-Tao; Block, Timothy M.

2012-01-01

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in viral infection and persistence and is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure even after extended treatment. Therefore, there is an urgent need for the development of novel therapeutic agents that directly target cccDNA formation and maintenance. By employing an innovative cell-based cccDNA assay in which secreted HBV e antigen is a cccDNA-dependent surrogate, we screened an in-house small-molecule library consisting of 85,000 drug-like compounds. Two structurally related disubstituted sulfonamides (DSS), termed CCC-0975 and CCC-0346, emerged and were confirmed as inhibitors of cccDNA production, with low micromolar 50% effective concentrations (EC50s) in cell culture. Further mechanistic studies demonstrated that DSS compound treatment neither directly inhibited HBV DNA replication in cell culture nor reduced viral polymerase activity in the in vitro endogenous polymerase assay but synchronously reduced the levels of HBV cccDNA and its putative precursor, deproteinized relaxed circular DNA (DP-rcDNA). However, DSS compounds did not promote the intracellular decay of HBV DP-rcDNA and cccDNA, suggesting that the compounds interfere primarily with rcDNA conversion into cccDNA. In addition, we demonstrated that CCC-0975 was able to reduce cccDNA biosynthesis in duck HBV-infected primary duck hepatocytes. This is the first attempt, to our knowledge, to identify small molecules that target cccDNA formation, and DSS compounds thus potentially serve as proof-of-concept drug candidates for development into therapeutics to eliminate cccDNA from chronic HBV infection. PMID:22644022

Synthesis, DNA binding, topoisomerase inhibition and cytotoxic properties of 2-chloroethylnitrosourea derivatives of hoechst 33258.

PubMed

Bielawski, Krzysztof; Bielawska, Anna; Anchim, Tomasz; Wołczyński, Sławomir

2005-06-01

A number of novel 2-chloroethylnitrosourea derivatives of Hoechst 33258 were synthesized and examined for cytotoxicity in breast cancer cell cultures and for inhibition of topoisomerases I and II. Evaluation of the cytotoxicity of these compounds employing a MTT assay and inhibition of [3H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that these compounds were more active than Hoechst 33258. The DNA-binding ability of these compounds was evaluated by an ultrafiltration method using calf thymus DNA, poly(dA-dT)2 and poly(dG-dC)2, indicated that these compounds as well as Hoechst 33258 well interact with AT base pair compared with GC pair. Binding studies indicate that these compounds bind more tightly to double-stranded DNA than the parent compound Hoechst 33258. The degree to which these compounds inhibited cell growth breast cancer cells was generally consistent with their relative DNA binding affinity. Mechanistic studies revealed that these compounds act as topoisomerase I (topo I) or topoisomerase II (topo II) inhibitors in plasmid relaxation assays.

Design, synthesis, molecular modeling and anti-proliferative evaluation of novel quinoxaline derivatives as potential DNA intercalators and topoisomerase II inhibitors.

PubMed

Ibrahim, M K; Taghour, M S; Metwaly, A M; Belal, A; Mehany, A B M; Elhendawy, M A; Radwan, M M; Yassin, A M; El-Deeb, N M; Hafez, E E; ElSohly, M A; Eissa, I H

2018-06-04

New series of [1,2,4]triazolo [4,3-a]quinoxaline and bis([1,2,4]triazolo)[4,3-a:3',4'-c]quinoxaline derivatives have been designed, synthesized and biologically evaluated for their cytotoxic activities against three tumor cell lines (HePG-2, Hep-2 and Caco-2). Compounds 16 e , 21, 25 a and 25 b exhibited the highest activities against the examined cell lines with IC 50 values ranging from 0.29 to 0.90 μM comparable to that of doxorubicin (IC 50 ranging from 0.51 to 0.73 μM). The most active members were further evaluated for their topoisomerase II (Topo II) inhibitory activities and DNA intercalating affinities as potential mechanisms for their anti-proliferative activities. Interestingly, the results of Topo II inhibition and DNA binding assays were consistent with that of the cytotoxicity data, where the most potent anti-proliferative derivatives exhibited good Topo II inhibitory activities and DNA binding affinities, comparable to that of doxorubicin. Moreover, the most active compound 25 a caused cell cycle arrest at G2/M phase and induced apoptosis in Caco-2 cells. In addition, Furthermore, molecular docking studies were performed for the novel compounds against DNA-Topo II complex to investigate their binding patterns. Based on these studies, it was concluded that DNA binding and/or Topo II inhibition may contribute to the observed cytotoxicity of the synthesized compounds. Copyright © 2018. Published by Elsevier Masson SAS.

Identification of genotoxic compounds using isogenic DNA repair deficient DT40 cell lines on a quantitative high throughput screening platform

PubMed Central

Nishihara, Kana; Huang, Ruili; Zhao, Jinghua; Shahane, Sampada A.; Witt, Kristine L.; Smith-Roe, Stephanie L.; Tice, Raymond R.; Takeda, Shunichi; Xia, Menghang

2016-01-01

DNA repair pathways play a critical role in maintaining cellular homeostasis by repairing DNA damage induced by endogenous processes and xenobiotics, including environmental chemicals. Induction of DNA damage may lead to genomic instability, disruption of cellular homeostasis and potentially tumours. Isogenic chicken DT40 B-lymphocyte cell lines deficient in DNA repair pathways can be used to identify genotoxic compounds and aid in characterising the nature of the induced DNA damage. As part of the US Tox21 program, we previously optimised several different DT40 isogenic clones on a high-throughput screening platform and confirmed the utility of this approach for detecting genotoxicants by measuring differential cytotoxicity in wild-type and DNA repair-deficient clones following chemical exposure. In the study reported here, we screened the Tox21 10K compound library against two isogenic DNA repair-deficient DT40 cell lines (KU70 −/−/RAD54 −/− and REV3 −/−) and the wild-type cell line using a cell viability assay that measures intracellular adenosine triphosphate levels. KU70 and RAD54 are genes associated with DNA double-strand break repair processes, and REV3 is associated with translesion DNA synthesis pathways. Active compounds identified in the primary screening included many well-known genotoxicants (e.g. adriamycin, melphalan) and several compounds previously untested for genotoxicity. A subset of compounds was further evaluated by assessing their ability to induce micronuclei and phosphorylated H2AX. Using this comprehensive approach, three compounds with previously undefined genotoxicity—2-oxiranemethanamine, AD-67 and tetraphenylolethane glycidyl ether—were identified as genotoxic. These results demonstrate the utility of this approach for identifying and prioritising compounds that may damage DNA. PMID:26243743

In vitro DNA binding, pBR322 plasmid cleavage and molecular modeling study of chiral benzothiazole Schiff-base-valine Cu(II) and Zn(II) complexes to evaluate their enantiomeric biological disposition for molecular target DNA

NASA Astrophysics Data System (ADS)

Alizadeh, Rahman; Afzal, Mohd; Arjmand, Farukh

2014-10-01

Bicyclic heterocyclic compounds viz. benzothiazoles are key components of deoxyribonucleic acid (DNA) molecules and participate directly in the encoding of genetic information. Benzothiazoles, therefore, represent a potent and selective class of antitumor compounds. The design and synthesis of chiral antitumor chemotherapeutic agents of Cu(II) and Zn(II), L- and -D benzothiazole Schiff base-valine complexes 1a &b and 2a &b, respectively were carried out and thoroughly characterized by spectroscopic and analytical techniques. Interaction of 1a and b and 2a and b with CT DNA by employing UV-vis, florescence, circular dichroic methods and cleavage studies of 1a with pBR322 plasmid, molecular docking were done in order to demonstrate their enantiomeric disposition toward the molecular drug target DNA. Interestingly, these studies unambiguously demonstrated the greater potency of L-enantiomer in comparison to D-enantiomer.

Synthesis, characterization and biological evaluation of novel α, β unsaturated amides.

PubMed

Esmailzadeh, K; Housaindokht, M R; Moradi, A; Esmaeili, A A; Sharifi, Z

2016-05-15

Three derivatives of α,β unsaturated amides have been successfully synthesized via Ugi-four component (U-4CR) reaction. The interactions of the amides with calf thymus deoxyribonucleic acid (ct-DNA) have been investigated in the Tris-HCl buffer (pH=7.4) using viscometric, spectroscopic, thermal denaturation studies, and also molecular docking. By UV-Vis absorption spectroscopy studies, adding CT-DNA to the compound solution caused the hypochromism indicates that there are interactions between the compounds and DNA base pairs. In competitive fluorescence with methylene blue as an intercalator probe, adding compounds to DNA-MB solution caused an increase in emission spectra of the complex. This could be because of compound replacing, with similar binding mode of MB, between the DNA base pairs due to release of bonded MB molecules from DNA-MB complex. Thermal denaturation studies and viscometric experiments also indicated that all three investigated compounds bind to CT-DNA by non-classical intercalation mode. Additionally, molecular docking technique predicted partial intercalation binding mode for the compounds. Also, the highest binding energy was obtained for compound 5a. These results are in agreement with results obtained by empirical methods. Copyright © 2016 Elsevier B.V. All rights reserved.

New phthalimide-appended Schiff bases: Studies of DNA binding, molecular docking and antioxidant activities.

PubMed

Nayab, Pattan Sirajuddin; Akrema; Ansari, Istikhar A; Shahid, Mohammad; Rahisuddin

2017-08-01

Herein, we investigated new phthalimide-based Schiff base molecules as promising DNA-binding and free radical scavenging agents. Physicochemical properties of these molecules were demonstrated on the basis of elemental analysis, ultraviolet-visible (UV-Vis), infra-red (IR), 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopy. All spectral data are agreed well with the proposed Schiff base framework. The DNA-binding potential of synthesized compounds were investigated by means of UV-visible, fluorescence, iodide quenching, circular dichroism, viscosity and thermal denaturation studies. The intrinsic binding constants (K b ) were calculated from absorption studies were found to be 1.1 × 10 4 and 1.0 × 10 4  M -1 for compounds 2a and 2b suggesting that compound 2a binding abilities with DNA were stronger than the compound 2b. Our studies showed that the presented compounds interact with DNA through groove binding. Molecular docking studies were carried out to predict the binding between Ct-DNA and test compounds. Interestingly, in silico predictions were corroborated with in vitro DNA-binding conclusions. Furthermore, the title compounds displayed remarkable antioxidant activity compared with reference standard. Copyright © 2016 John Wiley & Sons, Ltd.

Synthesis, crystal structure, DFT calculation and DNA binding studies of new water-soluble derivatives of dppz

NASA Astrophysics Data System (ADS)

Aminzadeh, Mohammad; Eslami, Abbas; Kia, Reza; Aleeshah, Roghayeh

2017-10-01

Diquaternarization of dipyrido-[2,3-a:2‧,3‧-c]-phenazine,(dppz) and its analogous dipyrido-[2,3-a:2‧,3‧-c]-dimethylphenazine,(dppx) using 1,3-dibromopropane afford new water-soluble derivatives of phenazine, propylene-bipyridyldiylium-phenazine (1) and propylene-bipyridyldiylium-dimethylphenazine (2). The compounds have been characterized by means of FT-IR, NMR, elemental analysis and conductometric measurements and their structure were determined by X-ray crystallography. The experimental studies on the compounds have been accompanied computationally by Density Functional Theory (DFT) calculations. The DNA binding properties of both compounds to calf thymus DNA (ctDNA) were investigated by UV-Vis absorption and emission methods. The expanded UV-Vis spectral data matrix was analyzed by multivariate curve resolution-alternating least squares (MCR-ALS) technique to obtain the concentration profile and pure spectra of all reaction species which existed in the interaction procedure. Multivariate curve resolution may help us to give a better understanding of the 1(Cl)2-ctDNA and 2(Cl)2-ctDNA interaction mechanism. The results suggest that both compounds bind tightly to DNA through intercalation mechanism and the DNA binding affinity of 2 is slightly lower than that of 1 due to steric hindrance of the methyl group. Also, thermal denaturation studies reveal that these compounds show strong affinity for binding with calf thymus DNA. The thermodynamic parameters of the DNA binding process were obtained from the temperature dependence of the binding constants and the results showed that binding of both compounds to DNA is an enthalpically driven process that is in agreement with proposed DNA intercalation capability of these compounds.

Importance of phenols structure on their activity as antinitrosating agents: A kinetic study

PubMed Central

Pessêgo, Márcia; Rosa da Costa, Ana M; Moreira, José A.

2011-01-01

Objective: Nitrosative deamination of DNA bases induced by reaction with reactive nitrogen species (RNS) has been pointed out as a probable cause of mutagenesis. (Poly)phenols, present in many food items from the Mediterranean diet, are believed to possess antinitrosating properties due to their RNS scavenging ability, which seems to be related to their structure. It has been suggested that phenolic compounds will react with the above-mentioned species more rapidly than most amino compounds, thus preventing direct nitrosation of the DNA bases and their transnitrosation from endogenous N-nitroso compounds, or most likely from the transient N-nitrosocompounds formed in vivo. Materials and Methods: In order to prove that assumption, a kinetic study of the nitroso group transfer from a N-methyl-N-nitrosobenzenesulfonamide (N-methyl-N-nitroso-4-methylbenzenesulfonamide, MeNMBS) to the DNA bases bearing an amine group and to a series of phenols was carried out. In the transnitrosation of phenols, the formation of nitrosophenol was monitored by Ultraviolet (UV) / Visible spectroscopy, and in the reactions of the DNA bases, the consumption of MeNMBS was followed by high performance liquid chromatography (HPLC). Results: The results obtained point to the transnitrosation of DNA bases being negligible, as well as that of phenols bearing electron-withdrawing groups. Phenols with methoxy substituents in positions 2, 4, and / or 6, although they seemed to react, did not afford the expected product. Phenols with electron-releasing substituents, unless these blocked the oxygen atom, reacted with our model compound at an appreciable rate. O-nitrosation of the phenolate ion followed by rearrangement of the C-nitrosophenol seemed to be involved. Conclusion: This study provided evidence that the above compounds might actually act as antinitrosating agents in vivo. PMID:21430963

Design checkpoint kinase 2 inhibitors by pharmacophore modeling and virtual screening techniques.

PubMed

Wang, Yen-Ling; Lin, Chun-Yuan; Shih, Kuei-Chung; Huang, Jui-Wen; Tang, Chuan-Yi

2013-12-01

Damage to DNA is caused by ionizing radiation, genotoxic chemicals or collapsed replication forks. When DNA is damaged or cells fail to respond, a mutation that is associated with breast or ovarian cancer may occur. Mammalian cells control and stabilize the genome using a cell cycle checkpoint to prevent damage to DNA or to repair damaged DNA. Checkpoint kinase 2 (Chk2) is one of the important kinases, which strongly affects DNA-damage and plays an important role in the response to the breakage of DNA double-strands and related lesions. Therefore, this study concerns Chk2. Its purpose is to find potential inhibitors using the pharmacophore hypotheses (PhModels) and virtual screening techniques. PhModels can identify inhibitors with high biological activities and virtual screening techniques are used to screen the database of the National Cancer Institute (NCI) to retrieve compounds that exhibit all of the pharmacophoric features of potential inhibitors with high interaction energy. Ten PhModels were generated using the HypoGen best algorithm. The established PhModel, Hypo01, was evaluated by performing a cost function analysis of its correlation coefficient (r), root mean square deviation (RMSD), cost difference, and configuration cost, with the values 0.955, 1.28, 192.51, and 16.07, respectively. The result of Fischer's cross-validation test for the Hypo01 model yielded a 95% confidence level, and the correlation coefficient of the testing set (rtest) had a best value of 0.81. The potential inhibitors were then chosen from the NCI database by Hypo01 model screening and molecular docking using the cdocker docking program. Finally, the selected compounds exhibited the identified pharmacophoric features and had a high interaction energy between the ligand and the receptor. Eighty-three potential inhibitors for Chk2 are retrieved for further study. Copyright © 2013 Elsevier Ltd. All rights reserved.

Odorous Compounds from Poultry Manure Induce DNA Damage, Nuclear Changes, and Decrease Cell Membrane Integrity in Chicken Liver Hepatocellular Carcinoma Cells

PubMed Central

Matusiak, Katarzyna; Gałęcki, Remigiusz; Borowski, Sebastian; Gutarowska, Beata

2017-01-01

Animal breeding and management of organic wastes pose a serious problem to the health of livestock and workers, as well as the nearby residents. The aim of the present study was to determine the mechanisms of toxicity of selected common odorous compounds from poultry manure, including ammonia, dimethylamine (DMA), trimethylamine (TMA), butyric acid, phenol, and indole. We measured their genotoxic and cytotoxic activity in the model chicken cell line (LMH), in vitro, by comet assay and lactate dehydrogenase assay, respectively. We also made microscopic observations of any morphological changes in these cells by DAPI staining. Four compounds, namely ammonia, DMA, TMA, and butyric acid increased DNA damage in a dose-dependent manner (p < 0.05), reaching genotoxicity as high as 73.2 ± 1.9%. Phenol and indole induced extensive DNA damage independent of the concentration used. Ammonia, DMA, and TMA caused a dose-dependent release of lactate dehydrogenase (p < 0.05). The IC50 values were 0.02%, 0.05%, and 0.1% for DMA, ammonia and TMA, respectively. These compounds also induced nuclear morphological changes, such as chromatin condensation, shrinkage, nuclear fragmentation (apoptotic bodies), and chromatin lysis. Our study exhibited the damaging effects of odorous compounds in chick LMH cell line. PMID:28820500

Zebrafish embryos as a screen for DNA methylation modifications after compound exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bouwmeester, Manon C.; Ruiter, Sander; Lommelaars, Tobias

Modified epigenetic programming early in life is proposed to underlie the development of an adverse adult phenotype, known as the Developmental Origins of Health and Disease (DOHaD) concept. Several environmental contaminants have been implicated as modifying factors of the developing epigenome. This underlines the need to investigate this newly recognized toxicological risk and systematically screen for the epigenome modifying potential of compounds. In this study, we examined the applicability of the zebrafish embryo as a screening model for DNA methylation modifications. Embryos were exposed from 0 to 72 h post fertilization (hpf) to bisphenol-A (BPA), diethylstilbestrol, 17α-ethynylestradiol, nickel, cadmium, tributyltin,more » arsenite, perfluoroctanoic acid, valproic acid, flusilazole, 5-azacytidine (5AC) in subtoxic concentrations. Both global and site-specific methylation was examined. Global methylation was only affected by 5AC. Genome wide locus-specific analysis was performed for BPA exposed embryos using Digital Restriction Enzyme Analysis of Methylation (DREAM), which showed minimal wide scale effects on the genome, whereas potential informative markers were not confirmed by pyrosequencing. Site-specific methylation was examined in the promoter regions of three selected genes vasa, vtg1 and cyp19a2, of which vasa (ddx4) was the most responsive. This analysis distinguished estrogenic compounds from metals by direction and sensitivity of the effect compared to embryotoxicity. In conclusion, the zebrafish embryo is a potential screening tool to examine DNA methylation modifications after xenobiotic exposure. The next step is to examine the adult phenotype of exposed embryos and to analyze molecular mechanisms that potentially link epigenetic effects and altered phenotypes, to support the DOHaD hypothesis. - Highlights: • Compound induced effects on DNA methylation in zebrafish embryos • Global methylation not an informative biomarker • Minimal genome wide site specific changes as detected with DREAM • Compound/class specific effects suggested by pyrosequence of specific targets • Zebrafish embryo may be a screening model for epigenetic effects.« less

Chiral halogenated Schiff base compounds: green synthesis, anticancer activity and DNA-binding study

NASA Astrophysics Data System (ADS)

Ariyaeifar, Mahnaz; Amiri Rudbari, Hadi; Sahihi, Mehdi; Kazemi, Zahra; Kajani, Abolghasem Abbasi; Zali-Boeini, Hassan; Kordestani, Nazanin; Bruno, Giuseppe; Gharaghani, Sajjad

2018-06-01

Eight enantiomerically pure halogenated Schiff base compounds were synthesized by reaction of halogenated salicylaldehydes with 3-Amino-1,2-propanediol (R or S) in water as green solvent at ambient temperature. All compounds were characterized by elemental analyses, NMR (1H and 13C), circular dichroism (CD) and FT-IR spectroscopy. FS-DNA binding studies of these compounds carried out by fluorescence quenching and UV-vis spectroscopy. The obtained results revealed that the ligands bind to DNA as: (Rsbnd ClBr) > (Rsbnd Cl2) > (Rsbnd Br2) > (Rsbnd I2) and (Ssbnd ClBr) > (Ssbnd Cl2) > (Ssbnd Br2) > (Ssbnd I2), indicating the effect of halogen on binding constant. In addition, DNA-binding constant of the Ssbnd and R-enantiomers are different from each other. The ligands can form halogen bonds with DNA that were confirmed by molecular docking. This method was also measured the bond distances and bond angles. The study of obtained data can have concluded that binding affinity of the ligands to DNA depends on strength of halogen bonds. The potential anticancer activity of ligands were also evaluated on MCF-7 and HeLa cancer cell lines by using MTT assay. The results showed that the anticancer activity and FS-DNA interaction is significantly dependent on the stereoisomers of Schiff base compounds as R-enantiomers displayed significantly higher activity than S-enantiomers. The molecular docking was also used to illustrate the specific DNA-binding of synthesized compounds and groove binding mode of DNA interaction was proposed for them. In addition, molecular docking results indicated that there are three types of bonds (Hsbnd and X-bond and hX-bond) between synthesized compounds and base pairs of DNA.

BINDING OF CARCINOGENS TO DNA AND COVALENT ADDUCTS DNA DAMAGE - PAH, AROMATIC AMINES, NITRO-AROMATIC COMPOUNDS, AND HALOGENATED COMPOUNDS

EPA Science Inventory

DNA adducts are the covalent addition products resulting from binding of reactive chemical species to DNA bases. The cancer initiating role of DNA adducts is well-established, and is clearly reflected in the high cancer incidence observed in individuals with deficiencies in any o...

Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines.

PubMed

Yamamoto, Kimiyo N; Hirota, Kouji; Kono, Koichi; Takeda, Shunichi; Sakamuru, Srilatha; Xia, Menghang; Huang, Ruili; Austin, Christopher P; Witt, Kristine L; Tice, Raymond R

2011-08-01

Included among the quantitative high throughput screens (qHTS) conducted in support of the US Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in seven isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis. Copyright © 2011 Wiley-Liss, Inc.

Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines

PubMed Central

Yamamoto, Kimiyo N.; Hirota, Kouji; Kono, Koichi; Takeda, Shunichi; Sakamuru, Srilatha; Xia, Menghang; Huang, Ruili; Austin, Christopher P.; Witt, Kristine L.; Tice, Raymond R.

2012-01-01

Included among the quantitative high throughput screens (qHTS) conducted in support of the U.S. Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in 7 isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis. PMID:21538559

Differential DNA methylation at conserved non-genic elements and evidence for transgenerational inheritance following developmental exposure to mono(2-ethylhexyl) phthalate and 5-azacytidine in zebrafish.

PubMed

Kamstra, Jorke H; Sales, Liana Bastos; Aleström, Peter; Legler, Juliette

2017-01-01

Exposure to environmental stressors during development may lead to latent and transgenerational adverse health effects. To understand the role of DNA methylation in these effects, we used zebrafish as a vertebrate model to investigate heritable changes in DNA methylation following chemical-induced stress during early development. We exposed zebrafish embryos to non-embryotoxic concentrations of the biologically active phthalate metabolite mono(2-ethylhexyl) phthalate (MEHP, 30 µM) and the DNA methyltransferase 1 inhibitor 5-azacytidine (5AC, 10 µM). Direct, latent and transgenerational effects on DNA methylation were assessed using global, genome-wide and locus-specific DNA methylation analyses. Following direct exposure in zebrafish embryos from 0 to 6 days post-fertilization, genome-wide analysis revealed a multitude of differentially methylated regions, strongly enriched at conserved non-genic elements for both compounds. Pathways involved in adipogenesis were enriched with the putative obesogenic compound MEHP. Exposure to 5AC resulted in enrichment of pathways involved in embryonic development and transgenerational effects on larval body length. Locus-specific methylation analysis of 10 differentially methylated sites revealed six of these loci differentially methylated in sperm sampled from adult zebrafish exposed during development to 5AC, and in first and second generation larvae. With MEHP, consistent changes were found at 2 specific loci in first and second generation larvae. Our results suggest a functional role for DNA methylation on cis-regulatory conserved elements following developmental exposure to compounds. Effects on these regions are potentially transferred to subsequent generations.

Comet assay in reconstructed 3D human epidermal skin models--investigation of intra- and inter-laboratory reproducibility with coded chemicals.

PubMed

Reus, Astrid A; Reisinger, Kerstin; Downs, Thomas R; Carr, Gregory J; Zeller, Andreas; Corvi, Raffaella; Krul, Cyrille A M; Pfuhler, Stefan

2013-11-01

Reconstructed 3D human epidermal skin models are being used increasingly for safety testing of chemicals. Based on EpiDerm™ tissues, an assay was developed in which the tissues were topically exposed to test chemicals for 3h followed by cell isolation and assessment of DNA damage using the comet assay. Inter-laboratory reproducibility of the 3D skin comet assay was initially demonstrated using two model genotoxic carcinogens, methyl methane sulfonate (MMS) and 4-nitroquinoline-n-oxide, and the results showed good concordance among three different laboratories and with in vivo data. In Phase 2 of the project, intra- and inter-laboratory reproducibility was investigated with five coded compounds with different genotoxicity liability tested at three different laboratories. For the genotoxic carcinogens MMS and N-ethyl-N-nitrosourea, all laboratories reported a dose-related and statistically significant increase (P < 0.05) in DNA damage in every experiment. For the genotoxic carcinogen, 2,4-diaminotoluene, the overall result from all laboratories showed a smaller, but significant genotoxic response (P < 0.05). For cyclohexanone (CHN) (non-genotoxic in vitro and in vivo, and non-carcinogenic), an increase compared to the solvent control acetone was observed only in one laboratory. However, the response was not dose related and CHN was judged negative overall, as was p-nitrophenol (p-NP) (genotoxic in vitro but not in vivo and non-carcinogenic), which was the only compound showing clear cytotoxic effects. For p-NP, significant DNA damage generally occurred only at doses that were substantially cytotoxic (>30% cell loss), and the overall response was comparable in all laboratories despite some differences in doses tested. The results of the collaborative study for the coded compounds were generally reproducible among the laboratories involved and intra-laboratory reproducibility was also good. These data indicate that the comet assay in EpiDerm™ skin models is a promising model for the safety assessment of compounds with a dermal route of exposure.

Probing cigarette smoke-induced DNA single-strand breaks and screening natural protective compounds by use of magnetic bead-based chemiluminescence.

PubMed

Chen, Hongjun; Yu, Zicheng; Cao, Zhijuan; Lau, Choiwan

2016-11-01

Magnetic bead (MB)-based chemiluminescence (CL) ELISA can be a sample-thrifty, time-saving tool for evaluation of cigarette smoke-induced DNA single-strand breaks (SSBs) with high specificity. This article describes a novel approach using immobilized oligonucleotide on MBs to determine cigarette smoke-induced DNA SSBs and screen some protective natural compounds. Typically, fluorescein-labeled DNA (FAM-DNA) was immobilized on the MBs and then oxidized by the smoke in the absence or presence of natural compounds, and a part of FAM-DNA was fragmented due to cigarette smoke-induced DNA SSB and then detached from MBs whereas other non-broken FAM-DNA still remained on MBs. Then, any broken FAM-DNA fragments, complex tobacco smoke matrix, and other stuff related with natural compounds were conveniently washed away by a magnetic force, and thus possible interfering substances were completely removed. Finally, those remaining non-broken FAM-DNA on MBs were reacted with HRP-labeled anti-fluorescein antibody and then detected by CL ELISA. CL signal was converted to molar concentrations of the FAM-DNA by interpolation from a pre-determined standard linear calibration curve. The level of DNA SSBs induced by cigarette smoke was thus calculated using the method. A library of 30 natural products was subsequently screened, and two among them were found to protect DNA from oxidative damage and thus may be promising compounds for the development of new drugs. The method developed will be useful for quantitative screening of drug genotoxicity in terms of induction of DNA SSBs. Graphical abstract ᅟ.

In vitro DNA binding, pBR322 plasmid cleavage and molecular modeling study of chiral benzothiazole Schiff-base-valine Cu(II) and Zn(II) complexes to evaluate their enantiomeric biological disposition for molecular target DNA.

PubMed

Alizadeh, Rahman; Afzal, Mohd; Arjmand, Farukh

2014-10-15

Bicyclic heterocyclic compounds viz. benzothiazoles are key components of deoxyribonucleic acid (DNA) molecules and participate directly in the encoding of genetic information. Benzothiazoles, therefore, represent a potent and selective class of antitumor compounds. The design and synthesis of chiral antitumor chemotherapeutic agents of Cu(II) and Zn(II), L- and -D benzothiazole Schiff base-valine complexes 1a &b and 2a &b, respectively were carried out and thoroughly characterized by spectroscopic and analytical techniques. Interaction of 1a and b and 2a and b with CT DNA by employing UV-vis, florescence, circular dichroic methods and cleavage studies of 1a with pBR322 plasmid, molecular docking were done in order to demonstrate their enantiomeric disposition toward the molecular drug target DNA. Interestingly, these studies unambiguously demonstrated the greater potency of L-enantiomer in comparison to D-enantiomer. Copyright © 2014 Elsevier B.V. All rights reserved.

A General Method for Discovering Inhibitors of Protein–DNA Interactions Using Photonic Crystal Biosensors

PubMed Central

Chan, Leo L.; Pineda, Maria; Heeres, James T.; Hergenrother, Paul J.; Cunningham, Brian T.

2009-01-01

Protein–DNA interactions are essential for fundamental cellular processes such as transcription, DNA damage repair, and apoptosis. As such, small molecule disruptors of these interactions could be powerful tools for investigation of these biological processes, and such compounds would have great potential as therapeutics. Unfortunately, there are few methods available for the rapid identification of compounds that disrupt protein–DNA interactions. Here we show that photonic crystal (PC) technology can be utilized to detect protein–DNA interactions, and can be used in a high-throughput screening mode to identify compounds that prevent protein–DNA binding. The PC technology is used to detect binding between protein–DNA interactions that are DNA-sequence-dependent (the bacterial toxin–antitoxin system MazEF) and those that are DNA-sequence-independent (the human apoptosis inducing factor (AIF)). The PC technology was further utilized in a screen for inhibitors of the AIF–DNA interaction, and through this screen aurin tricarboxylic acid was identified as the first in vitro inhibitor of AIF. The generality and simplicity of the photonic crystal method should enable this technology to find broad utility for identification of compounds that inhibit protein–DNA binding. PMID:18582039

New inhibitor targeting human transcription factor HSF1: effects on the heat shock response and tumor cell survival

PubMed Central

Vilaboa, Nuria; Boré, Alba; Martin-Saavedra, Francisco; Bayford, Melanie; Winfield, Natalie; Firth-Clark, Stuart; Kirton, Stewart B.

2017-01-01

Abstract Comparative modeling of the DNA-binding domain of human HSF1 facilitated the prediction of possible binding pockets for small molecules and definition of corresponding pharmacophores. In silico screening of a large library of lead-like compounds identified a set of compounds that satisfied the pharmacophoric criteria, a selection of which compounds was purchased to populate a biased sublibrary. A discriminating cell-based screening assay identified compound 001, which was subjected to systematic analysis of structure–activity relationships, resulting in the development of compound 115 (IHSF115). IHSF115 bound to an isolated HSF1 DNA-binding domain fragment. The compound did not affect heat-induced oligomerization, nuclear localization and specific DNA binding but inhibited the transcriptional activity of human HSF1, interfering with the assembly of ATF1-containing transcription complexes. IHSF115 was employed to probe the human heat shock response at the transcriptome level. In contrast to earlier studies of differential regulation in HSF1-naïve and -depleted cells, our results suggest that a large majority of heat-induced genes is positively regulated by HSF1. That IHSF115 effectively countermanded repression in a significant fraction of heat-repressed genes suggests that repression of these genes is mediated by transcriptionally active HSF1. IHSF115 is cytotoxic for a variety of human cancer cell lines, multiple myeloma lines consistently exhibiting high sensitivity. PMID:28369544

Method for performing site-specific affinity fractionation for use in DNA sequencing

DOEpatents

Mirzabekov, Andrei Darievich; Lysov, Yuri Petrovich; Dubley, Svetlana A.

1999-01-01

A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between said cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting said extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to said extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from said array.

Miniaturized reaction vessel system, method for performing site-specific biochemical reactions and affinity fractionation for use in DNA sequencing

DOEpatents

Mirzabekov, Andrei Darievich; Lysov, Yuri Petrovich; Dubley, Svetlana A.

2000-01-01

A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between said cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting said extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to said extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from said array.

Method for performing site-specific affinity fractionation for use in DNA sequencing

DOEpatents

Mirzabekov, A.D.; Lysov, Y.P.; Dubley, S.A.

1999-05-18

A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between the cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting the extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to the extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from the array. 14 figs.

Selective accumulation of harmful compounds by the DNA-inorganic hybrid-immobilized glass bead.

PubMed

Yamada, Masanori; Hamai, Akari

2009-08-11

Previously, we reported the DNA-inorganic hybrid material including double-stranded DNA by mixing the aqueous DNA solution and silane coupling reagents. Here, we immobilized the DNA-inorganic hybrid material onto the glass bead and prepared the DNA-immobilized glass bead column. The DNA-immobilized glass beads were stable in water and the amount of eluted DNA from the DNA-glass beads did not change for more than 1 week. Additionally, this DNA-immobilized column selectively accumulated the harmful compounds with the planar structure, such as dioxin- and polychlorinated biphenyl (PCB)-derivatives, and these accumulation percentages were 50-70%. Furthermore, the DNA-immobilized glass bead was recycled nine times by the application of ethanol solution and the accumulative ratio was maintained at more than 60% and did not appear to be decreasing. Therefore, these DNA-columns might have a potential for the selective removal and separation of DNA-intercalating molecules and harmful compounds with the planar structure from experimental or industrial drainages.

QSAR and docking studies on xanthone derivatives for anticancer activity targeting DNA topoisomerase IIα

PubMed Central

Alam, Sarfaraz; Khan, Feroz

2014-01-01

Due to the high mortality rate in India, the identification of novel molecules is important in the development of novel and potent anticancer drugs. Xanthones are natural constituents of plants in the families Bonnetiaceae and Clusiaceae, and comprise oxygenated heterocycles with a variety of biological activities along with an anticancer effect. To explore the anticancer compounds from xanthone derivatives, a quantitative structure activity relationship (QSAR) model was developed by the multiple linear regression method. The structure–activity relationship represented by the QSAR model yielded a high activity–descriptors relationship accuracy (84%) referred by regression coefficient (r2=0.84) and a high activity prediction accuracy (82%). Five molecular descriptors – dielectric energy, group count (hydroxyl), LogP (the logarithm of the partition coefficient between n-octanol and water), shape index basic (order 3), and the solvent-accessible surface area – were significantly correlated with anticancer activity. Using this QSAR model, a set of virtually designed xanthone derivatives was screened out. A molecular docking study was also carried out to predict the molecular interaction between proposed compounds and deoxyribonucleic acid (DNA) topoisomerase IIα. The pharmacokinetics parameters, such as absorption, distribution, metabolism, excretion, and toxicity, were also calculated, and later an appraisal of synthetic accessibility of organic compounds was carried out. The strategy used in this study may provide understanding in designing novel DNA topoisomerase IIα inhibitors, as well as for other cancer targets. PMID:24516330

Genotyping of Plant and Animal Samples without Prior DNA Purification

PubMed Central

Chum, Pak Y.; Haimes, Josh D.; André, Chas P.; Kuusisto, Pia K.; Kelley, Melissa L.

2012-01-01

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors. PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size. PMID:23051689

Markedly lowering the viscosity of aqueous solutions of DNA by additives.

PubMed

Elkin, Igor; Weight, Alisha K; Klibanov, Alexander M

2015-10-15

Aqueous solutions of DNAs, while relevant in drug delivery and as a target of therapies, are often very viscous making them difficult to use. Since less viscous solutions could enable targeted drug delivery and/or therapies, the purpose of the present work was to explore compounds capable of "thinning" such DNA solutions under pharmaceutically relevant conditions. To this end, viscosities of aqueous solutions of DNAs and model polyanions were examined at 25 °C in the absence and presence of a number of bulky organic salts (and related compounds) previously found to substantially lower the viscosities of concentrated protein solutions. Out of two dozen compounds tested, only three were found to be effective; the FDA-approved local anesthetics lidocaine, mepivacaine, and prilocaine at near-isotonic concentrations and pH 6.4 lowered solution viscosity of three different DNAs up to about 20 fold. The observed multi-fold viscosity reductions appear to be due to these bulky organic salts' structure-specific non-covalent binding to nucleotide bases resulting in denaturation (unwinding) to, and stabilization of, single-stranded DNA. Copyright © 2015 Elsevier B.V. All rights reserved.

Vitamin A and C compounds permitted in supplements differ in their abilities to affect cell viability, DNA and the DNA nucleoside deoxyguanosine.

PubMed

Bergström, Therese; Bergman, Jan; Möller, Lennart

2011-11-01

In accordance with the European Parliament and Council's directive, vitamin A and C supplements can include any of four (vitamin A) or five (vitamin C) specified compounds. This study focuses on these compounds and compares their abilities to affect the DNA and viability of cells in culture, but also their potencies to chemically oxidise the DNA nucleoside deoxyguanosine (dG). To study the vitamins' strict chemical oxidation potencies, dG was exposed to vitamin solution and the amount of the oxidation product 8'-hydroxydeoxyguanosine (8-oxodG) formed was estimated using a high-performance liquid chromatography system with electrochemical and ultraviolet detection. The vitamin's ability to cause DNA damage to promyelocytic leukaemia cells (HL-60), as detected by strand breaks, alkaline labile sites and formamido pyrimidine DNA glycosylase (FPG)-sensitive sites was, after vitamin exposure, measured using the comet assay and cytotoxicity was estimated using trypan blue staining. The results highlight that vitamin A and C compounds found in supplements do have different properties, chemically as well as in a cellular system. Among the vitamin C compounds, ascorbic acid, sodium ascorbate and calcium ascorbate stood out causing both oxidation to dG and cytotoxicity to cells. The vitamin A compounds retinol, retinyl acetate and retinal (a breakdown product found in vivo) caused oxidation of dG, while retinal was the only compound causing cytotoxicity, giving rise to an almost complete cell death. β-carotene caused, as the only vitamin compound, a small increase in FPG-sensitive sites. It is concluded that even though the compounds are found under the same name (vitamin A or C), they do have different properties linked to oxidation, cytotoxicity and DNA damage.

Balancing the Interactions of Ions, Water, and DNA in the Drude Polarizable Force Field

PubMed Central

2015-01-01

Recently we presented a first-generation all-atom Drude polarizable force field for DNA based on the classical Drude oscillator model, focusing on optimization of key dihedral angles followed by extensive validation of the force field parameters. Presently, we describe the procedure for balancing the electrostatic interactions between ions, water, and DNA as required for development of the Drude force field for DNA. The proper balance of these interactions is shown to impact DNA stability and subtler conformational properties, including the conformational equilibrium between the BI and BII states, and the A and B forms of DNA. The parametrization efforts were simultaneously guided by gas-phase quantum mechanics (QM) data on small model compounds and condensed-phase experimental data on the hydration and osmotic properties of biologically relevant ions and their solutions, as well as theoretical predictions for ionic distribution around DNA oligomer. In addition, fine-tuning of the internal base parameters was performed to obtain the final DNA model. Notably, the Drude model is shown to more accurately reproduce counterion condensation theory predictions of DNA charge neutralization by the condensed ions as compared to the CHARMM36 additive DNA force field, indicating an improved physical description of the forces dictating the ionic solvation of DNA due to the explicit treatment of electronic polarizability. In combination with the polarizable DNA force field, the availability of Drude polarizable parameters for proteins, lipids, and carbohydrates will allow for simulation studies of heterogeneous biological systems. PMID:24874104

Interaction of indole-papaverine with DNA in solutions of various ionic strength

NASA Astrophysics Data System (ADS)

Travkina, V. I.; Moroshkina, E. B.; Osinnikova, D. N.

2017-11-01

Interaction of synthetic alkaloid of isoquinoline series, which is an analogue of the biologically active compound papaverine, was studied by spectral, microcalorimetric, optical and hydrodynamic methods at different ionic strengths of medium. It was found that the investigated compound may interact with DNA in various ways depending on the ratio of ligand - DNA concentrations and ionic strength of solution (μ). When μ = 0.001, indole-papaverine intercalates into the double helix of DNA. The increase of μ resulted in a decrease of the affinity of the compound to DNA and a change its binding method.

Electrochemical study of quinone redox cycling: A novel application of DNA-based biosensors for monitoring biochemical reactions.

PubMed

Ensafi, Ali A; Jamei, Hamid Reza; Heydari-Bafrooei, Esmaeil; Rezaei, B

2016-10-01

This paper presents the results of an experimental investigation of voltammetric and impedimetric DNA-based biosensors for monitoring biological and chemical redox cycling reactions involving free radical intermediates. The concept is based on associating the amounts of radicals generated with the electrochemical signals produced, using differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). For this purpose, a pencil graphite electrode (PGE) modified with multiwall carbon nanotubes and poly-diallydimethlammonium chloride decorated with double stranded fish sperm DNA was prepared to detect DNA damage induced by the radicals generated from a redox cycling quinone (i.e., menadione (MD; 2-methyl-1,4-naphthoquinone)). Menadione was employed as a model compound to study the redox cycling of quinones. A direct relationship was found between free radical production and DNA damage. The relationship between MD-induced DNA damage and free radical generation was investigated in an attempt to identify the possible mechanism(s) involved in the action of MD. Results showed that DPV and EIS were appropriate, simple and inexpensive techniques for the quantitative and qualitative comparisons of different reducing reagents. These techniques may be recommended for monitoring DNA damages and investigating the mechanisms involved in the production of redox cycling compounds. Copyright © 2016 Elsevier B.V. All rights reserved.

Discovery and Optimization of Indolyl-Containing 4-Hydroxy-2-Pyridone Type II DNA Topoisomerase Inhibitors Active against Multidrug Resistant Gram-negative Bacteria.

PubMed

Gerasyuto, Aleksey I; Arnold, Michael A; Wang, Jiashi; Chen, Guangming; Zhang, Xiaoyan; Smith, Sean; Woll, Matthew G; Baird, John; Zhang, Nanjing; Almstead, Neil G; Narasimhan, Jana; Peddi, Srinivasa; Dumble, Melissa; Sheedy, Josephine; Weetall, Marla; Branstrom, Arthur A; Prasad, J V N; Karp, Gary M

2018-05-14

There exists an urgent medical need to identify new chemical entities (NCEs) targeting multidrug resistant (MDR) bacterial infections, particularly those caused by Gram-negative pathogens. 4-Hydroxy-2-pyridones represent a novel class of nonfluoroquinolone inhibitors of bacterial type II topoisomerases active against MDR Gram-negative bacteria. Herein, we report on the discovery and structure-activity relationships of a series of fused indolyl-containing 4-hydroxy-2-pyridones with improved in vitro antibacterial activity against fluoroquinolone resistant strains. Compounds 6o and 6v are representative of this class, targeting both bacterial DNA gyrase and topoisomerase IV (Topo IV). In an abbreviated susceptibility screen, compounds 6o and 6v showed improved MIC 90 values against Escherichia coli (0.5-1 μg/mL) and Acinetobacter baumannii (8-16 μg/mL) compared to the precursor compounds. In a murine septicemia model, both compounds showed complete protection in mice infected with a lethal dose of E. coli.

Cell and microsome mediated binding of 7,12-dimethylbenz(a)anthracene to DNA studied by fluorescence spectroscopy.

PubMed

Ivanovic, V; Geacintov, N E; Jeffrey, A M; Fu, P P; Harvey, R G; Weinstein, I B

1978-03-01

Fluorescence spectra of DNA isolated from hamster embryo cells incubated with 7,12-dimethylbenz(a)anthracene, or DNA modified in a microsomal system by reaction with this carcinogen or its 7-hydroxymethyl derivative, were compared to various model compounds. The spectra indicate that the DMBA derivative bound to DNA, in all 3 cases, has a 9,10-dimethylanthracene-like chromophore. They also provide the first evidence of the similarity in structure of the DNA-bound products between 7,12-dimethylbenz(a)anthracene and its 7-hydroxymethyl derivative. Our results are consistent with an activation mechanism that involves saturation of the 1,2,3,4-ring positions.

Genome-Wide Requirements for Resistance to Functionally Distinct DNA-Damaging Agents

PubMed Central

Proctor, Michael; Flaherty, Patrick; Jordan, Michael I; Arkin, Adam P; Davis, Ronald W; Nislow, Corey; Giaever, Guri

2005-01-01

The mechanistic and therapeutic differences in the cellular response to DNA-damaging compounds are not completely understood, despite intense study. To expand our knowledge of DNA damage, we assayed the effects of 12 closely related DNA-damaging agents on the complete pool of ~4,700 barcoded homozygous deletion strains of Saccharomyces cerevisiae. In our protocol, deletion strains are pooled together and grown competitively in the presence of compound. Relative strain sensitivity is determined by hybridization of PCR-amplified barcodes to an oligonucleotide array carrying the barcode complements. These screens identified genes in well-characterized DNA-damage-response pathways as well as genes whose role in the DNA-damage response had not been previously established. High-throughput individual growth analysis was used to independently confirm microarray results. Each compound produced a unique genome-wide profile. Analysis of these data allowed us to determine the relative importance of DNA-repair modules for resistance to each of the 12 profiled compounds. Clustering the data for 12 distinct compounds uncovered both known and novel functional interactions that comprise the DNA-damage response and allowed us to define the genetic determinants required for repair of interstrand cross-links. Further genetic analysis allowed determination of epistasis for one of these functional groups. PMID:16121259

Comet assay in reconstructed 3D human epidermal skin models—investigation of intra- and inter-laboratory reproducibility with coded chemicals

PubMed Central

Pfuhler, Stefan

2013-01-01

Reconstructed 3D human epidermal skin models are being used increasingly for safety testing of chemicals. Based on EpiDerm™ tissues, an assay was developed in which the tissues were topically exposed to test chemicals for 3h followed by cell isolation and assessment of DNA damage using the comet assay. Inter-laboratory reproducibility of the 3D skin comet assay was initially demonstrated using two model genotoxic carcinogens, methyl methane sulfonate (MMS) and 4-nitroquinoline-n-oxide, and the results showed good concordance among three different laboratories and with in vivo data. In Phase 2 of the project, intra- and inter-laboratory reproducibility was investigated with five coded compounds with different genotoxicity liability tested at three different laboratories. For the genotoxic carcinogens MMS and N-ethyl-N-nitrosourea, all laboratories reported a dose-related and statistically significant increase (P < 0.05) in DNA damage in every experiment. For the genotoxic carcinogen, 2,4-diaminotoluene, the overall result from all laboratories showed a smaller, but significant genotoxic response (P < 0.05). For cyclohexanone (CHN) (non-genotoxic in vitro and in vivo, and non-carcinogenic), an increase compared to the solvent control acetone was observed only in one laboratory. However, the response was not dose related and CHN was judged negative overall, as was p-nitrophenol (p-NP) (genotoxic in vitro but not in vivo and non-carcinogenic), which was the only compound showing clear cytotoxic effects. For p-NP, significant DNA damage generally occurred only at doses that were substantially cytotoxic (>30% cell loss), and the overall response was comparable in all laboratories despite some differences in doses tested. The results of the collaborative study for the coded compounds were generally reproducible among the laboratories involved and intra-laboratory reproducibility was also good. These data indicate that the comet assay in EpiDerm™ skin models is a promising model for the safety assessment of compounds with a dermal route of exposure. PMID:24150594

The effect of novel rhenium compounds on lymphosarcoma, PC-3 prostate and myeloid leukemia cancer cell lines and an investigation on the DNA binding properties of one of these compounds through electronic spectroscopy

PubMed Central

Parson, Carl; Smith, Valerie; Krauss, Christopher; Banerjee, Hirendra N.; Reilly, Christopher; Krause, Jeanette A.; Wachira, James M.; Giri, Dipak; Winstead, Angela; Mandal, Santosh K.

2014-01-01

Despite the tremendous success of cisplatin and other platinum-based anticancer drugs, severe toxicity and resistance to tumors limit their applications. It is believed that the coordination (formation of covalent bond) of the metal (platinum) to the nitrogen bases of DNA cause the ruptures of the cancer as well as normal cells. A search for anticancer drugs with different modes of action resulted in the synthesis of variety of novel compounds. Many of them are in clinical trials now. Recently we synthesized a series of novel rhenium pentylcarbonato compounds (PC1–PC6). The rhenium atom in each compound is coordinated (bonded) to a planar polypyridyl aromatic ligand, thereby forcing each compound to intercalate between the DNA bases. We have investigated the DNA binding properties of one of the PC-series of compounds (PC6) using electronic spectroscopy. The UV absorption titration of PC6 with DNA shows hypochromic effect with concomitant bathochromic shift of the charge transfer band at 290 nm. These results suggest that the compound PC6 binds to DNA through intercalation. It is therefore likely that the other PC-series of compounds will behave in a similar manner. Thus it is expected that these compounds will exhibit negligible or no side effect. We have observed that the PC-series of compounds are strong cytotoxic agents against lymphosarcoma (average GI50 ≈ 2±2.6 µM), PC-3 prostate (average GI50 ≈ 3±2.8 µM) and myeloid leukemia (average GI50 ≈ 3±2.8 µM) cancer cell lines. The average GI50 values of the PC-series of compounds are 2–3 less than the corresponding GI50 values of cisplatin. Also each of the PC-series of compounds exhibits less toxicity than cisplatin in the glomerular mesangial cells. PMID:25221731

π-Stacking between Casiopeinas® and DNA bases.

PubMed

Galindo-Murillo, Rodrigo; Hernandez-Lima, Joseelyne; González-Rendón, Mayra; Cortés-Guzmán, Fernando; Ruíz-Azuara, Lena; Moreno-Esparza, Rafael

2011-08-28

Casiopeínas® are copper complexes with the general formula [Cu(N-N)(N-O)]NO(3) and [Cu(N-N)(O-O)]NO(3) where N-N denotes a substituted bipyridine or phenanthroline, N-O indicates α-aminoacidate or peptide and O-O represents acetylacetonate or salicylaldehyde. This family of compounds has been evaluated in vitro and in vivo showing cytotoxic, genotoxic, and antineoplastic activity. The action mechanism is still not completely elucidated, but the possibility exists that these compounds interact with DNA by intercalation due to the aromatic moiety. In this work we found, using the properties of the electron density of a π-complex model base-Casiopeína®-base, that the stacking mechanism between Casiopeínas® and DNA bases is due to an electron density deficiency of the ligand of the Casiopeína® which is compensated for by an electron transfer from adenines by a π-π interaction.

Comparative analysis of topoisomerase IB inhibition and DNA intercalation by flavonoids and similar compounds: structural determinates of activity

PubMed Central

2004-01-01

Flavonoids and other polyphenolic compounds have been shown to inhibit human topoisomerase IB (topo I) through both inhibition of relaxation activity and through stabilization of the cleavable complex (poisoning). Some flavonoids have also been shown to intercalate DNA, and an association of topoisomerase inhibition with intercalation has been noted. We surveyed 34 polyphenolic compounds, primarily flavonoid glycones and aglycones, for their ability to inhibit topo I and to intercalate DNA using an in vitro gel electrophoresis method. We show that the most potent topo I poisons are the flavones and flavonols, and that these generally, but not always, are found to be DNA intercalators. There was no clear correlation, however, of topo-I-poisoning activity with the degree of DNA unwinding. Surprisingly, both DNA intercalation and topo I poisoning were shown to occur with some flavone glycones, including the C-glycosylflavone orientin. Inhibition of relaxation activity by flavonoids was found to be difficult to quantify and was most likely to be due to non-specific inhibition through flavonoid aggregation. As part of a structure–activity analysis, we also investigated the acid–base chemistry of flavonoids and determined that many flavonoids show acid–base activity with a pKa in the physiological pH region. For this reason, subtle pH changes can have significant effects on solution activity of flavonoids and their concomitant biological activity. In addition, these effects may be complicated by pH-dependent aggregation and oxidative degradation. Finally, we develop a simple model for the intercalation of flavonoids into DNA and discuss possible consequences of intercalation and topoisomerase inhibition on a number of cellular processes. PMID:15312049

Functional and structural analysis of AT-specific minor groove binders that disrupt DNA–protein interactions and cause disintegration of the Trypanosoma brucei kinetoplast

PubMed Central

Millan, Cinthia R.; Acosta-Reyes, Francisco J.; Lagartera, Laura; Ebiloma, Godwin U.; Lemgruber, Leandro; Nué Martínez, J. Jonathan; Saperas, Núria

2017-01-01

Abstract Trypanosoma brucei, the causative agent of sleeping sickness (Human African Trypanosomiasis, HAT), contains a kinetoplast with the mitochondrial DNA (kDNA), comprising of >70% AT base pairs. This has prompted studies of drugs interacting with AT-rich DNA, such as the N-phenylbenzamide bis(2-aminoimidazoline) derivatives 1 [4-((4,5-dihydro-1H-imidazol-2-yl)amino)-N-(4-((4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)benzamide dihydrochloride] and 2 [N-(3-chloro-4-((4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)-4-((4,5-dihydro-1H-imidazol-2-yl)amino)benzamide] as potential drugs for HAT. Both compounds show in vitro effects against T. brucei and in vivo curative activity in a mouse model of HAT. The main objective was to identify their cellular target inside the parasite. We were able to demonstrate that the compounds have a clear effect on the S-phase of T. brucei cell cycle by inflicting specific damage on the kinetoplast. Surface plasmon resonance (SPR)–biosensor experiments show that the drug can displace HMG box-containing proteins essential for kDNA function from their kDNA binding sites. The crystal structure of the complex of the oligonucleotide d[AAATTT]2 with compound 1 solved at 1.25 Å (PDB-ID: 5LIT) shows that the drug covers the minor groove of DNA, displaces bound water and interacts with neighbouring DNA molecules as a cross-linking agent. We conclude that 1 and 2 are powerful trypanocides that act directly on the kinetoplast, a structure unique to the order Kinetoplastida. PMID:28637278

Antibacterial and DNA cleavage activity of carbonyl functionalized N-heterocyclic carbene-silver(I) and selenium compounds

NASA Astrophysics Data System (ADS)

Haque, Rosenani A.; Iqbal, Muhammad Adnan; Mohamad, Faisal; Razali, Mohd R.

2018-03-01

The article describes syntheses and characterizations of carbonyl functionalized benzimidazolium salts, I-IV. While salts I-III are unstable at room temperature, salt IV remained stable and was further utilised to form N-heterocyclic carbene (NHC) compounds of silver(I), V and VI, and selenium compound, VII respectively. Compounds IV-VII were tested for their antibacterial potential against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Salt IV shows a very low inhibition potential (minimum inhibitory concentration, MIC 500 μg/mL) compared to the respective silver(I)-NHC, V and VI (MIC 31.25 μg/mL against both, E. coli and S. aureus) and selenium compound, VII (MIC 125 μg/mL against E. coli and 62.50 μg/mL against S. aureus). In DNA cleavage abilities, all the test compounds cleave DNA in which the VII cleaves the DNA at the faster rate. Meanwhile, the silver(I)-NHC complexes V and VI act at the same mode and pattern of DNA cleavage while VII is similar to IV.

Characterization of a Melamino Nitroheterocycle as a Potential Lead for the Treatment of Human African Trypanosomiasis

PubMed Central

Giordani, Federica; Buschini, Annamaria; Baliani, Alessandro; Kaiser, Marcel; Brun, Reto; Barrett, Michael P.; Pellacani, Claudia; Poli, Paola

2014-01-01

This paper reports an evaluation of a melamino nitroheterocycle, a potential lead for further development as an agent against human African trypanosomiasis (HAT). Studies on its efficacy, physicochemical and biopharmaceutical properties, and potential for toxicity are described. The compound previously had been shown to possess exceptional activity against Trypanosoma brucei in in vitro assays comparable to that of melarsoprol. Here, we demonstrate that the compound also was curative in the stringent acute mouse model T. brucei rhodesiense STIB 900 when given intraperitoneally at 40 mg/kg of body weight. Nevertheless, activity was only moderate when the oral route was used, and no cure was obtained when the compound was tested in a stage 2 rodent model of infection. Genotoxic profiling revealed that the compound induces DNA damage by a mechanism apparently independent from nitroreduction and involving the introduction of base pair substitutions (Ames test), possibly caused by oxidative damage of the DNA (comet test). No significant genotoxicity was observed at the chromosome level (micronucleus assay). The lack of suitable properties for oral and central nervous system uptake and the genotoxic liabilities prevent the progression of this melamine nitroheterocycle as a drug candidate for HAT. Further modification of the compound is required to improve the pharmacokinetic properties of the molecule and to separate the trypanocidal activity from the toxic potential. PMID:25022590

Spectroscopic, electrochemical DNA binding and in vivo anti-inflammatory studies on newly synthesized Schiff bases of 4-aminophenazone.

PubMed

Arshad, Nasima; Ahmad, Mukhtar; Ashraf, Muhammad Zaman; Nadeem, Humaira

2014-09-05

4-Aminophenazone (Ap-1) Schiff bases i.e., 4-{(3,4,5-trimethoxybenzylidine) amino}phenazone (Ap-2), 4-{(2-chlorobenzylidine) amino}phenazone (Ap-3) and 4-{(4-chlorobenzylidine)amino} phenazone (Ap-4) were synthesized and characterized by different spectroscopic techniques. Interaction of these compounds with ds.DNA was investigated through UV-Visible spectroscopy, fluorescence spectroscopy and cyclic voltammetry at stomach (4.7) and blood (7.4) pH under 37 °C (human body temperature). Instrumental findings were further quantified both kinetically and thermodynamically. Results obtained through these techniques inferred intercalative mode of binding of all the compounds with DNA. The binding constant data, "Kb", and free energy change, ΔG, indicated comparatively greater binding affinity and more spontaneity of binding of compounds with DNA at stomach pH (4.7), respectively. However, among these compounds, Ap-4 showed comparatively greater binding at both the pH. Formation of compound-DNA complex was further confirmed through the decrease in diffusion rates after the addition of DNA. The in vivo anti-inflammatory activity of the compounds was evaluated using the carrageenan-induced hind paw edema method. The results revealed that among all the compounds, Ap-4 showed greater percentage of edema inhibition compared to standard drug. Copyright © 2014 Elsevier B.V. All rights reserved.

Understanding DNA under oxidative stress and sensitization: the role of molecular modeling

PubMed Central

Dumont, Elise; Monari, Antonio

2015-01-01

DNA is constantly exposed to damaging threats coming from oxidative stress, i.e., from the presence of free radicals and reactive oxygen species. Sensitization from exogenous and endogenous compounds that strongly enhance the frequency of light-induced lesions also plays an important role. The experimental determination of DNA lesions, though a difficult subject, is somehow well established and allows to elucidate even extremely rare DNA lesions. In parallel, molecular modeling has become fundamental to clearly understand the fine mechanisms related to DNA defects induction. Indeed, it offers an unprecedented possibility to get access to an atomistic or even electronic resolution. Ab initio molecular dynamics may also describe the time-evolution of the molecular system and its reactivity. Yet the modeling of DNA (photo-)reactions does necessitate elaborate multi-scale methodologies to tackle a damage induction reactivity that takes place in a complex environment. The double-stranded DNA environment is first characterized by a very high flexibility, but also a strongly inhomogeneous electrostatic embedding. Additionally, one aims at capturing more subtle effects, such as the sequence selectivity which is of critical important for DNA damage. The structure and dynamics of the DNA/sensitizers complexes, as well as the photo-induced electron- and energy-transfer phenomena taking place upon sensitization, should be carefully modeled. Finally the factors inducing different repair ratios for different lesions should also be rationalized. In this review we will critically analyze the different computational strategies used to model DNA lesions. A clear picture of the complex interplay between reactivity and structural factors will be sketched. The use of proper multi-scale modeling leads to the in-depth comprehension of DNA lesions mechanisms and also to the rational design of new chemo-therapeutic agents. PMID:26236706

Nuclear aggregates of polyamines in a radiation-induced DNA damage model.

PubMed

Iacomino, Giuseppe; Picariello, Gianluca; Stillitano, Ilaria; D'Agostino, Luciano

2014-02-01

Polyamines (PA) are believed to protect DNA minimizing the effect of radiation damage either by inducing DNA compaction and aggregation or acting as scavengers of free radicals. Using an in vitro pDNA double strand breakage assay based on gel electrophoretic mobility, we compared the protective capability of PA against γ-radiation with that of compounds generated by the supramolecular self-assembly of nuclear polyamines and phosphates, named Nuclear Aggregates of Polyamines (NAPs). Both unassembled PA and in vitro produced NAPs (ivNAPs) were ineffective in conferring pDNA protection at the sub-mM concentration. Single PA showed an appreciable protective effect only at high (mM) concentrations. However, concentrations of spermine (4+) within a critical range (0.481 mM) induced pDNA precipitation, an event that was not observed with NAPs-pDNA interaction. We conclude that the interaction of individual PA is ineffective to assure DNA protection, simultaneously preserving the flexibility and charge density of the double strand. Furthermore, data obtained by testing polyamine and ivNAPS with the current radiation-induced DNA damage model support the concept that PA-phosphate aggregates are the only forms through which PA interact with DNA. Copyright © 2013 Elsevier Ltd. All rights reserved.

Synthesis of dicationic diaryltriazines nucleic acid binding agents

PubMed Central

Spychala, J; Wilson, WD; Zhao, M; Tidwell, RR; Dykstra, CC; Hall, JE; Jones, SK; Schinazi, RF

2010-01-01

Summary — The synthesis of 2,4-bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-1,3,5-triazine 6a and 2,4-bis[4-(1,4,5,6-tetrahydropyrimidin-2-yl)phenyl]-1,3,5-triazine 6b in 3 steps from either 4-bromobenzamidine or 4-(carbamoyl)benzamidine is reported. The synthesis of 4,6-bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-2-dimethylamino-1,3,5-triazine 9a and 4,6-bis[4-(1,4,5,6-tetrahydropyrimidin-2-yl)phenyl]-2-dimethylamino-1,3,5-triazine 9b in 2 steps from 1,4-dicyanobenzene is also described. The compounds 6b and 9b bind strongly to DNA model sequences and inhibit topoisomerase II from 2 microbial sources. Compounds 6a and 9a bind to both DNA and RNA model sequences whereas 6b and 9b essentially do not bind to the RNA model. PMID:21556310

Cyclometalated Iminophosphorane Gold(III) and Platinum(II) Complexes. A Highly Permeable Cationic Platinum(II) Compound with Promising Anticancer Properties

PubMed Central

2015-01-01

New organometallic gold(III) and platinum(II) complexes containing iminophosphorane ligands are described. Most of them are more cytotoxic to a number of human cancer cell lines than cisplatin. Cationic Pt(II) derivatives 4 and 5, which differ only in the anion, Hg2Cl62– or PF6– respectively, display almost identical IC50 values in the sub-micromolar range (25–335-fold more active than cisplatin on these cell lines). The gold compounds induced mainly caspase-independent cell death, as previously reported for related cycloaurated compounds containing IM ligands. Cycloplatinated compounds 3, 4, and 5 can also activate alternative caspase-independent mechanisms of death. However, at short incubation times cell death seems to be mainly caspase dependent, suggesting that the main mechanism of cell death for these compounds is apoptosis. Mercury-free compound 5 does not interact with plasmid (pBR322) DNA or with calf thymus DNA. Permeability studies of 5 by two different assays, in vitro Caco-2 monolayers and a rat perfusion model, have revealed a high permeability profile for this compound (comparable to that of metoprolol or caffeine) and an estimated oral fraction absorbed of 100%, which potentially makes it a good candidate for oral administration. PMID:26147404

A small organic compound enhances the religation reaction of human topoisomerase I and identifies crucial elements for the religation mechanism

PubMed Central

Arnò, Barbara; Coletta, Andrea; Tesauro, Cinzia; Zuccaro, Laura; Fiorani, Paola; Lentini, Sara; Galloni, Pierluca; Conte, Valeria; Floris, Barbara; Desideri, Alessandro

2013-01-01

The different steps of the human Top1 (topoisomerase I) catalytic cycle have been analysed in the presence of a pentacyclic-diquinoid synthetic compound. The experiments indicate that it efficiently inhibits the cleavage step of the enzyme reaction, fitting well into the catalytic site. Surprisingly the compound, when incubated with the binary topoisomerase–DNA cleaved complex, helps the enzyme to remove itself from the cleaved DNA and close the DNA gap, increasing the religation rate. The compound also induces the religation of the stalled enzyme–CPT (camptothecin)–DNA ternary complex. Analysis of the molecule docked over the binary complex, together with its chemical properties, suggests that the religation enhancement is due to the presence on the compound of two oxygen atoms that act as hydrogen acceptors. This property facilitates the deprotonation of the 5′ DNA end, suggesting that this is the limiting step in the topoisomerase religation mechanism. PMID:23368812

Screening and analysis of bioactive compounds with biofingerprinting chromatogram analysis of traditional Chinese medicines targeting DNA by microdialysis/HPLC.

PubMed

Su, Xingye; Kong, Liang; Li, Xin; Chen, Xueguo; Guo, Ming; Zou, Hanfa

2005-05-27

Biofingerprinting chromatogram analysis, which is defined as the comparison of fingerprinting chromatograms of the extract of traditional Chinese medicines (TCMs) before and after the interaction with biological systems (DNA, protein, cell, etc.), was proposed for screening and analysis of the multiple bioactive compounds in TCMs. A method of microdialysis sampling combined with high performance liquid chromatography (HPLC) was applied to the study of DNA-binding property for the extracts of TCMs. Seven compounds were found to bind to calf thymus DNA (ct-DNA) from the TCMs of Coptis chinensis Franch (Coptis), but only three ones from Phellodendron amurense Rupr. (Phellodendron) and none from Sophoraflavescens Ait. (Sophora) to bind to ct-DNA, respectively. Three of them were identified as berberine, palmatine and jatrorrhizine and their association constants (K) to ct-DNA were determined by microdialysis/HPLC. Competitive binding behaviors of them to ct-DNA were also investigated.

Zebrafish as a model to study the role of DNA methylation in environmental toxicology.

PubMed

Kamstra, Jorke H; Aleström, Peter; Kooter, Jan M; Legler, Juliette

2015-11-01

Environmental epigenetics is a rapidly growing field which studies the effects of environmental factors such as nutrition, stress, and exposure to compounds on epigenetic gene regulation. Recent studies have shown that exposure to toxicants in vertebrates is associated with changes in DNA methylation, a major epigenetic mechanism affecting gene transcription. Zebra fish, a well-known model in toxicology and developmental biology, are emerging as a model species in environmental epigenetics despite their evolutionary distance to rodents and humans. In this review, recent insights in DNA methylation during zebra fish development are discussed and compared to mammalian models in order to evaluate zebra fish as a model to study the role of DNA methylation in environmental toxicology. Differences exist in DNA methylation reprogramming during early development, whereas in later developmental stages, tissue distribution of both 5-methylcytosine and 5-hydroxymethylcytosine seems more conserved between species, as well as basic DNA (de)methylation mechanisms. All DNA methyl transferases identified so far in mammals are present in zebra fish, as well as a number of major demethylation pathways. However, zebra fish appear to lack some methylation pathways present in mammals, such as parental imprinting. Several studies report effects on DNA methylation in zebra fish following exposure to environmental contaminants, such as arsenic, benzo[a]pyrene, and tris(1,3-dichloro-2-propyl)phosphate. Though more research is needed to examine heritable effects of contaminant exposure on DNA methylation, recent data suggests the usefulness of the zebra fish as a model in environmental epigenetics.

C8-Linked Pyrrolobenzodiazepine Monomers with Inverted Building Blocks Show Selective Activity against Multidrug Resistant Gram-Positive Bacteria.

PubMed

Andriollo, Paolo; Hind, Charlotte K; Picconi, Pietro; Nahar, Kazi S; Jamshidi, Shirin; Varsha, Amrit; Clifford, Melanie; Sutton, J Mark; Rahman, Khondaker Miraz

2018-02-09

Antimicrobial resistance has become a major global concern. Development of novel antimicrobial agents for the treatment of infections caused by multidrug resistant (MDR) pathogens is an urgent priority. Pyrrolobenzodiazepines (PBDs) are a promising class of antibacterial agents initially discovered and isolated from natural sources. Recently, C8-linked PBD biaryl conjugates have been shown to be active against some MDR Gram-positive strains. To explore the role of building block orientations on antibacterial activity and obtain structure activity relationship (SAR) information, four novel structures were synthesized in which the building blocks of previously reported compounds were inverted, and their antibacterial activity was studied. The compounds showed minimum inhibitory concentrations (MICs) in the range of 0.125-32 μg/mL against MDR Gram-positive strains with a bactericidal mode of action. The results showed that a single inversion of amide bonds reduces the activity while the double inversion restores the activity against MDR pathogens. All inverted compounds did not stabilize DNA and lacked eukaryotic toxicity. The compounds inhibit DNA gyrase in vitro, and the most potent compound was equally active against both wild-type and mutant DNA gyrase in a biochemical assay. The observed activity of the compounds against methicillin resistant S. aureus (MRSA) strains with equivalent gyrase mutations is consistent with gyrase inhibition being the mechanism of action in vivo, although this has not been definitively confirmed in whole cells. This conclusion is supported by a molecular modeling study showing interaction of the compounds with wild-type and mutant gyrases. This study provides important SAR information about this new class of antibacterial agents.

A new class of antibacterials, the imidazopyrazinones, reveal structural transitions involved in DNA gyrase poisoning and mechanisms of resistance.

PubMed

Germe, Thomas; Vörös, Judit; Jeannot, Frederic; Taillier, Thomas; Stavenger, Robert A; Bacqué, Eric; Maxwell, Anthony; Bax, Benjamin D

2018-05-04

Imidazopyrazinones (IPYs) are a new class of compounds that target bacterial topoisomerases as a basis for their antibacterial activity. We have characterized the mechanism of these compounds through structural/mechanistic studies showing they bind and stabilize a cleavage complex between DNA gyrase and DNA ('poisoning') in an analogous fashion to fluoroquinolones, but without the requirement for the water-metal-ion bridge. Biochemical experiments and structural studies of cleavage complexes of IPYs compared with an uncleaved gyrase-DNA complex, reveal conformational transitions coupled to DNA cleavage at the DNA gate. These involve movement at the GyrA interface and tilting of the TOPRIM domains toward the scissile phosphate coupled to capture of the catalytic metal ion. Our experiments show that these structural transitions are involved generally in poisoning of gyrase by therapeutic compounds and resemble those undergone by the enzyme during its adenosine triphosphate-coupled strand-passage cycle. In addition to resistance mutations affecting residues that directly interact with the compounds, we characterized a mutant (D82N) that inhibits formation of the cleavage complex by the unpoisoned enzyme. The D82N mutant appears to act by stabilizing the binary conformation of DNA gyrase with uncleaved DNA without direct interaction with the compounds. This provides general insight into the resistance mechanisms to antibiotics targeting bacterial type II topoisomerases.

A new class of antibacterials, the imidazopyrazinones, reveal structural transitions involved in DNA gyrase poisoning and mechanisms of resistance

PubMed Central

Germe, Thomas; Vörös, Judit; Jeannot, Frederic; Taillier, Thomas; Stavenger, Robert A; Bacqué, Eric; Bax, Benjamin D

2018-01-01

Abstract Imidazopyrazinones (IPYs) are a new class of compounds that target bacterial topoisomerases as a basis for their antibacterial activity. We have characterized the mechanism of these compounds through structural/mechanistic studies showing they bind and stabilize a cleavage complex between DNA gyrase and DNA (‘poisoning’) in an analogous fashion to fluoroquinolones, but without the requirement for the water–metal–ion bridge. Biochemical experiments and structural studies of cleavage complexes of IPYs compared with an uncleaved gyrase–DNA complex, reveal conformational transitions coupled to DNA cleavage at the DNA gate. These involve movement at the GyrA interface and tilting of the TOPRIM domains toward the scissile phosphate coupled to capture of the catalytic metal ion. Our experiments show that these structural transitions are involved generally in poisoning of gyrase by therapeutic compounds and resemble those undergone by the enzyme during its adenosine triphosphate-coupled strand-passage cycle. In addition to resistance mutations affecting residues that directly interact with the compounds, we characterized a mutant (D82N) that inhibits formation of the cleavage complex by the unpoisoned enzyme. The D82N mutant appears to act by stabilizing the binary conformation of DNA gyrase with uncleaved DNA without direct interaction with the compounds. This provides general insight into the resistance mechanisms to antibiotics targeting bacterial type II topoisomerases. PMID:29538767

Unusually Strong Binding to the DNA Minor Groove by a Highly Twisted Benzimidazole-Diphenylether: Induced Fit and Bound Water†

PubMed Central

Tanious, Farial A.; Laine, William; Peixoto, Paul; Bailly, Christian; Goodwin, Kristie D.; Lewis, Mark A.; Long, Eric C.; Georgiadis, Millie M.; Tidwell, Richard R.; Wilson, W. David

2008-01-01

RT29 is a dicationic diamidine derivative that does not obey the classical “rules” for shape and functional group placement that are expected to result in strong binding and specific recognition of the DNA minor groove. The compound contains a benzimidazole-diphenyl ether core that is flanked by the amidine cations. The diphenyl ether is highly twisted and gives the entire compound too much curvature to fit well to the shape of the minor groove. DNaseI footprinting, fluorescence intercalator displacement studies and circular dichroism spectra, however, indicate that the compound is an AT specific minor groove binding agent. Even more surprisingly, quantitative biosensor-surface plasmon resonance and isothermal titration calorimetric results indicate that the compound binds with exceptional strength to certain AT sequences in DNA with a large negative enthalpy of binding. Crystallographic results for the DNA complex of RT29 compared to calculated results for the free compound show that the compound undergoes significant conformational changes to enhance its minor groove interactions. In addition, a water molecule is incorporated directly into the complex to complete the compound-DNA interface and it forms an essential link between the compound and base pair edges at the floor of the minor groove. The calculated ΔCp value for complex formation is substantially less than the experimentally observed value in support of water being an intrinsic part of the complex with a major contribution to the ΔCp value. Both the induced fit conformational changes of the compound and the bound water are essential for strong binding to DNA by RT29. PMID:17506529

DNA as a Binary Code: How the Physical Structure of Nucleotide Bases Carries Information

ERIC Educational Resources Information Center

McCallister, Gary

2005-01-01

The DNA triplet code also functions as a binary code. Because double-ring compounds cannot bind to double-ring compounds in the DNA code, the sequence of bases classified simply as purines or pyrimidines can encode for smaller groups of possible amino acids. This is an intuitive approach to teaching the DNA code. (Contains 6 figures.)

Multiply Intercalator-Substituted Cu(II) Cyclen Complexes as DNA Condensers and DNA/RNA Synthesis Inhibitors.

PubMed

Hormann, Jan; Malina, Jaroslav; Lemke, Oliver; Hülsey, Max J; Wedepohl, Stefanie; Potthoff, Jan; Schmidt, Claudia; Ott, Ingo; Keller, Bettina G; Brabec, Viktor; Kulak, Nora

2018-05-07

Many drugs that are applied in anticancer therapy such as the anthracycline doxorubicin contain DNA-intercalating 9,10-anthraquinone (AQ) moieties. When Cu(II) cyclen complexes were functionalized with up to three (2-anthraquinonyl)methyl substituents, they efficiently inhibited DNA and RNA synthesis resulting in high cytotoxicity (selective for cancer cells) accompanied by DNA condensation/aggregation phenomena. Molecular modeling suggests an unusual bisintercalation mode with only one base pair between the two AQ moieties and the metal complex as a linker. A regioisomer, in which the AQ moieties point in directions unfavorable for such an interaction, had a much weaker biological activity. The ligands alone and corresponding Zn(II) complexes (used as redox inert control compounds) also exhibited lower activity.

High-Content Analysis Provides Mechanistic Insights into the Testicular Toxicity of Bisphenol A and Selected Analogues in Mouse Spermatogonial Cells.

PubMed

Liang, Shenxuan; Yin, Lei; Shengyang Yu, Kevin; Hofmann, Marie-Claude; Yu, Xiaozhong

2017-01-01

Bisphenol A (BPA), an endocrine-disrupting compound, was found to be a testicular toxicant in animal models. Bisphenol S (BPS), bisphenol AF (BPAF), and tetrabromobisphenol A (TBBPA) were recently introduced to the market as alternatives to BPA. However, toxicological data of these compounds in the male reproductive system are still limited so far. This study developed and validated an automated multi-parametric high-content analysis (HCA) using the C18-4 spermatogonial cell line as a model. We applied these validated HCA, including nuclear morphology, DNA content, cell cycle progression, DNA synthesis, cytoskeleton integrity, and DNA damage responses, to characterize and compare the testicular toxicities of BPA and 3 selected commercial available BPA analogues, BPS, BPAF, and TBBPA. HCA revealed BPAF and TBBPA exhibited higher spermatogonial toxicities as compared with BPA and BPS, including dose- and time-dependent alterations in nuclear morphology, cell cycle, DNA damage responses, and perturbation of the cytoskeleton. Our results demonstrated that this specific culture model together with HCA can be utilized for quantitative screening and discriminating of chemical-specific testicular toxicity in spermatogonial cells. It also provides a fast and cost-effective approach for the identification of environmental chemicals that could have detrimental effects on reproduction. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A Novel Spirooxindole Derivative Inhibits the Growth of Leishmania donovani Parasites both In Vitro and In Vivo by Targeting Type IB Topoisomerase

PubMed Central

Saha, Sourav; Acharya, Chiranjit; Pal, Uttam; Chowdhury, Somenath Roy; Sarkar, Kahini; Maiti, Nakul C.

2016-01-01

Visceral leishmaniasis is a fatal parasitic disease, and there is an emergent need for development of effective drugs against this neglected tropical disease. We report here the development of a novel spirooxindole derivative, N-benzyl-2,2′α-3,3′,5′,6′,7′,7α,α′-octahydro-2methoxycarbonyl-spiro[indole-3,3′-pyrrolizidine]-2-one (compound 4c), which inhibits Leishmania donovani topoisomerase IB (LdTopIB) and kills the wild type as well as drug-resistant parasite strains. This compound inhibits catalytic activity of LdTopIB in a competitive manner. Unlike camptothecin (CPT), the compound does not stabilize the DNA-topoisomerase IB cleavage complex; rather, it hinders drug-DNA-enzyme covalent complex formation. Fluorescence studies show that the stoichiometry of this compound binding to LdTopIB is 2:1 (mole/mole), with a dissociation constant of 6.65 μM. Molecular docking with LdTopIB using the stereoisomers of compound 4c produced two probable hits for the binding site, one in the small subunit and the other in the hinge region of the large subunit of LdTopIB. This spirooxindole is highly cytotoxic to promastigotes of L. donovani and also induces apoptosis-like cell death in the parasite. Treatment with compound 4c causes depolarization of mitochondrial membrane potential, formation of reactive oxygen species inside parasites, and ultimately fragmentation of nuclear DNA. Compound 4c also effectively clears amastigote forms of wild-type and drug-resistant parasites from infected mouse peritoneal macrophages but has less of an effect on host macrophages. Moreover, compound 4c showed strong antileishmanial efficacies in the BALB/c mouse model of leishmaniasis. This compound potentially can be used as a lead for developing excellent antileishmanial agents against emerging drug-resistant strains of the parasite. PMID:27503653

Improved model of hydrated calcium ion for molecular dynamics simulations using classical biomolecular force fields.

PubMed

Yoo, Jejoong; Wilson, James; Aksimentiev, Aleksei

2016-10-01

Calcium ions (Ca(2+) ) play key roles in various fundamental biological processes such as cell signaling and brain function. Molecular dynamics (MD) simulations have been used to study such interactions, however, the accuracy of the Ca(2+) models provided by the standard MD force fields has not been rigorously tested. Here, we assess the performance of the Ca(2+) models from the most popular classical force fields AMBER and CHARMM by computing the osmotic pressure of model compounds and the free energy of DNA-DNA interactions. In the simulations performed using the two standard models, Ca(2+) ions are seen to form artificial clusters with chloride, acetate, and phosphate species; the osmotic pressure of CaAc2 and CaCl2 solutions is a small fraction of the experimental values for both force fields. Using the standard parameterization of Ca(2+) ions in the simulations of Ca(2+) -mediated DNA-DNA interactions leads to qualitatively wrong outcomes: both AMBER and CHARMM simulations suggest strong inter-DNA attraction whereas, in experiment, DNA molecules repel one another. The artificial attraction of Ca(2+) to DNA phosphate is strong enough to affect the direction of the electric field-driven translocation of DNA through a solid-state nanopore. To address these shortcomings of the standard Ca(2+) model, we introduce a custom model of a hydrated Ca(2+) ion and show that using our model brings the results of the above MD simulations in quantitative agreement with experiment. Our improved model of Ca(2+) can be readily applied to MD simulations of various biomolecular systems, including nucleic acids, proteins and lipid bilayer membranes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 752-763, 2016. © 2016 Wiley Periodicals, Inc.

A selective plasmin inhibitor, trans-aminomethylcyclohexanecarbonyl-L-(O-picolyl)tyrosine-octylamide (YO-2), induces thymocyte apoptosis.

PubMed

Lee, Eibai; Enomoto, Riyo; Takemura, Kazu; Tsuda, Yuko; Okada, Yoshio

2002-04-01

The treatment of rat thymocytes with YO-2, a novel inhibitor of plasmin, resulted in an increase in DNA fragmentation. DNA fragmentation was also induced by another YO compounds such as YO-0, -3, -4 and -5. These YO compounds are the inhibitor of plasmin activity. On the other hand, YO-1, -6 and -8 that hardly inhibit plasmin activity had no effect on DNA fragmentation. Analysis of fragmented DNA from thymocytes treated with YO-2 by agarose gel electrophoresis revealed that the compound caused internucleosomal DNA fragmentation. In addition, judging from a laser scanning microscopy, annexin V-positive and propidium iodide-negative cells were increased by the treatment of the cells with the compound. Moreover, chromatin condensation was observed in thymocytes treated with the compound. These results demonstrated that YO-2 induces thymocyte apoptosis. There seemed to be some correlation between the apoptosis induced by YO compounds and their plasmin inhibitory effect. However, because the other protease inhibitors including pepstatin A, leupeptin, AEBSF, DFP and E-64-d did not affect DNA fragmentation, YO compounds are likely to have unique mechanism on plasmin or to show the effect on the other plasmin-like proteases. The plasmin inhibitory activity may have an important role in YO-2-induced apoptosis. Furthermore, the stimulations of caspase-8, -9 and -3-like activities were observed in thymocytes treated with YO-2. These results suggest that YO-2 induces thymocyte apoptosis via activation of caspase cascade.

Synthesis of galactosyl compounds for targeted gene delivery.

PubMed

Ren, T; Zhang, G; Liu, D

2001-11-01

Cell-specific DNA delivery offers a great potential for targeted gene therapy. Toward this end, we have synthesized a series of compounds carrying galactose residues as a targeting ligand for asialoglycoprotein receptors of hepatocytes and primary amine groups as a functional domain for DNA binding. Biological activity of these galactosyl compounds in DNA delivery was evaluated in HepG2 and BL-6 cells and compared with respect to the number of galactose residues as well as primary amine groups in each molecule. Transfection experiments using a firefly luciferase gene as a reporter revealed that compounds with multivalent binding properties were more active in DNA delivery. An optimal transfection activity in HepG2 cells requires seven primary amine groups and a minimum of two galactose residues in each molecule. The transfection activity of compounds carrying multi-galactose residues can be inhibited by asialofetuin, a natural substrate for asialoglycoprotein receptors of hepatocytes, suggesting that gene transfer by these galactosyl compounds is asialoglycoprotein receptor-mediated. These results provide direct evidence in support of our new strategy for the use of small and synthetic compounds for cell specific and targeted gene delivery.

Risk assessment of DNA-reactive carcinogens in food.

PubMed

Jeffrey, A M; Williams, G M

2005-09-01

Risk assessment of DNA-reactive carcinogens in food requires knowledge of the extent of DNA damage in the target organ which results from the competition between DNA adduct formation and repair. Estimates of DNA adduct levels can be made by direct measurement or indirectly as a consequence of their presence, for example, by tumor formation in animal models or exposed populations epidemiologically. Food-borne DNA-reactive carcinogens are present from a variety of sources. They are generally not intrinsically DNA-reactive but require bioactivation to DNA-reactive metabolites a process which may be modulated by the compound itself or the presence of other xenobiotics. A single DNA reactant may form several distinct DNA adducts each undergoing different rates of repair. Some DNA reactants may be photochemically activated or produce reactive oxygen species and thus indirect oxidative DNA damage. The levels of DNA adducts arising from exposures influenced by variations in the doses, the frequency with which an individual is exposed, and rates of DNA repair for specific adducts. Each adduct has a characteristic efficiency with which it induces mutations. Based on experience with the well-studied DNA-reactive food carcinogen aflatoxin B(1) (AFB(1)), a limit of 20 ppb or approximately 30 microg/day has been set and is considered a tolerable daily intake (TDI). Since AFB(1) is considered a potent carcinogen, doses of <1.5 microg of unknown compounds are considered TDIs. Most DNA-reactants, including acrylamide, heterocyclic amines, and alpha,beta-unsaturated carbonyl are below this value. Above that value, measurement of actual DNA adducts levels in either experimental animals with a risk assessment, or, when this occurs, exposed humans are needed. A number of approaches to undertake this are described including immunological, mass spectrometric and (32)P-postlabeling or the use of surrogates such as hemoglobin adducts, together with approaches to evaluate the results. A discussion of approaches to estimating possible threshold effects for DNA-reactive carcinogens is made.

Risk assessment of DNA-reactive carcinogens in food

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeffrey, A.M.; Williams, G.M.

2005-09-01

Risk assessment of DNA-reactive carcinogens in food requires knowledge of the extent of DNA damage in the target organ which results from the competition between DNA adduct formation and repair. Estimates of DNA adduct levels can be made by direct measurement or indirectly as a consequence of their presence, for example, by tumor formation in animal models or exposed populations epidemiologically. Food-borne DNA-reactive carcinogens are present from a variety of sources. They are generally not intrinsically DNA-reactive but require bioactivation to DNA-reactive metabolites a process which may be modulated by the compound itself or the presence of other xenobiotics. Amore » single DNA reactant may form several distinct DNA adducts each undergoing different rates of repair. Some DNA reactants may be photochemically activated or produce reactive oxygen species and thus indirect oxidative DNA damage. The levels of DNA adducts arising from exposures influenced by variations in the doses, the frequency with which an individual is exposed, and rates of DNA repair for specific adducts. Each adduct has a characteristic efficiency with which it induces mutations. Based on experience with the well-studied DNA-reactive food carcinogen aflatoxin B{sub 1} (AFB{sub 1}), a limit of 20 ppb or {approx}30 {mu}g/day has been set and is considered a tolerable daily intake (TDI). Since AFB{sub 1} is considered a potent carcinogen, doses of <1.5 {mu}g of unknown compounds are considered TDIs. Most DNA-reactants, including acrylamide, heterocyclic amines, and {alpha},{beta}-unsaturated carbonyl are below this value. Above that value, measurement of actual DNA adducts levels in either experimental animals with a risk assessment, or, when this occurs, exposed humans are needed. A number of approaches to undertake this are described including immunological, mass spectrometric and {sup 32}P-postlabeling or the use of surrogates such as hemoglobin adducts, together with approaches to evaluate the results. A discussion of approaches to estimating possible threshold effects for DNA-reactive carcinogens is made.« less

Action mechanism of 6, 6'-dihydroxythiobinupharidine from Nuphar japonicum, which showed anti-MRSA and anti-VRE activities.

PubMed

Okamura, Shinya; Nishiyama, Eri; Yamazaki, Tomohiro; Otsuka, Nao; Taniguchi, Shoko; Ogawa, Wakano; Hatano, Tsutomu; Tsuchiya, Tomofusa; Kuroda, Teruo

2015-06-01

Multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE), cause serious infections at clinical sites, for which the development of new drugs is necessary. We screened candidates for new antibiotics and investigated its action mechanism. An antimicrobial compound was isolated from an extract of Nuphar japonicum. Its chemical structure was determined by NMR, MS, and optical rotation. We measured its minimum inhibitory concentration (MIC) using the microdilution method. The effects of the compound on DNA gyrase and DNA topoisomerase IV were investigated with DNA supercoiling, decatenation, and cleavage assay. We isolated and identified 6,6'-dihydroxythiobinupharidine as the antimicrobial compound. The MIC of this compound was 1-4 μg/mL against various MRSA and VRE strains. We also demonstrated that this compound inhibited DNA topoisomerase IV (IC50 was 10-15 μM), but not DNA gyrase in S. aureus, both of which are known to be the targets of quinolone antibiotics and necessary for DNA replication. However, this compound only exhibited slight cross-resistance to norfloxacin-resistant S. aureus, which indicated that DTBN might inhibit other targets besides topoisomerase IV. These results suggest that 6,6'-dihydroxythiobinupharidine may be a potent candidate or seed for novel antibacterial agents. DTBN from N. japonicum showed anti-MRSA and anti-VRE activities. DTBN might be involved in the inhibition of DNA topoisomerase IV. DTBN might be useful as a seed compound. The information on the inhibition mechanism of DTBN will be useful for the modification of DTBN towards developing novel anti-MRSA and anti-VRE drug. Copyright © 2015 Elsevier B.V. All rights reserved.

Study of interaction of antimutagenic 1,4-dihydropyridine AV-153-Na with DNA-damaging molecules and its impact on DNA repair activity.

PubMed

Leonova, Elina; Rostoka, Evita; Sauvaigo, Sylvie; Baumane, Larisa; Selga, Turs; Sjakste, Nikolajs

2018-01-01

1,4-dihydropyridines (1,4-DHP) possesses important biochemical and pharmacological properties, including antioxidant and antimutagenic activities. It was shown that the antimutagenic 1,4-dihydropyridine AV-153-Na interacts with DNA. The aim of the current study was to test the capability of the compound to scavenge peroxynitrite and hydroxyl radical, to test intracellular distribution of the compound, and to assess the ability of the compound to modify the activity of DNA repair enzymes and to protect the DNA in living cells against peroxynitrite-induced damage. Peroxynitrite decomposition was assayed by UV spectroscopy, hydroxyl radical scavenging-by EPR spectroscopy. DNA breakage was determined by the "comet method", activity of DNA repair enzymes-using Glyco-SPOT and ExSy-SPOT assays. Intracellular distribution of the compound was studied by laser confocal scanning fluorescence microscopy. Fluorescence spectroscopy titration and circular dichroism spectroscopy were used to study interactions of the compound with human serum albumin. Some ability to scavenge hydroxyl radical by AV-153-Na was detected by the EPR method, but it turned out to be incapable of reacting chemically with peroxynitrite. However, AV-153-Na effectively decreased DNA damage produced by peroxynitrite in cultured HeLa cells. The Glyco-SPOT test essentially revealed an inhibition by AV-153-Na of the enzymes involved thymine glycol repair. Results with ExSy-SPOT chip indicate that AV-153-Na significantly stimulates excision/synthesis repair of 8-oxoguanine (8-oxoG), abasic sites (AP sites) and alkylated bases. Laser confocal scanning fluorescence microscopy demonstrated that within the cells AV-153-Na was found mostly in the cytoplasm; however, a stain in nucleolus was also detected. Binding to cytoplasmic structures might occur due to high affinity of the compound to proteins revealed by spectroscopical methods. Activation of DNA repair enzymes after binding to DNA appears to be the basis for the antimutagenic effects of AV-153-Na.

Evaluation of Acridine Orange Derivatives as DNA-Targeted Radiopharmaceuticals for Auger Therapy: Influence of the Radionuclide and Distance to DNA

PubMed Central

Pereira, Edgar; do Quental, Letícia; Palma, Elisa; Oliveira, Maria Cristina; Mendes, Filipa; Raposinho, Paula; Correia, Isabel; Lavrado, João; Di Maria, Salvatore; Belchior, Ana; Vaz, Pedro; Santos, Isabel; Paulo, António

2017-01-01

A new family of 99mTc(I)- tricarbonyl complexes and 125I-heteroaromatic compounds bearing an acridine orange (AO) DNA targeting unit was evaluated for Auger therapy. Characterization of the DNA interaction, performed with the non-radioactive Re and 127I congeners, confirmed that all compounds act as DNA intercalators. Both classes of compounds induce double strand breaks (DSB) in plasmid DNA but the extent of DNA damage is strongly dependent on the linker between the Auger emitter (99mTc or 125I) and the AO moiety. The in vitro evaluation was complemented with molecular docking studies and Monte Carlo simulations of the energy deposited at the nanometric scale, which corroborated the experimental data. Two of the tested compounds, 125I-C5 and 99mTc-C3, place the corresponding radionuclide at similar distances to DNA and produce comparable DSB yields in plasmid and cellular DNA. These results provide the first evidence that 99mTc can induce DNA damage with similar efficiency to that of 125I, when both are positioned at comparable distances to the double helix. Furthermore, the high nuclear retention of 99mTc-C3 in tumoral cells suggests that 99mTc-labelled AO derivatives are more promising for the design of Auger-emitting radiopharmaceuticals than the 125I-labelled congeners. PMID:28211920

The water soluble peripherally tetra-substituted zinc(ii), manganese(iii) and copper(ii) phthalocyanines as new potential anticancer agents.

PubMed

Barut, Burak; Sofuoğlu, Ayşenur; Biyiklioglu, Zekeriya; Özel, Arzu

2016-09-28

In this study, [2-(2-morpholin-4-ylethoxy)ethoxy] group substituted zinc(ii), manganese(iii) and copper(ii) phthalocyanines 2-4 and their water soluble derivatives 2a, 3a and 4a were synthesized and the interactions of compounds 2a, 3a and 4a with CT-DNA and supercoiled pBR322 plasmid DNA were investigated. The results of binding experiments showed that these compounds were able to interact with CT-DNA via intercalative mode with a strong binding affinity in the order 3a > 2a > 4a. DNA-photocleavage activities of compounds 2a, 3a and 4a were determined. These compounds cleaved supercoiled pBR322 plasmid DNA efficiently under irradiation at 650 nm for 2a and 4a, and at 750 nm for 3a. These compounds displayed remarkable inhibitory activities against topoisomerase I enzyme in a dose-dependent manner. All of these results suggest that these phthalocyanines might be suitable anticancer agents due to their strong binding affinities, significant cleavage activities and effective topoisomerase I inhibition.

Curcumin-I Knoevenagel's condensates and their Schiff's bases as anticancer agents: synthesis, pharmacological and simulation studies.

PubMed

Ali, Imran; Haque, Ashanul; Saleem, Kishwar; Hsieh, Ming Fa

2013-07-01

Pyrazolealdehydes (4a-d), Knoevenagel's condensates (5a-d) and Schiff's bases (6a-d) of curcumin-I were synthesized, purified and characterized. Hemolysis assays, cell line activities, DNA bindings and docking studies were carried out. These compounds were lesser hemolytic than standard drug doxorubicin. Minimum cell viability (MCF-7; wild) observed was 59% (1.0 μg/mL) whereas the DNA binding constants ranged from 1.4×10(3) to 8.1×10(5) M(-1). The docking energies varied from -7.30 to -13.4 kcal/mol. It has been observed that DNA-compound adducts were stabilized by three governing forces (Van der Wall's, H-bonding and electrostatic attractions). It has also been observed that compounds 4a-d preferred to enter minor groove while 5a-d and 6a-d interacted with major grooves of DNA. The anticancer activities of the reported compounds might be due to their interactions with DNA. These results indicated the bright future of the reported compounds as anticancer agents. Copyright © 2013 Elsevier Ltd. All rights reserved.

Investigation of the electrostatic and hydration properties of DNA minor groove-binding by a heterocyclic diamidine by osmotic pressure.

PubMed

Erlitzki, Noa; Huang, Kenneth; Xhani, Suela; Farahat, Abdelbasset A; Kumar, Arvind; Boykin, David W; Poon, Gregory M K

2017-12-01

Previous investigations of sequence-specific DNA binding by model minor groove-binding compounds showed that the ligand/DNA complex was destabilized in the presence of compatible co-solutes. Inhibition was interpreted in terms of osmotic stress theory as the uptake of significant numbers of excess water molecules from bulk solvent upon complex formation. Here, we interrogated the AT-specific DNA complex formed with the symmetric heterocyclic diamidine DB1976 as a model for minor groove DNA recognition using both ionic (NaCl) and non-ionic cosolutes (ethylene glycol, glycine betaine, maltose, nicotinamide, urea). While the non-ionic cosolutes all destabilized the ligand/DNA complex, their quantitative effects were heterogeneous in a cosolute- and salt-dependent manner. Perturbation with NaCl in the absence of non-ionic cosolute showed that preferential hydration water was released upon formation of the DB1976/DNA complex. As salt probes counter-ion release from charged groups such as the DNA backbone, we propose that the preferential hydration uptake in DB1976/DNA binding observed in the presence of osmolytes reflects the exchange of preferentially bound cosolute with hydration water in the environs of the bound DNA, rather than a net uptake of hydration waters by the complex. Copyright © 2017 Elsevier B.V. All rights reserved.

Lemon balm extract (Melissa officinalis, L.) promotes melanogenesis and prevents UVB-induced oxidative stress and DNA damage in a skin cell model.

PubMed

Pérez-Sánchez, Almudena; Barrajón-Catalán, Enrique; Herranz-López, María; Castillo, Julián; Micol, Vicente

2016-11-01

Solar ultraviolet (UV) radiation is one of the main causes of a variety of cutaneous disorders, including photoaging and skin cancer. Its UVB component (280-315nm) leads to oxidative stress and causes inflammation, DNA damage, p53 induction and lipid and protein oxidation. Recently, an increase in the use of plant polyphenols with antioxidant and anti-inflammatory properties has emerged to protect human skin against the deleterious effects of sunlight. This study evaluates the protective effects of lemon balm extract (LBE) (Melissa Officinalis, L) and its main phenolic compound rosmarinic acid (RA) against UVB-induced damage in human keratinocytes. The LBE composition was determined by HPLC analysis coupled to photodiode array detector and ion trap mass spectrometry with electrospray ionization (HPLC-DAD-ESI-IT-MS/MS). Cell survival, ROS generation and DNA damage were determined upon UVB irradiation in the presence of LBE. The melanogenic capacity of LBE was also determined. RA and salvianolic acid derivatives were the major compounds, but caffeic acid and luteolin glucuronide were also found in LBE. LBE and RA significantly increased the survival of human keratinocytes upon UVB radiation, but LBE showed a stronger effect. LBE significantly decreased UVB-induced intracellular ROS production. Moreover, LBE reduced UV-induced DNA damage and the DNA damage response (DDR), which were measured as DNA strand breaks in the comet assay and histone H2AX activation, respectively. Finally, LBE promoted melanogenesis in the cell model. These results suggest that LBE may be considered as a candidate for the development of oral/topical photoprotective ingredients against UVB-induced skin damage. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Design, synthesis and biological evaluation of berberine-benzimidazole hybrids as new type of potentially DNA-targeting antimicrobial agents.

PubMed

Jeyakkumar, Ponmani; Zhang, Ling; Avula, Srinivasa Rao; Zhou, Cheng-He

2016-10-21

A series of novel berberine-benzimidazole derivatives were conveniently and efficiently synthesized and characterized by NMR, IR, MS and HRMS spectra. Most of the prepared compounds showed effective antimicrobial activities in contrast with clinical norfloxacin, chloromycin and fluconazole. Especially, compound 5d exhibited good anti-MRSA, anti-Escherichia coli, and anti-Salmonella typhi activity with low MIC values of 2-8 μg/mL, which were comparable or even superior to reference drugs. The preliminarily interactive investigation revealed that the most active compound 5d could effectively intercalate into DNA to form 5d-DNA complex and cleavage DNA by agarose gel electrophoresis experiments. It was also found that compound 5d was able to efficiently permeabilize the membranes of both Gram-positive (MRSA) and Gram-negative (E. coli DH52) bacteria. Experiments and molecular docking both showed that human serum albumin (HSA) could effectively transport compound 5d and hydrophobic interactions and hydrogen bonds play important roles in the association of compound 5d with HSA. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Conjugation of Benzylvanillin and Benzimidazole Structure Improves DNA Binding with Enhanced Antileukemic Properties

PubMed Central

Al-Mudarris, Ban A.; Chen, Shih-Hsun; Liang, Po-Huang; Osman, Hasnah; Jamal Din, Shah Kamal Khan; Abdul Majid, Amin M. S.

2013-01-01

Benzyl-o-vanillin and benzimidazole nucleus serve as important pharmacophore in drug discovery. The benzyl vanillin (2-(benzyloxy)-3-methoxybenzaldehyde) compound shows anti-proliferative activity in HL60 leukemia cancer cells and can effect cell cycle progression at G2/M phase. Its apoptosis activity was due to disruption of mitochondrial functioning. In this study, we have studied a series of compounds consisting of benzyl vanillin and benzimidazole structures. We hypothesize that by fusing these two structures we can produce compounds that have better anticancer activity with improved specificity particularly towards the leukemia cell line. Here we explored the anticancer activity of three compounds namely 2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2MP, N-1-(2-benzyloxy-3-methoxybenzyl)-2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2XP, and (R) and (S)-1-(2-benzyloxy-3-methoxyphenyl)-2, 2, 2-trichloroethyl benzenesulfonate, 3BS and compared their activity to 2-benzyloxy-3-methoxybenzaldehyde, (Bn1), the parent compound. 2XP and 3BS induces cell death of U937 leukemic cell line through DNA fragmentation that lead to the intrinsic caspase 9 activation. DNA binding study primarily by the equilibrium binding titration assay followed by the Viscosity study reveal the DNA binding through groove region with intrinsic binding constant 7.39 µM/bp and 6.86 µM/bp for 3BS and 2XP respectively. 2XP and 3BS showed strong DNA binding activity by the UV titration method with the computational drug modeling showed that both 2XP and 3BS failed to form any electrostatic linkages except via hydrophobic interaction through the minor groove region of the nucleic acid. The benzylvanillin alone (Bn1) has weak anticancer activity even after it was combined with the benzimidazole (2MP), but after addition of another benzylvanillin structure (2XP), stronger activity was observed. Also, the combination of benzylvanillin with benzenesulfonate (3BS) significantly improved the anticancer activity of Bn1. The present study provides a new insight of benzyl vanillin derivatives as potential anti-leukemic agent. PMID:24260527

Identification of a Hydrophobic Cleft in the LytTR Domain of AgrA as a Locus for Small Molecule Interactions that Inhibit DNA Binding

PubMed Central

Leonard, Paul G.; Bezar, Ian F.; Sidote, David J.; Stock, Ann M.

2012-01-01

The AgrA transcription factor regulates the quorum-sensing response in Staphylococcus aureus, controlling the production of hemolysins and other virulence factors. AgrA binds to DNA via its C-terminal LytTR domain, a domain not found in humans but common in many pathogenic bacteria, making it a potential target for antimicrobial development. We have determined the crystal structure of the apo AgrA LytTR domain and screened a library of 500 fragment compounds to find inhibitors of AgrA DNA-binding activity. Using NMR, the binding site for five compounds has been mapped to a common locus at the C-terminal end of the LytTR domain, a site known to be important for DNA-binding activity. Three of these compounds inhibit AgrA DNA binding. These results provide the first evidence that LytTR domains can be targeted by small organic compounds. PMID:23181972

Studies on DNA damage: discordant responses of rate of DNA disentanglement (viscosimetrically evaluated) and alkaline elution rate, obtained for several compounds. Possible explanations of the discrepancies.

PubMed

Parodi, S; Balbi, C; Abelmoschi, M L; Pala, M; Russo, P; Santi, L

1983-12-01

Alkaline elution is a well-known method for detecting DNA damage. Recently we have developed a viscosimetric method that is even more sensitive than alkaline elution. Here we report that the two methods, although apparently both revealing alkaline DNA fragmentation, can give dramatically different results for a significant series of compounds. We suspect that alkaline elution might reveal not only DNA fragmentation but also the extent of disentanglement of chromatin structure, whereas this DNA disentanglement rate, when evaluated viscosimetrically , is more strictly correlated with the initiation of DNA unwinding.

SOS response in bacteria: Inhibitory activity of lichen secondary metabolites against Escherichia coli RecA protein.

PubMed

Bellio, Pierangelo; Di Pietro, Letizia; Mancini, Alisia; Piovano, Marisa; Nicoletti, Marcello; Brisdelli, Fabrizia; Tondi, Donatella; Cendron, Laura; Franceschini, Nicola; Amicosante, Gianfranco; Perilli, Mariagrazia; Celenza, Giuseppe

2017-06-15

RecA is a bacterial multifunctional protein essential to genetic recombination, error-prone replicative bypass of DNA damages and regulation of SOS response. The activation of bacterial SOS response is directly related to the development of intrinsic and/or acquired resistance to antimicrobials. Although recent studies directed towards RecA inactivation via ATP binding inhibition described a variety of micromolar affinity ligands, inhibitors of the DNA binding site are still unknown. Twenty-seven secondary metabolites classified as anthraquinones, depsides, depsidones, dibenzofurans, diphenyl-butenolides, paraconic acids, pseudo-depsidones, triterpenes and xanthones, were investigated for their ability to inhibit RecA from Escherichia coli. They were isolated in various Chilean regions from 14 families and 19 genera of lichens. The ATP hydrolytic activity of RecA was quantified detecting the generation of free phosphate in solution. The percentage of inhibition was calculated fixing at 100µM the concentration of the compounds. Deeper investigations were reserved to those compounds showing an inhibition higher than 80%. To clarify the mechanism of inhibition, the semi-log plot of the percentage of inhibition vs. ATP and vs. ssDNA, was evaluated. Only nine compounds showed a percentage of RecA inhibition higher than 80% (divaricatic, perlatolic, alpha-collatolic, lobaric, lichesterinic, protolichesterinic, epiphorellic acids, sphaerophorin and tumidulin). The half-inhibitory concentrations (IC 50 ) calculated for these compounds were ranging from 14.2µM for protolichesterinic acid to 42.6µM for sphaerophorin. Investigations on the mechanism of inhibition showed that all compounds behaved as uncompetitive inhibitors for ATP binding site, with the exception of epiphorellic acid which clearly acted as non-competitive inhibitor of the ATP site. Further investigations demonstrated that epiphorellic acid competitively binds the ssDNA binding site. Kinetic data were confirmed by molecular modelling binding predictions which shows that epiphorellic acid is expected to bind the ssDNA site into the L2 loop of RecA protein. In this paper the first RecA ssDNA binding site ligand is described. Our study sets epiphorellic acid as a promising hit for the development of more effective RecA inhibitors. In our drug discovery approach, natural products in general and lichen in particular, represent a successful source of active ligands and structural diversity. Copyright © 2017 Elsevier GmbH. All rights reserved.

Antioxidant and prooxidant effects of polyphenol compounds on copper-mediated DNA damage.

PubMed

Perron, Nathan R; García, Carla R; Pinzón, Julio R; Chaur, Manuel N; Brumaghim, Julia L

2011-05-01

Inhibition of copper-mediated DNA damage has been determined for several polyphenol compounds. The 50% inhibition concentration values (IC(50)) for most of the tested polyphenols are between 8 and 480 μM for copper-mediated DNA damage prevention. Although most tested polyphenols were antioxidants under these conditions, they generally inhibited Cu(I)-mediated DNA damage less effectively than Fe(II)-mediated damage, and some polyphenols also displayed prooxidant activity. Because semiquinone radicals and hydroxyl radical adducts were detected by EPR spectroscopy in solutions of polyphenols, Cu(I), and H(2)O(2), it is likely that weak polyphenol-Cu(I) interactions permit a redox-cycling mechanism, whereby the necessary reactants to cause DNA damage (Cu(I), H(2)O(2), and reducing agents) are regenerated. The polyphenol compounds that prevent copper-mediated DNA damage likely follow a radical scavenging pathway as determined by EPR spectroscopy. Copyright © 2011 Elsevier Inc. All rights reserved.

Effect of selected strains of Debaryomyces hansenii on the volatile compound production of dry fermented sausage "salchichón".

PubMed

Andrade, M A Jesús; Córdoba, Juan José; Casado, Eva M A; Córdoba, María G; Rodríguez, Mar

2010-06-01

Different biotypes of Debaryomyces hansenii, characterized by mitochondrial DNA (mtDNA) restriction analysis, were inoculated in dry fermented sausages to evaluate their influence as single starter culture on volatile compound generation throughout the ripening process. Similar evolution of physicochemical parameters and microbial population was observed in both uninoculated and inoculated sausages. The tested biotypes modified the volatile compound profile of sausages specially in esters, branched alcohols and aldehydes. The biotype of D. hansenii with the E mtDNA restriction pattern is the most suitable to be used as starter culture since it produced volatile compounds involved in flavour development of dry-cured meat products such as 3-methylbutanol, 3-methylbutanal and 2-propanone. Moreover, the use of D. hansenii strains with the B, C2 and E mtDNA restriction patterns, as a mixed starter culture, should be also considered to generate low amount of sulphur compounds in dry-cured meat products. Copyright 2010 Elsevier Ltd. All rights reserved.

Testing chemical carcinogenicity by using a transcriptomics HepaRG-based model?

PubMed Central

Doktorova, T. Y.; Yildirimman, Reha; Ceelen, Liesbeth; Vilardell, Mireia; Vanhaecke, Tamara; Vinken, Mathieu; Ates, Gamze; Heymans, Anja; Gmuender, Hans; Bort, Roque; Corvi, Raffaella; Phrakonkham, Pascal; Li, Ruoya; Mouchet, Nicolas; Chesne, Christophe; van Delft, Joost; Kleinjans, Jos; Castell, Jose; Herwig, Ralf; Rogiers, Vera

2014-01-01

The EU FP6 project carcinoGENOMICS explored the combination of toxicogenomics and in vitro cell culture models for identifying organotypical genotoxic- and non-genotoxic carcinogen-specific gene signatures. Here the performance of its gene classifier, derived from exposure of metabolically competent human HepaRG cells to prototypical non-carcinogens (10 compounds) and hepatocarcinogens (20 compounds), is reported. Analysis of the data at the gene and the pathway level by using independent biostatistical approaches showed a distinct separation of genotoxic from non-genotoxic hepatocarcinogens and non-carcinogens (up to 88 % correct prediction). The most characteristic pathway responding to genotoxic exposure was DNA damage. Interlaboratory reproducibility was assessed by blindly testing of three compounds, from the set of 30 compounds, by three independent laboratories. Subsequent classification of these compounds resulted in correct prediction of the genotoxicants. As expected, results on the non-genotoxic carcinogens and the non-carcinogens were less predictive. In conclusion, the combination of transcriptomics with the HepaRG in vitro cell model provides a potential weight of evidence approach for the evaluation of the genotoxic potential of chemical substances. PMID:26417288

Identification of hydrolyzable tannins (punicalagin, punicalin and geraniin) as novel inhibitors of hepatitis B virus covalently closed circular DNA

PubMed Central

Liu, Chunlan; Cai, Dawei; Zhang, Lin; Tang, Wei; Yan, Ran

2017-01-01

The development of new agents to target HBV cccDNA is urgently needed because of the limitations of current available drugs for treatment of hepatitis B. By using a cell-based assay in which the production of HBeAg is in a cccDNA-dependent manner, we screened a compound library derived from Chinese herbal remedies for inhibitors against HBV cccDNA. Three hydrolyzable tannins, specifically punicalagin, punicalin and geraniin, emerged as novel anti-HBV agents. These compounds significantly reduced the production of secreted HBeAg and cccDNA in a dose-dependent manner in our assay, without dramatic alteration of viral DNA replication. Furthermore, punicalagin did not affect precore/core promoter activity, pgRNA transcription, core protein expression, or HBsAg secretion. By employing the cell-based cccDNA accumulation and stability assay, we found that these tannins significantly inhibited the establishment of cccDNA and modestly facilitated the degradation of preexisting cccDNA. Collectively, our results suggest that hydrolyzable tannins inhibit HBV cccDNA production via a dual mechanism through preventing the formation of cccDNA and promoting cccDNA decay, although the latter effect is rather minor. These hydrolyzable tannins may serve as lead compounds for the development of new agents to cure HBV infection. PMID:27591143

Analysis of the DNA damage produced by a platinum-acridine antitumor agent and its effects in NCI-H460 lung cancer cells.

PubMed

Qiao, Xin; Zeitany, Alexandra E; Wright, Marcus W; Essader, Amal S; Levine, Keith E; Kucera, Gregory L; Bierbach, Ulrich

2012-07-01

High-performance liquid chromatography in conjunction with electrospray mass spectrometry (LC-ESMS) was used to structurally characterize the adducts formed by the platinum-acridine agent [PtCl(en)(N-(2-(acridin-9-ylamino)ethyl)-N-methylpropionimidamide)](NO(3))(2) (compound 1) in cell-free DNA. Compound 1 forms monofunctional adducts exclusively with guanine, based on the fragments identified in enzymatic digests (dG*, dGMP*, dApG*, and dTpG*, where the asterisk denotes bound drug). The time course of accumulation and DNA adduct formation of compound 1 and the clinical drug cisplatin in NCI-H460 lung cancer cells at physiologically relevant drug concentrations (0.1 μM) was studied by inductively-coupled plasma mass spectrometry (ICP-MS). Compound 1 accumulates rapidly in cells and reaches intracellular levels of up to 60-fold higher than those determined for cisplatin. The hybrid agent shows unusually high DNA binding levels: while cisplatin adducts form at a maximum frequency of 5 adducts per 10(6) nucleotides, compound 1 produces 25 adducts per 10(6) nucleotides after only 3 h of continuous incubation with the lung cancer cells. The high overall levels of compound 1 in the cells and in cellular DNA over the entire 12-h treatment period translate into a rapid decrease in cell viability. Possible implications of these findings for the mechanism of action of compound 1 and the agent's potential to overcome tumor resistance to cisplatin are discussed.

G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV

PubMed Central

Madireddy, Advaitha; Purushothaman, Pravinkumar; Loosbroock, Christopher P.; Robertson, Erle S.; Schildkraut, Carl L.; Verma, Subhash C.

2016-01-01

Kaposi's sarcoma associated herpesvirus (KSHV) establishes life-long latent infection by persisting as an extra-chromosomal episome in the infected cells and by maintaining its genome in dividing cells. KSHV achieves this by tethering its epigenome to the host chromosome by latency associated nuclear antigen (LANA), which binds in the terminal repeat (TR) region of the viral genome. Sequence analysis of the TR, a GC-rich DNA element, identified several potential Quadruplex G-Rich Sequences (QGRS). Since quadruplexes have the tendency to obstruct DNA replication, we used G-quadruplex stabilizing compounds to examine their effect on latent DNA replication and the persistence of viral episomes. Our results showed that these G-quadruplex stabilizing compounds led to the activation of dormant origins of DNA replication, with preferential bi-directional pausing of replications forks moving out of the TR region, implicating the role of the G-rich TR in the perturbation of episomal DNA replication. Over time, treatment with PhenDC3 showed a loss of viral episomes in the infected cells. Overall, these data show that G-quadruplex stabilizing compounds retard the progression of replication forks leading to a reduction in DNA replication and episomal maintenance. These results suggest a potential role for G-quadruplex stabilizers in the treatment of KSHV-associated diseases. PMID:26837574

Differentiation of yeasts growing on dry-cured Iberian ham by mitochondrial DNA restriction analysis, RAPD-PCR and their volatile compounds production.

PubMed

Andrade, M J; Rodríguez, M; Casado, E M; Bermúdez, E; Córdoba, J J

2009-09-01

The efficiency of mitochondrial DNA (mtDNA) restriction analysis, RAPD-PCR and volatile compounds analysis to differentiate yeast biotypes involved in flavour development of dry-cured Iberian ham throughout the ripening process is evaluated. For this purpose, 86 yeasts isolated from Iberian hams in the main ripening stages at different industries of the four Protected Designations of Origin of this product, were used. The combination of mtDNA restriction analysis and RAPD-PCR using the primer (GACA)4 showed a higher variability in the yeast species detected than obtained using only mtDNA restriction analysis. Only two species, Debaryomyces hansenii and Candida zeylanoides, were identified throughout the whole ripening process and a wide diversity of biotypes was found in these two species, with those of D. hansenii predominating. Clear differences between biotypes were detected in the generation of volatile compounds, with the biotype C2-2 of D. hansenii showing the highest concentrations of volatiles. The combined use of mtDNA restriction analysis and RAPD-PCR distinguishes yeast biotypes with different production of volatile compounds. In addition, analysis of the production profile of volatile compounds is needed to differentiate yeast strains of the same biotype recovered at different stages of ripening. Thus, the combination of these three methods could be very useful to select or monitor yeasts as starter cultures in dry-cured meat products.

N7-platinated ribonucleotides are not incorporated by RNA polymerases. New perspectives for a rational design of platinum antitumor drugs.

PubMed

Benedetti, Michele; Romano, Alessandro; De Castro, Federica; Girelli, Chiara R; Antonucci, Daniela; Migoni, Danilo; Verri, Tiziano; Fanizzi, Francesco P

2016-10-01

In this work, we assessed the capacity of RNA polymerases to use platinated ribonucleotides as substrates for RNA synthesis by testing the incorporation of the model compound [Pt(dien)(N7-5'-GTP)] (dien=diethylenetriamine; GTP=5'-guanosine triphosphate) into a natural RNA sequence. The yield of in vitro transcription operated by T7 RNA polymerase, on the LacZ (Escherichia coli gene encoding for β-galactosidase) sequence, decreases progressively with decreasing the concentration of natural GTP, in favor of the platinated nucleotide, [Pt(dien)(N7-5'-GTP)]. Comparison of the T7 RNA polymerase transcription activities for [Pt(dien)(N7-5'-GTP)] compound incorporation reaction test, with respect to the effect of a decreasing concentration of natural GTP, showed no major differences. A specific inhibitory effect of compound [Pt(dien)(N7-5'-GTP)] (which may pair the complementary base on the DNA strand, without being incorporated in the RNA by the T7 RNA polymerase) was evidenced. Our findings therefore suggest that RNA polymerases, unlike DNA polymerases, are unable to incorporate N7-platinated nucleotides into newly synthesized nucleic acids. In this respect, specifically designed N7-platinated nucleotides based compounds could be used in alternative to the classical platinum based drugs. This approach may offer a possible strategy to target specifically DNA, without affecting RNA, and is potentially able to better modulate pharmacological activity. Copyright © 2016 Elsevier Inc. All rights reserved.

Synthesis, DNA/RNA affinity and antitumour activity of new aromatic diamidines linked by 3,4-ethylenedioxythiophene.

PubMed

Stolić, Ivana; Mišković, Katarina; Piantanida, Ivo; Lončar, Mirela Baus; Glavaš-Obrovac, Ljubica; Bajić, Miroslav

2011-02-01

A series of novel 2,5-bis(amidinophenyl)-3,4-ethylenedioxythiophenes (5-10 and 15) has been synthesized. Compounds 5-10 bind to the DNA minor groove as the dominant binding site and strongly stabilize the double helix of ct-DNA. Surprisingly, the same compounds also thermally stabilize ds-RNA, whereby most of them form stacked dimers along the RNA double helix. The only exception is compound 15 which, due to its structural features, showed no interaction with DNA or RNA. Compounds 5-10 have shown a moderate to strong cytotoxic effect (GI50=1.5-9.0 μM) on a panel of seven tumour cell lines. The diimidazoline derivative 9, due to its highest inhibitory potential on the growth of all tested tumour cell lines, was investigated in more detail by testing its ability to enter into cells and influence the cell cycle. Compound 9 (5 μM) was internalized successfully in cell cytoplasm during a 30-min incubation period, followed by nuclear localization upon 90-min incubation. Significant arrest in HeLa cells in the G2/M phase, shown by cell cycle analysis at an equitoxic (50 μM) concentration, suggests interaction of a studied compound with cellular DNA as the main mode of biological action. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Yeast as a model system to study metabolic impact of selenium compounds

PubMed Central

Herrero, Enrique; Wellinger, Ralf E.

2015-01-01

Inorganic Se forms such as selenate or selenite (the two more abundant forms in nature) can be toxic in Saccharomyces cerevisiae cells, which constitute an adequate model to study such toxicity at the molecular level and the functions participating in protection against Se compounds. Those Se forms enter the yeast cell through other oxyanion transporters. Once inside the cell, inorganic Se forms may be converted into selenide through a reductive pathway that in physiological conditions involves reduced glutathione with its consequent oxidation into diglutathione and alteration of the cellular redox buffering capacity. Selenide can subsequently be converted by molecular oxygen into elemental Se, with production of superoxide anions and other reactive oxygen species. Overall, these events result in DNA damage and dose-dependent reversible or irreversible protein oxidation, although additional oxidation of other cellular macromolecules cannot be discarded. Stress-adaptation pathways are essential for efficient Se detoxification, while activation of DNA damage checkpoint and repair pathways protects against Se-mediated genotoxicity. We propose that yeast may be used to improve our knowledge on the impact of Se on metal homeostasis, the identification of Se-targets at the DNA and protein levels, and to gain more insights into the mechanism of Se-mediated apoptosis. PMID:28357286

Design, synthesis and DNA-binding study of some novel morpholine linked thiazolidinone derivatives

NASA Astrophysics Data System (ADS)

War, Javeed Ahmad; Srivastava, Santosh Kumar; Srivastava, Savitri Devi

2017-02-01

The emergence of multiple drug resistance amongst bacterial strains resulted in many clinical drugs to be ineffective. Being vulnerable to bacterial infections any lack in the development of new antimicrobial drugs could pose a serious threat to public health. Here we report design and synthesis of a novel class of morpholine linked thiazolidinone hybrid molecules. The compounds were characterized by FT-IR, NMR and HRMS techniques. Susceptibility tests showed that most of the synthesized molecules were highly active against multiple bacterial strains. Compound 3f displayed MIC values which were better than the standard drug for most of the tested strains. DNA being a well defined target for many antimicrobial drugs was probed as possible target for these synthetic molecules. DNA-binding study of 3f with sm-DNA was probed through UV-vis absorption, fluorescence quenching, gel electrophoresis and molecular docking techniques. The studies revealed that compound 3f has strong affinity towards DNA and binds at the minor groove. The docking studies revealed that the compound 3f shows preferential binding towards A/T residues.

Design, synthesis and DNA-binding study of some novel morpholine linked thiazolidinone derivatives.

PubMed

War, Javeed Ahmad; Srivastava, Santosh Kumar; Srivastava, Savitri Devi

2017-02-15

The emergence of multiple drug resistance amongst bacterial strains resulted in many clinical drugs to be ineffective. Being vulnerable to bacterial infections any lack in the development of new antimicrobial drugs could pose a serious threat to public health. Here we report design and synthesis of a novel class of morpholine linked thiazolidinone hybrid molecules. The compounds were characterized by FT-IR, NMR and HRMS techniques. Susceptibility tests showed that most of the synthesized molecules were highly active against multiple bacterial strains. Compound 3f displayed MIC values which were better than the standard drug for most of the tested strains. DNA being a well defined target for many antimicrobial drugs was probed as possible target for these synthetic molecules. DNA-binding study of 3f with sm-DNA was probed through UV-vis absorption, fluorescence quenching, gel electrophoresis and molecular docking techniques. The studies revealed that compound 3f has strong affinity towards DNA and binds at the minor groove. The docking studies revealed that the compound 3f shows preferential binding towards A/T residues. Copyright © 2016 Elsevier B.V. All rights reserved.

Equilibrium location for spherical DNA and toroidal cyclodextrin

NASA Astrophysics Data System (ADS)

Sarapat, Pakhapoom; Baowan, Duangkamon; Hill, James M.

2018-05-01

Cyclodextrin comprises a ring structure composed of glucose molecules with an ability to form complexes of certain substances within its central cavity. The compound can be utilised for various applications including food, textiles, cosmetics, pharmaceutics, and gene delivery. In gene transfer, the possibility of forming complexes depends upon the interaction energy between cyclodextrin and DNA molecules which here are modelled as a torus and a sphere, respectively. Our proposed model is derived using the continuum approximation together with the Lennard-Jones potential, and the total interaction energy is obtained by integrating over both the spherical and toroidal surfaces. The results suggest that the DNA prefers to be symmetrically situated about 1.2 Å above the centre of the cyclodextrin to minimise its energy. Furthermore, an optimal configuration can be determined for any given size of torus and sphere.

Design, Synthesis, and Evaluation of Novel Tyrosine-Based DNA Gyrase B Inhibitors.

PubMed

Cotman, Andrej E; Trampuž, Marko; Brvar, Matjaž; Kikelj, Danijel; Ilaš, Janez; Peterlin-Mašič, Lucija; Montalvão, Sofia; Tammela, Päivi; Frlan, Rok

2017-08-01

The discovery and synthesis of new tyrosine-based inhibitors of DNA gyrase B (GyrB), which target its ATPase subunit, is reported. Twenty-four compounds were synthesized and evaluated for activity against DNA gyrase and DNA topoisomerase IV. The antibacterial properties of selected GyrB inhibitors were demonstrated by their activity against Staphylococcus aureus and Enterococcus faecalis in the low micromolar range. The most promising compounds, 8a and 13e, inhibited Escherichia coli and S. aureus GyrB with IC 50 values of 40 and 30 µM. The same compound also inhibited the growth of S. aureus and E. faecalis with minimal inhibitory concentrations (MIC 90 ) of 14 and 28 µg/mL, respectively. © 2017 Deutsche Pharmazeutische Gesellschaft.

Identification of hydrolyzable tannins (punicalagin, punicalin and geraniin) as novel inhibitors of hepatitis B virus covalently closed circular DNA.

PubMed

Liu, Chunlan; Cai, Dawei; Zhang, Lin; Tang, Wei; Yan, Ran; Guo, Haitao; Chen, Xulin

2016-10-01

The development of new agents to target HBV cccDNA is urgently needed because of the limitations of current available drugs for treatment of hepatitis B. By using a cell-based assay in which the production of HBeAg is in a cccDNA-dependent manner, we screened a compound library derived from Chinese herbal remedies for inhibitors against HBV cccDNA. Three hydrolyzable tannins, specifically punicalagin, punicalin and geraniin, emerged as novel anti-HBV agents. These compounds significantly reduced the production of secreted HBeAg and cccDNA in a dose-dependent manner in our assay, without dramatic alteration of viral DNA replication. Furthermore, punicalagin did not affect precore/core promoter activity, pgRNA transcription, core protein expression, or HBsAg secretion. By employing the cell-based cccDNA accumulation and stability assay, we found that these tannins significantly inhibited the establishment of cccDNA and modestly facilitated the degradation of preexisting cccDNA. Collectively, our results suggest that hydrolyzable tannins inhibit HBV cccDNA production via a dual mechanism through preventing the formation of cccDNA and promoting cccDNA decay, although the latter effect is rather minor. These hydrolyzable tannins may serve as lead compounds for the development of new agents to cure HBV infection. Copyright © 2016 Elsevier B.V. All rights reserved.

A new compound, withangulatin A, promotes type II DNA topoisomerase-mediated DNA damage.

PubMed

Juang, J K; Huang, H W; Chen, C M; Liu, H J

1989-03-31

Withangulatin A, a new compound with a known chemical structure and from the antitumor Chinese herb Physalis angulata L, was found to act on topoisomerase II to induce topoisomerase II-mediated DNA damage in vitro. It has two effective dosage ranges of approximate 0.5 and 20 microM, with about one-third the activity of 20 microM VM-26.

Structure-activity studies of dicationically substituted bis-benzimidazoles against Giardia lamblia: correlation of antigiardial activity with DNA binding affinity and giardial topoisomerase II inhibition.

PubMed Central

Bell, C A; Dykstra, C C; Naiman, N A; Cory, M; Fairley, T A; Tidwell, R R

1993-01-01

Nine dicationically substituted bis-benzimidazoles were examined for their in vitro activities against Giardia lamblia WB (ATCC 30957). The potential mechanisms of action of these compounds were evaluated by investigating the relationship among in vitro antigiardial activity and the affinity of the molecules for DNA and their ability to inhibit the activity of giardial topoisomerase II. Each compound demonstrated antigiardial activity, as measured by assessing the incorporation of [methyl-3H]thymidine by giardial trophozoites exposed to the test agents. Three compounds exhibited excellent in vitro antigiardial activities, with 50% inhibitory concentrations which compared very favorably with those of two currently used drugs, quinacrine HCl and metronidazole. Putative mechanisms of action for these compounds were suggested by the strong correlation observed among in vitro antigiardial activity and the affinity of the molecules for natural and synthetic DNA and their ability to inhibit the relaxation activity of giardial topoisomerase II. A strong correlation between the DNA binding affinity of these compounds and their inhibition of giardial topoisomerase II activity was also observed. Images PMID:8109934

Neutral Porphyrin Derivative Exerts Anticancer Activity by Targeting Cellular Topoisomerase I (Top1) and Promotes Apoptotic Cell Death without Stabilizing Top1-DNA Cleavage Complexes

PubMed Central

2017-01-01

Camptothecin (CPT) selectively traps topoisomerase 1-DNA cleavable complexes (Top1cc) to promote anticancer activity. Here, we report the design and synthesis of a new class of neutral porphyrin derivative 5,10-bis(4-carboxyphenyl)-15, 20-bis(4-dimethylaminophenyl)porphyrin (compound 8) as a potent catalytic inhibitor of human Top1. In contrast to CPT, compound 8 reversibly binds with the free enzyme and inhibits the formation of Top1cc and promotes reversal of the preformed Top1cc with CPT. Compound 8 induced inhibition of Top1cc formation in live cells was substantiated by fluorescence recovery after photobleaching (FRAP) assays. We established that MCF7 cells treated with compound 8 trigger proteasome-mediated Top1 degradation, accumulate higher levels of reactive oxygen species (ROS), PARP1 cleavage, oxidative DNA fragmentation, and stimulate apoptotic cell death without stabilizing apoptotic Top1-DNA cleavage complexes. Finally, compound 8 shows anticancer activity by targeting cellular Top1 and preventing the enzyme from directly participating in the apoptotic process. PMID:29290109

Inhibition of radical-induced DNA strand breaks by water-soluble constituents of coffee: phenolics and caffeine metabolites.

PubMed

Rathod, M A; Patel, D; Das, A; Tipparaju, S R; Shinde, S S; Anderson, R F

2013-07-01

Epidemiological studies have associated coffee consumption with an inverse risk of developing Parkinson's disease, hepatocellular carcinoma and cirrhosis. The molecular mechanisms by which low concentrations of the constituents of coffee measured in human plasma can reduce the incidence of such diseases are not clear. Using an in vitro plasmid DNA system and radiolytically generated reactive oxygen species under constant radical scavenging conditions, we have shown that coffee chlorogenic acid, its derivatives and certain metabolites of caffeine reduce some of the free radical damage sustained to the DNA. A reduction in the amount of prompt DNA single-strand breaks (SSBs) was observed for all compounds whose radical one-electron reduction potential is < 1.0 V. However, except for chlorogenic acid, the compounds were found to be inactive in reducing the amount of radical damage to the DNA bases. These results support a limited antioxidant role for such compounds in their interaction with DNA radicals.

Lymphocyte DNA damage in Turkish asphalt workers detected by the comet assay.

PubMed

Bacaksiz, Aysegul; Kayaalti, Zeliha; Soylemez, Esma; Tutkun, Engin; Soylemezoglu, Tulin

2014-01-01

Asphalt has a highly complex structure and it contains several organic compounds including polycyclic aromatic hydrocarbons and heterocyclic compounds. In this study, comet assay was used to detect the DNA damage in blood lymphocytes of 30 workers exposed to asphalt fumes and 30 nonexposed controls. This is the first report on Turkish asphalt workers' investigated DNA damage using the alkaline single cell gel electrophoresis (SCGE). The DNA damage was evaluated by the percentage of DNA in the comet tail (% tail DNA) for each cell. According to our results, workers exposed to asphalt fumes had higher DNA damage than the control group (p < 0.01). The present study showed that asphalt fumes caused a significant increase in DNA damage and the comet assay is a suitable method for determining DNA damage in asphalt workers.

Competition among Li+, Na+, K+ and Rb+ Monovalent Ions for DNA in Molecular Dynamics Simulations using the Additive CHARMM36 and Drude Polarizable Force Fields

PubMed Central

Savelyev, Alexey; MacKerell, Alexander D.

2015-01-01

In the present study we report on interactions of and competition between monovalent ions for two DNA sequences in MD simulations. Efforts included the development and validation of parameters for interactions among the first-group monovalent cations, Li+, Na+, K+ and Rb+, and DNA in the Drude polarizable and additive CHARMM36 force fields (FF). The optimization process targeted gas-phase QM interaction energies of various model compounds with ions and osmotic pressures of bulk electrolyte solutions of chemically relevant ions. The optimized ionic parameters are validated against counterion condensation theory and buffer exchange-atomic emission spectroscopy measurements providing quantitative data on the competitive association of different monovalent ions with DNA. Comparison between experimental and MD simulation results demonstrates that, compared to the additive CHARMM36 model, the Drude FF provides an improved description of the general features of the ionic atmosphere around DNA and leads to closer agreement with experiment on the ionic competition within the ion atmosphere. Results indicate the importance of extended simulation systems on the order of 25 Å beyond the DNA surface to obtain proper convergence of ion distributions. PMID:25751286

G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV.

PubMed

Madireddy, Advaitha; Purushothaman, Pravinkumar; Loosbroock, Christopher P; Robertson, Erle S; Schildkraut, Carl L; Verma, Subhash C

2016-05-05

Kaposi's sarcoma associated herpesvirus (KSHV) establishes life-long latent infection by persisting as an extra-chromosomal episome in the infected cells and by maintaining its genome in dividing cells. KSHV achieves this by tethering its epigenome to the host chromosome by latency associated nuclear antigen (LANA), which binds in the terminal repeat (TR) region of the viral genome. Sequence analysis of the TR, a GC-rich DNA element, identified several potential Quadruplex G-Rich Sequences (QGRS). Since quadruplexes have the tendency to obstruct DNA replication, we used G-quadruplex stabilizing compounds to examine their effect on latent DNA replication and the persistence of viral episomes. Our results showed that these G-quadruplex stabilizing compounds led to the activation of dormant origins of DNA replication, with preferential bi-directional pausing of replications forks moving out of the TR region, implicating the role of the G-rich TR in the perturbation of episomal DNA replication. Over time, treatment with PhenDC3 showed a loss of viral episomes in the infected cells. Overall, these data show that G-quadruplex stabilizing compounds retard the progression of replication forks leading to a reduction in DNA replication and episomal maintenance. These results suggest a potential role for G-quadruplex stabilizers in the treatment of KSHV-associated diseases. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Experimental and DFT studies on DNA binding and photocleavage of two cationic porphyrins. Effects of the introduction of a carboxyphenyl into pyridinium porphyrin.

PubMed

Zhao, Ping; Xu, Lian-Cai; Huang, Jin-Wang; Liu, Jie; Yu, Han-Cheng; Zheng, Kang-Cheng; Ji, Liang-Nian

2008-12-15

The DNA-binding affinities and DNA photocleavage abilities of cationic porphyrin, 5-(4-carboxyphenyl)-10,15,20-tris(4-methylpyridiniumyl)porphyrin (CTMPyP), and its reference compound meso-tetrakis(N-methyl-4-pyridiniumyl)porphyrin (H2TMPyP) have been investigated. The DNA-binding behaviors of the two compounds in NaH2PO4 buffer were compared systematically by using absorption, fluorescence and circular dichroism (CD) spectra, thermal denaturation as well as viscosity measurements. The experimental results show that CTMPyP binds to DNA in an outside binding mode, while H2TMPyP in an intercalative mode. Photocleavage experiments reveal that both two compounds employ 1O2-mediated mechanism in cleaving DNA and H2TMPyP can cleave DNA more efficiently than CTMPyP. Theoretical calculations were carried out with the density functional theory (DFT), and the calculated results indicate that the character and energies of some frontier orbitals of CTMPyP are quite different from those of H2TMPyP. These theoretical results can be used to explain their different DNA-binding modes and affinities to a certain extent.

Spectroscopic, molecular docking and structural activity studies of (E)-N‧-(substituted benzylidene/methylene) isonicotinohydrazide derivatives for DNA binding and their biological screening

NASA Astrophysics Data System (ADS)

Arshad, Nasima; Perveen, Fouzia; Saeed, Aamer; Channar, Pervaiz Ali; Farooqi, Shahid Iqbal; Larik, Fayaz Ali; Ismail, Hammad; Mirza, Bushra

2017-07-01

Acid catalyzed condensation of isoniazid with a number of suitably substituted aromatic and heterocyclic aldehydes was carried out in dry ethanol to afford the title (E)-N‧-(substituted benzylidene/methylene) isonicotinohydrazides (SF 1 - SF 4) in good yields. These compounds were characterized and further investigated for their binding with ds.DNA using UV- spectroscopy and molecular docking and for antitumor and antimicrobial potentials. A good correlation was found among spectroscopic, theoretical and biological results. UV- spectra in the presence of DNA concentrations and their data interpretation in terms binding constant "Kb" and free energy change (ΔG) provided evidences for the significant and spontaneous binding of the compounds with DNA. Molecular docking studies and structural analysis further supported the UV-findings and indicated that the modes of interactions between bromo- (SF 1) and flouro- (SF 4) substituted isonicotinohydrazides is intercalation while methoxy- (SF 2) and hydroxy- (SF 3) substituted isonicotinohydrazides interact with DNA helix via groove binding. SF 1 exhibited comparatively higher Kb value (UV-; 8.07 × 103 M-1, docking; 8.11 × 103 M-1) which inferred that the respective compound muddles to DNA most powerfully. SF 1 has shown the lowest IC50 (345.3 μg/mL) value among all the compounds indicating its comparatively highest activity towards tumor inhibition. None of the compound has shown perceptible antibacterial and antifungal activities.

Ketone-DNA: a versatile postsynthetic DNA decoration platform.

PubMed

Dey, S; Sheppard, T L

2001-12-13

[reaction: see text] A general strategy for the functional diversification of DNA oligonucleotides under physiological conditions was developed. We describe the synthesis of DNA molecules bearing ketone ports (ketone-DNA) and the efficient postsynthetic decoration of ketone-DNA with structurally diverse aminooxy compounds.

Development of propidium iodide as a fluorescence probe for the on-line screening of non-specific DNA-intercalators in Fufang Banbianlian Injection.

PubMed

Niu, Yanyan; Li, Sensen; Lin, Zongtao; Liu, Meixian; Wang, Daidong; Wang, Hong; Chen, Shizhong

2016-09-09

Fufang Banbianlian Injection (FBI) has been widely used as an anti-inflammatory and anti-tumor prescription. To understand the relationships between its bioactive ingredients and pharmacological efficacies, our previous study has been successfully identified some DNA-binding compounds in FBI using an established on-line screening system, in which 4',6-diamidino-2-phenylindole (DAPI) was developed as a probe. However, DAPI can be only used to screen ATT-specific DNA minor groove binders, leaving the potential active intercalators unknown in FBI. As a continuation of our studies on FBI, here we present a sensitive analytical method for rapid identification and evaluation of DNA-intercalators using propidium iodide (PI) as a fluorescent probe. We have firstly established the technique of high-performance liquid chromatography-diode-array detector-multistage mass spectrometry-deoxyribonucleic acid-propidium iodide-fluorescence detector (HPLC-DAD-MS(n)-DNA-PI-FLD) system. As a result, 38 of 58 previously identified compounds in FBI were DNA-intercalation active. Interestingly, all previously reported DNA-binders also showed intercalative activities, suggesting they are dual-mode DNA-binders. Quantitative study showed that flavonoid glycosides and chlorogenic acids were the main active compounds in FBI, and displayed similar DNA-binding ability using either DAPI or PI. In addition, 13 active compounds were used to establish the structure-activity relationships. In this study, PI was developed into an on-line method for identifying DNA-intercalators for the first time, and thus it will be a useful high-throughput screening technique for other related samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Toxicity prediction of PHDDs and phenols in the light of nucleic acid bases and DNA base pair interaction.

PubMed

Mondal Roy, Sutapa; Roy, Debesh R; Sahoo, Suban K

2015-11-01

The applicability of Density Functional Theory (DFT) based descriptors for the development of quantitative structure-toxicity relationships (QSTR) is assessed for two different series of toxic aromatic compounds, viz., polyhalogenated dibenzo-p-dioxins (PHDDs) and phenols (PHs). A series of 20 compounds each for PHDDs and PHs with their experimental toxicities (IC50 and IGC50) is chosen in the present study to develop DFT based efficient quantum chemical parameters (QCPs) for explaining the toxin potential of the considered compounds. A systematic analysis to find out the electron donation/acceptance nature of these selected compounds with the considered model biosystems, viz., nucleic acid (NA) bases and DNA base pairs, is performed to identify potential QCPs. Accordingly, PHDDs is found to be electron acceptors whereas phenols as donors, during their interaction with biosystems. Two parameter regression model is carried out comprising global charge transfer (ΔN), and local Fukui Function's for nucleophilic attack (fk(+)) for PHDDs and the same for electrophilic attack (fk(-)) in case of PHs. It is heartening to note that our chosen descriptors, viz, charge transfer (ΔN) and Fukui Function (fk(±)) plays a crucial role by explaining more than 90% of the observed toxic behavior (in terms of correlation-coefficient, R) of PHDDs and PHs. The developed QCPs, viz., ΔN and fk(±) can be added as the new descriptors in the QSTR parlance. Copyright © 2015 Elsevier Inc. All rights reserved.

Camptosorus sibiricus rupr aqueous extract prevents lung tumorigenesis via dual effects against ROS and DNA damage.

PubMed

He, Shugui; Ou, Rilan; Wang, Wensheng; Ji, Liyan; Gao, Hui; Zhu, Yuanfeng; Liu, Xiaomin; Zheng, Hongming; Liu, Zhongqiu; Wu, Peng; Lu, Linlin

2018-06-28

Camptosorus sibiricus Rupr (CSR) is a widely used herbal medicine with antivasculitis, antitrauma, and antitumor effects. However, the effect of CSR aqueous extract on B[a]P-initiated tumorigenesis and the underlying mechanism remain unclear. Moreover, the compounds in CSR aqueous extract need to be identified and structurally characterized. We aim to investigate the chemopreventive effect of CSR and the underlying molecular mechanism. A B[a]P-stimulated normal cell model (BEAS.2B) and lung adenocarcinoma animal model were established on A/J mice. In B[a]P-treated BEAS.2B cells, the protective effects of CSR aqueous extract on B[a]P-induced DNA damage and ROS production were evaluated through flow cytometry, Western blot, real-time quantitative PCR, single-cell gel electrophoresis, and immunofluorescence. Moreover, a model of B[a]P-initiated lung adenocarcinoma was established on A/J mice to determine the chemopreventive effect of CSR in vivo. The underlying mechanism was analyzed via immunohistochemistry and microscopy. Furthermore, the new compounds in CSR aqueous extract were isolated and structurally characterized using IR, HR-ESI-MS, and 1D and 2D NMR spectroscopy. CSR effectively suppressed ROS production by re-activating Nrf2-mediated reductases HO-1 and NQO-1. Simultaneously, CSR attenuated the DNA damage of BEAS.2B cells in the presence of B[a]P. Moreover, CSR at 1.5 and 3 g/kg significantly suppressed tumorigenesis with tumor inhibition ratios of 36.65% and 65.80%, respectively. The tumor volume, tumor size, and multiplicity of B[a]P-induced lung adenocarcinoma were effectively decreased by CSR in vivo. After extracting and identifying the compounds in CSR aqueous extract, three new triterpene saponins were isolated and characterized structurally. CSR aqueous extract prevents lung tumorigenesis by exerting dual effects against ROS and DNA damage, suggesting that CSR is a novel and effective agent for B[a]P-induced carcinogenesis. Moreover, by isolating and structurally characterizing three new triterpene saponins, our study further standardized the quality of CSR aqueous extract, which could widen CSR clinical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Synthesis of isatin thiosemicarbazones derivatives: in vitro anti-cancer, DNA binding and cleavage activities.

PubMed

Ali, Amna Qasem; Teoh, Siang Guan; Salhin, Abdussalam; Eltayeb, Naser Eltaher; Khadeer Ahamed, Mohamed B; Abdul Majid, A M S

2014-05-05

New derivatives of thiosemicarbazone Schiff base with isatin moiety were synthesized L1-L6. The structures of these compounds were characterized based on the spectroscopic techniques. Compound L6 was further characterized by XRD single crystal. The interaction of these compounds with calf thymus (CT-DNA) exhibited high intrinsic binding constant (k(b)=5.03-33.00×10(5) M(-1)) for L1-L3 and L5 and (6.14-9.47×10(4) M(-1)) for L4 and L6 which reflect intercalative activity of these compounds toward CT-DNA. This result was also confirmed by the viscosity data. The electrophoresis studies reveal the higher cleavage activity of L1-L3 than L4-L6. The in vitro anti-proliferative activity of these compounds against human colon cancer cell line (HCT 116) revealed that the synthesized compounds (L3, L6 and L2) exhibited good anticancer potency. Copyright © 2014 Elsevier B.V. All rights reserved.

Sequence-specific DNA binding Pyrrole-imidazole polyamides and their applications.

PubMed

Kawamoto, Yusuke; Bando, Toshikazu; Sugiyama, Hiroshi

2018-05-01

Pyrrole-imidazole polyamides (Py-Im polyamides) are cell-permeable compounds that bind to the minor groove of double-stranded DNA in a sequence-specific manner without causing denaturation of the DNA. These compounds can be used to control gene expression and to stain specific sequences in cells. Here, we review the history, structural variations, and functional investigations of Py-Im polyamides. Copyright © 2018 Elsevier Ltd. All rights reserved.

Computational fishing of new DNA methyltransferase inhibitors from natural products.

PubMed

Maldonado-Rojas, Wilson; Olivero-Verbel, Jesus; Marrero-Ponce, Yovani

2015-07-01

DNA methyltransferase inhibitors (DNMTis) have become an alternative for cancer therapies. However, only two DNMTis have been approved as anticancer drugs, although with some restrictions. Natural products (NPs) are a promising source of drugs. In order to find NPs with novel chemotypes as DNMTis, 47 compounds with known activity against these enzymes were used to build a LDA-based QSAR model for active/inactive molecules (93% accuracy) based on molecular descriptors. This classifier was employed to identify potential DNMTis on 800 NPs from NatProd Collection. 447 selected compounds were docked on two human DNA methyltransferase (DNMT) structures (PDB codes: 3SWR and 2QRV) using AutoDock Vina and Surflex-Dock, prioritizing according to their score values, contact patterns at 4 Å and molecular diversity. Six consensus NPs were identified as virtual hits against DNMTs, including 9,10-dihydro-12-hydroxygambogic, phloridzin, 2',4'-dihydroxychalcone 4'-glucoside, daunorubicin, pyrromycin and centaurein. This method is an innovative computational strategy for identifying DNMTis, useful in the identification of potent and selective anticancer drugs. Copyright © 2015 Elsevier Inc. All rights reserved.

Design, synthesis and biological evaluation of 5-fluorouracil-derived benzimidazoles as novel type of potential antimicrobial agents.

PubMed

Fang, Xue-Jie; Jeyakkumar, Ponmani; Avula, Srinivasa Rao; Zhou, Qian; Zhou, Cheng-He

2016-06-01

A series of 5-fluorouracil benzimidazoles as novel type of potential antimicrobial agents were designed and synthesized for the first time. Bioactive assay manifested that some of the prepared compounds exhibited good or even stronger antibacterial and antifungal activities against the tested strains in comparison with reference drugs norfloxacin, chloromycin and fluconazole. Noticeably, 3-fluorobenzyl benzimidazole derivative 5c gave remarkable antimicrobial activities against Saccharomyces cerevisiae, MRSA and Bacillus proteus with MIC values of 1, 2 and 4μg/mL, respectively. Experimental research revealed that compound 5c could effectively intercalate into calf thymus DNA to form compound 5c-DNA complex which might block DNA replication and thus exert antimicrobial activities. Molecular docking indicated that compound 5c should bind with DNA topoisomerase IA through three hydrogen bonds by the use of fluorine atom and oxygen atoms in 5-fluorouracil with the residue Lys 423. Copyright © 2016 Elsevier Ltd. All rights reserved.

Synthesis, DNA binding ability and anticancer activity of 2-heteroaryl substituted benzimidazoles linked pyrrolo[2,1-c][1,4]benzodiazepine conjugates.

PubMed

Kamal, Ahmed; Pogula, Praveen Kumar; Khan, Mohammed Naseer Ahmed; Seshadri, Bobburi Naga; Sreekanth, Kokkonda

2013-08-01

As a continuation of our efforts to develop the benzimidazole-PBD conjugates as potential anticancer agents, a series of heteroaryl substituted benzimidazole linked PBD conjugates has been synthesized and evaluated for their anticancer potential in 60 human cancer cell lines. Most of the compounds exhibited promising anticancer activity and interestingly, compounds 4c and 4d displayed significant activity in most of the cell lines tested. Whereas, compound 4e showed selectivity in renal cancer cells with GI50 values of <10 and 70 nM against RXF 393 and UO-31 cell lines, respectively. Further, these compounds also showed significant DNA-binding affinity by thermal denaturation study using duplex form of calf thymus (CT) DNA.

Synthesis, spectroscopic characterization and structural investigations of new adduct compound of carbazole with picric acid: DNA binding and antimicrobial studies

NASA Astrophysics Data System (ADS)

Saravanabhavan, Munusamy; Sathya, Krishnan; Puranik, Vedavati G.; Sekar, Marimuthu

2014-01-01

Carbazole picrate (CP), a new organic compound has been synthesized, characterized by various analytical and spectroscopic technique such as FT-IR, UV-Vis, 1H and 13C NMR spectroscopy. An orthorhombic geometry was proposed based on single crystal XRD study. The thermal stability of the crystal was studied by using thermo-gravimetric and differential thermal analyses and found that it was stable up to 170 °C. Further, the newly synthesized title compound was tested for its in vitro antibacterial and antifungal activity against various bacterial and fungal species. Also, the compound was tested for its binding activity with Calf thymus (CT) DNA and the results show a considerable interaction between CP and CT-DNA.

Nitrite curing of chicken, pork, and beef inhibits oxidation but does not affect N-nitroso compound (NOC)-specific DNA adduct formation during in vitro digestion.

PubMed

Van Hecke, Thomas; Vanden Bussche, Julie; Vanhaecke, Lynn; Vossen, Els; Van Camp, John; De Smet, Stefaan

2014-02-26

Uncured and nitrite-cured chicken, pork, and beef were used as low, medium, and high sources of heme-Fe, respectively, and exposed to an in vitro digestion model simulating the mouth, stomach, duodenum, and colon. With increasing content of iron compounds, up to 25-fold higher concentrations of the toxic lipid oxidation products malondialdehyde, 4-hydroxy-2-nonenal, and other volatile aldehydes were formed during digestion, together with increased protein carbonyl compounds as measurement of protein oxidation. Nitrite curing of all meats lowered lipid and protein oxidation to the level of oxidation in uncured chicken. Strongly depending on the individual fecal inoculum, colonic digestion of beef resulted in significantly higher concentrations of the NOC-specific DNA adduct O(6)-carboxymethyl-guanine compared to chicken and pork, whereas nitrite curing had no significant effect. This study confirms previously reported evidence that heme-Fe is involved in the epidemiological association between red meat consumption and colorectal cancer, but questions the role of nitrite curing in this association.

Chemical Biology Probes from Advanced DNA-encoded Libraries.

PubMed

Salamon, Hazem; Klika Škopić, Mateja; Jung, Kathrin; Bugain, Olivia; Brunschweiger, Andreas

2016-02-19

The identification of bioactive compounds is a crucial step toward development of probes for chemical biology studies. Screening of DNA-encoded small molecule libraries (DELs) has emerged as a validated technology to interrogate vast chemical space. DELs consist of chimeric molecules composed of a low-molecular weight compound that is conjugated to a DNA identifier tag. They are screened as pooled libraries using selection to identify "hits." Screening of DELs has identified numerous bioactive compounds. Some of these molecules were instrumental in gaining a deeper understanding of biological systems. One of the main challenges in the field is the development of synthesis methodology for DELs.

Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

PubMed Central

de Almeida, Sinara Mônica Vitalino; Lafayette, Elizabeth Almeida; Gomes da Silva, Lúcia Patrícia Bezerra; Amorim, Cézar Augusto da Cruz; de Oliveira, Tiago Bento; Gois Ruiz, Ana Lucia Tasca; de Carvalho, João Ernesto; de Moura, Ricardo Olímpio; Beltrão, Eduardo Isidoro Carneiro; de Lima, Maria do Carmo Alves; de Carvalho Júnior, Luiz Bezerra

2015-01-01

In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a–h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 104 to 1.0 × 106 M−1 and quenching constants from −0.2 × 104 to 2.18 × 104 M−1 indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N-(4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties. PMID:26068233

Application of an in vitro DNA protection assay to visualize stress mediation properties of the Dps protein.

PubMed

Karas, Vlad O; Westerlaken, Ilja; Meyer, Anne S

2013-05-31

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage. The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

Hepatoprotective effect of fermented ginseng and its major constituent compound K in a rat model of paracetamol (acetaminophen)-induced liver injury.

PubMed

Igami, Kentaro; Shimojo, Yosuke; Ito, Hisatomi; Miyazaki, Toshitsugu; Kashiwada, Yoshiki

2015-04-01

This work aimed at evaluating the effect of fermented ginseng (FG) and fermented red ginseng (FRG) against rat liver injury caused by paracetamol (acetaminophen (APAP)). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum and histopathological changes in the liver were analysed to determine the degree of liver injury. Deoxyribonucleic acid (DNA) microarray analysis was performed to compare gene expression levels altered in the rat livers. Phosphorylated Jun-N-terminal kinase (JNK) in human hepatocellular carcinoma (HepG2) cells were detected using western blot analysis to investigate the anti-inflammatory activity of compound K. Pretreatment with FG, containing compound K at high concentration, attenuated AST as well as ALT levels in rats, while no obvious effect was observed in the group that received FRG, whose content of compound K was lower than that of FG. In addition, the results of our histopathological analysis were consistent with changes in the serum biochemical analysis. DNA microarray analysis indicated that JNK- and glutathione S-transferase (GST)-related genes were involved in the hepatotoxicity. Notably, compound K, a major ginsenoside in FG, inhibited the phosphorylation of JNK in HepG2 cells. FG was shown to possess hepatoprotective activity against paracetamol (APAP)-induced liver injury better than FRG. Compound K might play an important role for an anti-inflammatory activity of FG by inhibiting JNK signalling in the liver. © 2014 Royal Pharmaceutical Society.

A selective chemosensor for fluoride ion and its interaction with Calf Thymus DNA

NASA Astrophysics Data System (ADS)

Ghosh, Soumen; Al Masum, Abdulla; Ganguly, Aniruddha; Islam, Md. Maidul; Alam, Md. Akhtarul; Guchhait, Nikhil

2017-05-01

The amido-Schiff base 1 (N1, N3-bis (2-nitrobenzylidene)benzene-1,3-dicabohydrazide) containing a sbnd CONHsbnd group and sbnd CHdbnd Nsbnd linkage has been synthesized by the condensation between isophthalic acid dihydrazide and o-nitrobenzaldehyde. This molecule can act as a fluoride ion sensor with high selectivity and sensitivity. Presence of nitro group in the phenyl ring may be responsible for the detection of fluoride ion visually with a dramatic color change from colorless to deep red in aqueous dimethyl sulphoxide solution. This Schiff base can be used as test kit for sensing of fluoride ion in the solid state. Compound 1 can detect fluoride also in commercially available toothpaste. As the compound has adequate solubility in DMSO-water mixture (7:93, v/v) and having some hydrogen bond donor and acceptor centers, we have investigated its nature of binding with Calf Thymus-DNA (CT-DNA) using theoretical molecular modelling and other experimental methods like UV-vis spectroscopy, circular dichroic and thermal melting studies. Thermodynamic parameters have been obtained using the well known Van't Hoff's equation. From both theoretical and experimental findings it has been observed that it can interact effectively with CT-DNA with binding energy - 7.55 kcal/mol to - 7.50 kcal/mol.

Effect of seven Indian plant extracts on Fenton reaction-mediated damage to DNA constituents.

PubMed

Kar, Indrani; Chattopadhyaya, Rajagopal

2017-11-01

The influences of substoichiometric amounts of seven plant extracts in the Fenton reaction-mediated damage to deoxynucleosides, deoxynucleoside monophosphates, deoxynucleoside triphosphates, and supercoiled plasmid DNA were studied to rationalize anticancer properties reported in some of these extracts. Extracts from Acacia catechu, Emblica officinalis, Spondias dulcis, Terminalia belerica, Terminalia chebula, as well as gallic acid, epicatechin, chebulagic acid and chebulinic acid enhance the extent of damage in Fenton reactions with all monomeric substrates but protect supercoiled plasmid DNA, compared to standard Fenton reactions. The damage to pyrimidine nucleosides/nucleotides is enhanced by these extracts and compounds to a greater extent than for purine ones in a concentration dependent manner. Dolichos biflorus and Hemidesmus indicus extracts generally do not show this enhancement for the monomeric substrates though they protect plasmid DNA. Compared to standard Fenton reactions for deoxynucleosides with ethanol, the presence of these five plant extracts render ethanol scavenging less effective as the radical is generated in the vicinity of the target. Since substoichiometric amounts of these extracts and the four compounds produce this effect, a catalytic mechanism involving the presence of a ternary complex of the nucleoside/nucleotide substrate, a plant compound and the hydroxyl radical is proposed. Such a mechanism cannot operate for plasmid DNA as the planar rings in the extract compounds cannot stack with the duplex DNA bases. These plant extracts, by enhancing Fenton reaction-mediated damage to deoxynucleoside triphosphates, slow down DNA replication in rapidly dividing cancer cells, thus contributing to their anticancer properties.

Common Chemical Inductors of Replication Stress:  Focus on Cell-Based Studies.

PubMed

Vesela, Eva; Chroma, Katarina; Turi, Zsofia; Mistrik, Martin

2017-02-21

DNA replication is a highly demanding process regarding the energy and material supply and must be precisely regulated, involving multiple cellular feedbacks. The slowing down or stalling of DNA synthesis and/or replication forks is referred to as replication stress (RS). Owing to the complexity and requirements of replication, a plethora of factors may interfere and challenge the genome stability, cell survival or affect the whole organism. This review outlines chemical compounds that are known inducers of RS and commonly used in laboratory research. These compounds act on replication by direct interaction with DNA causing DNA crosslinks and bulky lesions (cisplatin), chemical interference with the metabolism of deoxyribonucleotide triphosphates (hydroxyurea), direct inhibition of the activity of replicative DNA polymerases (aphidicolin) and interference with enzymes dealing with topological DNA stress (camptothecin, etoposide). As a variety of mechanisms can induce RS, the responses of mammalian cells also vary. Here, we review the activity and mechanism of action of these compounds based on recent knowledge, accompanied by examples of induced phenotypes, cellular readouts and commonly used doses.

Common Chemical Inductors of Replication Stress: Focus on Cell-Based Studies

PubMed Central

Vesela, Eva; Chroma, Katarina; Turi, Zsofia; Mistrik, Martin

2017-01-01

DNA replication is a highly demanding process regarding the energy and material supply and must be precisely regulated, involving multiple cellular feedbacks. The slowing down or stalling of DNA synthesis and/or replication forks is referred to as replication stress (RS). Owing to the complexity and requirements of replication, a plethora of factors may interfere and challenge the genome stability, cell survival or affect the whole organism. This review outlines chemical compounds that are known inducers of RS and commonly used in laboratory research. These compounds act on replication by direct interaction with DNA causing DNA crosslinks and bulky lesions (cisplatin), chemical interference with the metabolism of deoxyribonucleotide triphosphates (hydroxyurea), direct inhibition of the activity of replicative DNA polymerases (aphidicolin) and interference with enzymes dealing with topological DNA stress (camptothecin, etoposide). As a variety of mechanisms can induce RS, the responses of mammalian cells also vary. Here, we review the activity and mechanism of action of these compounds based on recent knowledge, accompanied by examples of induced phenotypes, cellular readouts and commonly used doses. PMID:28230817

Composite material of DNA and cyclodextrin-immobilized poly(ethyleneimine): Accumulation of harmful compounds from multi-component solution.

PubMed

Yamada, Masanori; Hori, Minako; Tabuchi, Shinya

2010-08-01

Water-soluble beta-cyclodextrin-immobilized poly(ethyleneimine) (PEICD) was synthesized by the grafting of beta-cyclodextrin to the branched poly(ethyleneimine). In an aqueous solution, this PEICD polymer could encapsulate bisphenol A, known to be a harmful compound. Additionally, the stability constant of bisphenol A to the PEICD polymer was 1.1 x 10(4)M(-1). However, the water-solubility of PEICD has been making it difficult to utilize it as an environmental material. Therefore, we prepared the DNA-PEICD composite material by mixing the double-stranded DNA and PEICD. This DNA-PEICD composite material was extremely stable in water and possessed both properties of the intercalation into the double-stranded DNA and the encapsulation into the CD cavity. As a result, this material can accumulate various harmful compounds, such as dioxin- and polychlorobiphenyl (PCB)-derivatives and bisphenol A, from a multi-component solution. Therefore, the DNA-PEICD composite material may have the potential to be used as an environmental material. Copyright 2010 Elsevier B.V. All rights reserved.

Synthesis and characterization of azo-guanidine based alcoholic media naked eye DNA sensor

PubMed Central

Hashmat, Uzma; Yousaf, Muhammad; Lal, Bhajan; Ullah, Shafiq; Holder, Alvin A.; Badshah, Amin

2016-01-01

DNA sensing always has an open meadow of curiosity for biotechnologists and other researchers. Recently, in this field, we have introduced an emerging class of molecules containing azo and guanidine functionalities. In this study, we have synthesized three new compounds (UA1, UA6 and UA7) for potential application in DNA sensing in alcoholic medium. The synthesized materials were characterized by elemental analysis, FTIR, UV-visible, 1H NMR and 13C NMR spectroscopies. Their DNA sensing potential were investigated by UV-visible spectroscopy. The insight of interaction with DNA was further investigated by electrochemical (cyclic voltammetry) and hydrodynamic (viscosity) studies. The results showed that compounds have moderate DNA binding properties, with the binding constants range being 7.2 × 103, 2.4 × 103 and 0.2 × 103 M−1, for UA1, UA6 and UA7, respectively. Upon binding with DNA, there was a change in colour (a blue shift in the λmax value) which was observable with a naked eye. These results indicated the potential of synthesized compounds as DNA sensors with detection limit 1.8, 5.8 and 4.0 ng µl−1 for UA1, UA6 and UA7, respectively. PMID:28018613

Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX-Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model.

PubMed

Zareba, Ilona; Surazynski, Arkadiusz; Chrusciel, Marcin; Miltyk, Wojciech; Doroszko, Milena; Rahman, Nafis; Palka, Jerzy

2017-01-01

The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7shPRODH/POX) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7shPRODH/POX cells. All studied compounds decreased cell viability in MCF-7 and MCF-7shPRODH/POX cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7shPRODH/POX and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7shPRODH/POX cells and contributed to the induction of pro-survival mode only in MCF-7shPRODH/POX cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7shPRODH/POX cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways. © 2017 The Author(s). Published by S. Karger AG, Basel.

Structural Mechanism of Replication Stalling on a Bulky Amino-Polycyclic Aromatic Hydrocarbon DNA Adduct by a Y Family DNA Polymerase

PubMed Central

Kirouac, Kevin N.; Basu, Ashis K.; Ling, Hong

2013-01-01

Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties. PMID:23876706

Structural mechanism of replication stalling on a bulky amino-polycyclic aromatic hydrocarbon DNA adduct by a y family DNA polymerase.

PubMed

Kirouac, Kevin N; Basu, Ashis K; Ling, Hong

2013-11-15

Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties. © 2013.

Synthesis, DNA-binding affinity and cytotoxicity of the dinuclear platinum(II) complexes with berenil and amines ligands.

PubMed

Bielawski, Krzysztof; Bielawska, Anna; Popławska, Bozena; Bołkun-Skórnicka, Urszula

2008-01-01

A series of platinium(II) complexes of formula [Pt2L4(berenil)2]Cl4.4HCl.2H2O where L is piperidine (1), 4-picoline (2), 3-picoline (3) or isopropylamine (4) was prepared and their cytotoxicity have been tested against the growth of human breast cancer cells. Evaluation of the cytotoxicity of these compounds employing a MTT assay and inhibition of [3H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that these compounds were more active than cisplatin. Data from the ethidium displacement assay indicated that these compounds show moderate specificity for AT base pairs of DNA. Compounds 1-4 were also potent topoisomerase II inhibitors, with 50% inhibitory concentrations (IC50) ranging from 5 to 50 microM.

Synthesis, spectroscopic characterization and structural investigations of new adduct compound of carbazole with picric acid: DNA binding and antimicrobial studies.

PubMed

Saravanabhavan, Munusamy; Sathya, Krishnan; Puranik, Vedavati G; Sekar, Marimuthu

2014-01-24

Carbazole picrate (CP), a new organic compound has been synthesized, characterized by various analytical and spectroscopic technique such as FT-IR, UV-Vis, (1)H and (13)C NMR spectroscopy. An orthorhombic geometry was proposed based on single crystal XRD study. The thermal stability of the crystal was studied by using thermo-gravimetric and differential thermal analyses and found that it was stable up to 170°C. Further, the newly synthesized title compound was tested for its in vitro antibacterial and antifungal activity against various bacterial and fungal species. Also, the compound was tested for its binding activity with Calf thymus (CT) DNA and the results show a considerable interaction between CP and CT-DNA. Copyright © 2013 Elsevier B.V. All rights reserved.

Interaction of cholinesterase modulators with DNA and their cytotoxic activity.

PubMed

Janockova, Jana; Gulasova, Zuzana; Plsikova, Jana; Musilek, Kamil; Kuca, Kamil; Mikes, Jaromir; Culka, Lubomir; Fedorocko, Peter; Kozurkova, Maria

2014-03-01

This research was focused on a study of the binding properties of a series of cholinesterase reactivators compounds K075 (1), K027 (2) and inhibitors compounds K524, K009 and 7-MEOTA (3-5) with calf thymus DNA. The nature of the interactions between compounds 1-5 and DNA were studied using spectroscopic techniques (UV-vis, fluorescence spectroscopy and circular dichroism). The binding constants for complexes of cholinesterase modulators with DNA were determined from UV-vis spectroscopic titrations (K=0.5 × 10(4)-8.9 × 10(5)M(-1)). The ability of the prepared analogues to relax topoisomerase I was studied with electrophoretic techniques and it was proved that ligands 4 and 5 inhibited this enzyme at a concentration of 30 μM. The biological activity of the novel compounds was assessed through an examination of changes in cell cycle distribution, mitochondrial membrane potential and cellular viability. Inhibitors 3-5 exhibited a cytotoxic effect on HL-60 (human acute promyelocytic leukaemia) cell culture, demonstrated a tendency to affect mitochondrial physiology and viability, and also forced cells to accumulate in the G1/G0-phase of the cell cycle. The cholinesterase reactivators 1 and 2 were found relatively save from the point of view of DNA binding, whereas cholinesterase inhibitors 3-5 resulted as strong DNA binding agents that limit their plausible use. Copyright © 2013 Elsevier B.V. All rights reserved.

Analysis of Active Methylotrophic Communities: When DNA-SIP Meets High-Throughput Technologies.

PubMed

Taubert, Martin; Grob, Carolina; Howat, Alexandra M; Burns, Oliver J; Chen, Yin; Neufeld, Josh D; Murrell, J Colin

2016-01-01

Methylotrophs are microorganisms ubiquitous in the environment that can metabolize one-carbon (C1) compounds as carbon and/or energy sources. The activity of these prokaryotes impacts biogeochemical cycles within their respective habitats and can determine whether these habitats act as sources or sinks of C1 compounds. Due to the high importance of C1 compounds, not only in biogeochemical cycles, but also for climatic processes, it is vital to understand the contributions of these microorganisms to carbon cycling in different environments. One of the most challenging questions when investigating methylotrophs, but also in environmental microbiology in general, is which species contribute to the environmental processes of interest, or "who does what, where and when?" Metabolic labeling with C1 compounds substituted with (13)C, a technique called stable isotope probing, is a key method to trace carbon fluxes within methylotrophic communities. The incorporation of (13)C into the biomass of active methylotrophs leads to an increase in the molecular mass of their biomolecules. For DNA-based stable isotope probing (DNA-SIP), labeled and unlabeled DNA is separated by isopycnic ultracentrifugation. The ability to specifically analyze DNA of active methylotrophs from a complex background community by high-throughput sequencing techniques, i.e. targeted metagenomics, is the hallmark strength of DNA-SIP for elucidating ecosystem functioning, and a protocol is detailed in this chapter.

Investigation of antimicrobial activities, DNA interaction, structural and spectroscopic properties of 2-chloro-6-(trifluoromethyl)pyridine

NASA Astrophysics Data System (ADS)

Evecen, Meryem; Kara, Mehmet; Idil, Onder; Tanak, Hasan

2017-06-01

2-Chloro-6-(trifluoromethyl)pyridine has been characterized by FT-IR, 1H and 13C NMR experiment. FT-IR spectra of the molecule has been recorded in the 4000-400 cm-1 region. The molecular structural parameters and vibrational frequencies were computed using the HF and DFT (B3LYP, B3PW91) methods with the 6-31+G(d,p) and 6-311++G(d,p) basis sets. 1H and 13C NMR Gauge Including Atomic Orbital (GIAO) chemical shifts of the compound were calculated using the density functional method (B3LYP) with the 6-311++G(d,p) basis set. The vibrational wavenumbers and chemical shifts were compared with the experimental data of the compound. Using the TD-DFT methodology, electronic absorption spectra of the compound have been computed. Besides, solvent effects on the excitation energies and chemical shifts were carried out using the integral equation formalism of the polarisable continuum model (IEF-PCM). DFT calculations of the compound, Mulliken's charges, molecular electrostatic potential (MEP), natural bond orbital (NBO) and thermodynamic properties were also obtained theoretically. In addition, the antimicrobial activities were tested by using minimal inhibitory concentration method (MIC) and also the effect of the molecule on pBR322 plasmid DNA was monitored byagarose gel electrophoresis experiments.

Novel selection methods for DNA-encoded chemical libraries

PubMed Central

Chan, Alix I.; McGregor, Lynn M.; Liu, David R.

2015-01-01

Driven by the need for new compounds to serve as biological probes and leads for therapeutic development and the growing accessibility of DNA technologies including high-throughput sequencing, many academic and industrial groups have begun to use DNA-encoded chemical libraries as a source of bioactive small molecules. In this review, we describe the technologies that have enabled the selection of compounds with desired activities from these libraries. These methods exploit the sensitivity of in vitro selection coupled with DNA amplification to overcome some of the limitations and costs associated with conventional screening methods. In addition, we highlight newer techniques with the potential to be applied to the high-throughput evaluation of DNA-encoded chemical libraries. PMID:25723146

Synthesis, spectroscopic, physicochemical and structural characterization of tetrandrine-based macrocycles functionalized with acridine and anthracene groups: DNA binding and anti-proliferative activity.

PubMed

Calvillo-Páez, Viviana; Sotelo-Mundo, Rogerio R; Leyva-Peralta, Mario; Gálvez-Ruiz, Juan Carlos; Corona-Martínez, David; Moreno-Corral, Ramón; Escobar-Picos, Raymundo; Höpfl, Herbert; Juárez-Sánchez, Octavio; Lara, Karen Ochoa

2018-04-25

In this work, we report on the synthesis of two new mono-alkylated tetrandrine derivatives with acridine and anthracene units, MAcT and MAnT. The compounds were fully characterized by physicochemical techniques and single-crystal X-ray diffraction analysis. In addition, both derivatives were studied as nucleotide receptors and double-stranded DNA binders in aqueous phosphate buffer at pH = 7.2 using UV-vis and fluorescence spectroscopy. According to the molecular recognition studies, MAcT and MAnT exhibit high affinity (K ∼ 10 5  M -1 ) and selectivity for ds-DNA, presumably in an intercalation mode. Finally, the anti-proliferative effects of the tetrandrine derivatives on different cancer cell lines were explored, revealing promising activities. Particularly, the mono-anthracene tetrandrine derivative MAnT showed an IC 50 of 2.74 μg/mL on the HeLa cervical cancer cell line, representing a value 3.3 times smaller than that obtained for unsubstituted tetrandrine. Examination of the cytotoxic effects on the HeLa cell line by inverted microscopy suggests that the cell death mechanism consists basically in apoptosis. The molecular modelling of three ds-DNA-MAcT complexes, suggested that the macrocycles may use an intercalation binding mode towards DNA. MAcT is predicted to bind into the major groove of the ds-DNA providing non-covalent interactions such as electrostatic, van der Waals and hydrophobic interactions that lead to selectivity. Overall experimental data supports the mode of action of MAnT and MAcT as cytotoxic compounds against cancer cell lines via a DNA interaction mechanism. Copyright © 2018 Elsevier B.V. All rights reserved.

DNA binding properties and biological evaluation of dihydropyrimidinones derivatives as potential antitumor agents.

PubMed

Wang, Gongke; Li, Xiangrong; Gou, Yaping; Chen, Yuhan; Yan, Changling; Lu, Yan

2013-10-01

The binding properties of two medicinally important dihydropyrimidinones derivatives 5-(Ethoxycarbonyl)-6-methyl-4-phenyl-3,4-dihydropyrimidin-2(1H)-one (EMPD) and 5-(Ethoxycarbonyl)-6-methyl-4-(4-chlorophenyl)-3,4-dihydropyrimidin-2(1H)-one (EMCD) with calf-thymus DNA (ctDNA) were investigated by spectroscopy, viscosity, isothermal titration calorimetry (ITC) and molecular modeling techniques. Simultaneously, their biological activities were evaluated with MTT assay method. The binding constants determined with spectroscopic titration and ITC were found to be in the same order of 10(4)M(-1). According to the results of viscosity studies, fluorescence competitive binding experiment and ITC investigations, intercalative binding was evaluated as the dominant binding modes between the two compounds and ctDNA. Furthermore, the results of molecular modeling corroborated those obtained from spectroscopic, viscosimetric and ITC investigations. Evaluation of the antitumor activities of the two derivatives against different tumor cell lines proved that they exhibited significant tumor cell inhibition rate, accordingly blocking DNA transcription and replication. The present results favor the development of potential drugs related with dihydropyrimidinones derivatives in the treatment of some diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

Current developments in forensic interpretation of mixed DNA samples (Review).

PubMed

Hu, Na; Cong, Bin; Li, Shujin; Ma, Chunling; Fu, Lihong; Zhang, Xiaojing

2014-05-01

A number of recent improvements have provided contemporary forensic investigations with a variety of tools to improve the analysis of mixed DNA samples in criminal investigations, producing notable improvements in the analysis of complex trace samples in cases of sexual assult and homicide. Mixed DNA contains DNA from two or more contributors, compounding DNA analysis by combining DNA from one or more major contributors with small amounts of DNA from potentially numerous minor contributors. These samples are characterized by a high probability of drop-out or drop-in combined with elevated stutter, significantly increasing analysis complexity. At some loci, minor contributor alleles may be completely obscured due to amplification bias or over-amplification, creating the illusion of additional contributors. Thus, estimating the number of contributors and separating contributor genotypes at a given locus is significantly more difficult in mixed DNA samples, requiring the application of specialized protocols that have only recently been widely commercialized and standardized. Over the last decade, the accuracy and repeatability of mixed DNA analyses available to conventional forensic laboratories has greatly advanced in terms of laboratory technology, mathematical models and biostatistical software, generating more accurate, rapid and readily available data for legal proceedings and criminal cases.

Current developments in forensic interpretation of mixed DNA samples (Review)

PubMed Central

HU, NA; CONG, BIN; LI, SHUJIN; MA, CHUNLING; FU, LIHONG; ZHANG, XIAOJING

2014-01-01

A number of recent improvements have provided contemporary forensic investigations with a variety of tools to improve the analysis of mixed DNA samples in criminal investigations, producing notable improvements in the analysis of complex trace samples in cases of sexual assult and homicide. Mixed DNA contains DNA from two or more contributors, compounding DNA analysis by combining DNA from one or more major contributors with small amounts of DNA from potentially numerous minor contributors. These samples are characterized by a high probability of drop-out or drop-in combined with elevated stutter, significantly increasing analysis complexity. At some loci, minor contributor alleles may be completely obscured due to amplification bias or over-amplification, creating the illusion of additional contributors. Thus, estimating the number of contributors and separating contributor genotypes at a given locus is significantly more difficult in mixed DNA samples, requiring the application of specialized protocols that have only recently been widely commercialized and standardized. Over the last decade, the accuracy and repeatability of mixed DNA analyses available to conventional forensic laboratories has greatly advanced in terms of laboratory technology, mathematical models and biostatistical software, generating more accurate, rapid and readily available data for legal proceedings and criminal cases. PMID:24748965

Controlling the surface‐mediated release of DNA using ‘mixed multilayers’

PubMed Central

Appadoo, Visham; Carter, Matthew C. D.

2016-01-01

Abstract We report the design of erodible ‘mixed multilayer’ coatings fabricated using plasmid DNA and combinations of both hydrolytically degradable and charge‐shifting cationic polymer building blocks. Films fabricated layer‐by‐layer using combinations of a model poly(β‐amino ester) (polymer 1) and a model charge‐shifting polymer (polymer 2) exhibited DNA release profiles that were substantially different than those assembled using DNA and either polymer 1 or polymer 2 alone. In addition, the order in which layers of these two cationic polymers were deposited during assembly had a profound impact on DNA release profiles when these materials were incubated in physiological buffer. Mixed multilayers ∼225 nm thick fabricated by depositing layers of polymer 1/DNA onto films composed of polymer 2/DNA released DNA into solution over ∼60 days, with multi‐phase release profiles intermediate to and exhibiting some general features of polymer 1/DNA or polymer 2/DNA films (e.g., a period of rapid release, followed by a more extended phase). In sharp contrast, ‘inverted’ mixed multilayers fabricated by depositing layers of polymer 2/DNA onto films composed of polymer 1/DNA exhibited release profiles that were almost completely linear over ∼60‐80 days. These and other results are consistent with substantial interdiffusion and commingling (or mixing) among the individual components of these compound materials. Our results reveal this mixing to lead to new, unanticipated, and useful release profiles and provide guidance for the design of polymer‐based coatings for the local, surface‐mediated delivery of DNA from the surfaces of topologically complex interventional devices, such as intravascular stents, with predictable long‐term release profiles. PMID:27981243

DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis.

PubMed

MacConnell, Andrew B; McEnaney, Patrick J; Cavett, Valerie J; Paegel, Brian M

2015-09-14

The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the "structure elucidation problem": the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS's utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 10(4) molecules/bead and sequencing allowed for elucidation of each compound's synthetic history. We applied DESPS to the combinatorial synthesis of a 75,645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and PCR make implementation of DESPS straightforward, and may prompt the chemistry community to revisit the synthesis of more complex and diverse libraries.

Synergy between Prkdc and Trp53 regulates stem cell proliferation and GI-ARS after irradiation.

PubMed

Gurley, Kay E; Ashley, Amanda K; Moser, Russell D; Kemp, Christopher J

2017-11-01

Ionizing radiation (IR) is one of the most widely used treatments for cancer. However, acute damage to the gastrointestinal tract or gastrointestinal acute radiation syndrome (GI-ARS) is a major dose-limiting side effect, and the mechanisms that underlie this remain unclear. Here we use mouse models to explore the relative roles of DNA repair, apoptosis, and cell cycle arrest in radiation response. IR induces DNA double strand breaks and DNA-PK mutant Prkdc scid/scid mice are sensitive to GI-ARS due to an inability to repair these breaks. IR also activates the tumor suppressor p53 to trigger apoptotic cell death within intestinal crypt cells and p53 deficient mice are resistant to apoptosis. To determine if DNA-PK and p53 interact to govern radiosensitivity, we compared the response of single and compound mutant mice to 8 Gy IR. Compound mutant Prkdc scid/scid /Trp53 -/- mice died earliest due to severe GI-ARS. While both Prkdc scid/scid and Prkdc scid/scid /Trp53 -/- mutant mice had higher levels of IR-induced DNA damage, particularly within the stem cell compartment of the intestinal crypt, in Prkdc scid/scid /Trp53 -/- mice these damaged cells abnormally progressed through the cell cycle resulting in mitotic cell death. This led to a loss of Paneth cells and a failure to regenerate the differentiated epithelial cells required for intestinal function. IR-induced apoptosis did not correlate with radiosensitivity. Overall, these data reveal that DNA repair, mediated by DNA-PK, and cell cycle arrest, mediated by p53, cooperate to protect the stem cell niche after DNA damage, suggesting combination approaches to modulate both pathways may be beneficial to reduce GI-ARS. As many cancers harbor p53 mutations, this also suggests targeting DNA-PK may be effective to enhance sensitivity of p53 mutant tumors to radiation.

Radiosensitization of biologically active DNA in cellular extracts by oxygen. Evidence that the presence of SH-compounds is not required

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vanhemmen, J.J.; Meuling, W.J.A.; Bleichrodt, J.F.

1974-01-01

The radiosensitization by oxygen of biological active bacteriophage DNA in bacterial extracts was studied. The oxygen effect in such a system appeared not to be due or due only to a minor extent to the presence of endogenous sulfhydryl compounds. The components in a cell extract which enable oxygen and other sensitizers to sensitize DNA could not be destroyed by extremely high doses of gamma radiation. (Author) (GRA)

Antibacterial Drug Leads: DNA and Enzyme Multitargeting

DOE PAGES

Zhu, Wei; Wang, Yang; Li, Kai; ...

2015-01-09

Here, we report the results of an investigation of the activity of a series of amidine and bisamidine compounds against Staphylococcus aureus and Escherichia coli. The most active compounds bound to an AT-rich DNA dodecamer (CGCGAATTCGCG) 2 and using DSC were found to increase the melting transition by up to 24 °C. Several compounds also inhibited undecaprenyl diphosphate synthase (UPPS) with IC 50 values of 100–500 nM, and we found good correlations (R 2 = 0.89, S. aureus; R 2 = 0.79, E. coli) between experimental and predicted cell growth inhibition by using DNA ΔT m and UPPS IC 50more » experimental results together with one computed descriptor. Finally, we also solved the structures of three bisamidines binding to DNA as well as three UPPS structures. Overall, the results are of general interest in the context of the development of resistance-resistant antibiotics that involve multitargeting.« less

Discovery, SAR, and X-ray Binding Mode Study of BCATm Inhibitors from a Novel DNA-Encoded Library

PubMed Central

2015-01-01

As a potential target for obesity, human BCATm was screened against more than 14 billion DNA encoded compounds of distinct scaffolds followed by off-DNA synthesis and activity confirmation. As a consequence, several series of BCATm inhibitors were discovered. One representative compound (R)-3-((1-(5-bromothiophene-2-carbonyl)pyrrolidin-3-yl)oxy)-N-methyl-2′-(methylsulfonamido)-[1,1′-biphenyl]-4-carboxamide (15e) from a novel compound library synthesized via on-DNA Suzuki–Miyaura cross-coupling showed BCATm inhibitory activity with IC50 = 2.0 μM. A protein crystal structure of 15e revealed that it binds to BCATm within the catalytic site adjacent to the PLP cofactor. The identification of this novel inhibitor series plus the establishment of a BCATm protein structure provided a good starting point for future structure-based discovery of BCATm inhibitors. PMID:26288694

Application of virtual screening and molecular dynamics for the analysis of selectivity of inhibitors of HU proteins targeted to the DNA-recognition site

NASA Astrophysics Data System (ADS)

Talyzina, A. A.; Agapova, Yu. K.; Podshivalov, D. D.; Timofeev, V. I.; Sidorov-Biryukov, D. D.; Rakitina, T. V.

2017-11-01

DNA-Binding HU proteins are essential for the maintenance of genomic DNA supercoiling and compaction in prokaryotic cells and are promising pharmacological targets for the design of new antibacterial agents. The virtual screening for low-molecular-weight compounds capable of specifically interacting with the DNA-recognition loop of the HU protein from the mycoplasma Spiroplasma melliferum was performed. The ability of the initially selected ligands to form stable complexes with the protein target was assessed by molecular dynamics simulation. One compound, which forms an unstable complex, was eliminated by means of a combination of computational methods, resulting in a decrease in the number of compounds that will pass to the experimental test phase. This approach can be used to solve a wide range of problems related to the search for and validation of low-molecular-weight inhibitors specific for a particular protein target.

A Novel Chemical Compound for Inhibition of SARS Coronavirus Helicase.

PubMed

Lee, Jin-Moo; Cho, Jin-Beom; Ahn, Hee-Chul; Jung, Woong; Jeong, Yong-Joo

2017-11-28

We have discovered a novel chemical compound, (E)-3-(furan-2-yl)- N -(4-sulfamoylphenyl) acrylamide, that suppresses the enzymatic activities of SARS coronavirus helicase. To determine the inhibitory effect, ATP hydrolysis and double-stranded DNA unwinding assays were performed in the presence of various concentrations of the compound. Through these assays, we obtained IC 50 values of 2.09 ± 0.30 µM (ATP hydrolysis) and 13.2 ± 0.9 µM (DNA unwinding), respectively. Moreover, we found that the compound did not have any significant cytotoxicity when 40 µM of it was used. Our results showed that the compound might be useful to be developed as an inhibitor against SARS coronavirus.

Radioprotection and Cell Cycle Arrest of Intestinal Epithelial Cells by Darinaparsin, a Tumor Radiosensitizer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Junqiang; Doi, Hiroshi; Saar, Matthias

2013-12-01

Purpose: It was recently reported that the organic arsenic compound darinaparsin (DPS) is a cytotoxin and radiosensitizer of tumor cells in vitro and in subcutaneous xenograft tumors. Surprisingly, it was also found that DPS protects normal intestinal crypt epithelial cells (CECs) from clonogenic death after ionizing radiation (IR). Here we tested the DPS radiosensitizing effect in a clinically relevant model of prostate cancer and explored the radioprotective effect and mechanism of DPS on CECs. Methods and Materials: The radiation modification effect of DPS was tested in a mouse model of orthotopic xenograft prostate cancer and of IR-induced acute gastrointestinal syndrome.more » The effect of DPS on CEC DNA damage and DNA damage responses was determined by immunohistochemistry. Results: In the mouse model of IR-induced gastrointestinal syndrome, DPS treatment before IR accelerated recovery from body weight loss and increased animal survival. DPS decreased post-IR DNA damage and cell death, suggesting that the radioprotective effect was mediated by enhanced DNA damage repair. Shortly after DPS injection, significant cell cycle arrest was observed in CECs at both G1/S and G2/M checkpoints, which was accompanied by the activation of cell cycle inhibitors p21 and growth arrest and DNA-damage-inducible protein 45 alpha (GADD45A). Further investigation revealed that DPS activated ataxia telangiectasia mutated (ATM), an important inducer of DNA damage repair and cell cycle arrest. Conclusions: DPS selectively radioprotected normal intestinal CECs and sensitized prostate cancer cells in a clinically relevant model. This effect may be, at least in part, mediated by DNA damage response activation and has the potential to significantly increase the therapeutic index of radiation therapy.« less

Diphenylmethyl selenocyanate attenuates malachite green induced oxidative injury through antioxidation & inhibition of DNA damage in mice

PubMed Central

Das, Jayanta Kumar; Sarkar, Sibani; Hossain, Sk Ugir; Chakraborty, Pramita; Das, Rajat Kumar; Bhattacharya, Sudin

2013-01-01

Background & objectives: Malachite green (MG), an environmentally hazardous material, is used as a non permitted food colouring agent, especially in India. Selenium (Se) is an essential nutritional trace element required for animals and humans to guard against oxidative stress induced by xenobiotic compounds of diverse nature. In the present study, the role of the selenium compound diphenylmethyl selenocyanate (DMSE) was assessed on the oxidative stress (OS) induced by a food colouring agent, malachite green (MG) in vivo in mice. Methods: Swiss albino mice (Mus musculus) were intraperitoneally injected with MG at a standardized dose of 100 μg/ mouse for 30 days. DMSE was given orally at an optimum dose of 3 mg/kg b.w. in pre (15 days) and concomitant treatment schedule throughout the experimental period. The parameters viz. ALT, AST, LPO, GSH, GST, SOD, CAT, GPx, TrxR, CA, MN, MI and DNA damage have been evaluated. Results: The DMSE showed its potential to protect against MG induced hepatotoxicity by controlling the serum alanine aminotransferase and aspartate amino transferase (ALT and AST) levels and also ameliorated oxidative stress by modulating hepatic lipid peroxidation and different detoxifying and antioxidative enzymes such as glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), and also the selenoenzymes such as glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) and reduced glutathione level which in turn reduced DNA damage. Interpretation & conclusions: The organo-selenium compound DMSE showed significant protection against MG induced heptotoxicity and DNA damage in murine model. Better protection was observed in pretreatment group than in the concomitant group. Further studies need to be done to understand the mechanism of action. PMID:23852297

A novel carbohydrate derived compound FCP5 causes DNA strand breaks and oxidative modifications of DNA bases in cancer cells.

PubMed

Czubatka, Anna; Sarnik, Joanna; Lucent, Del; Blasiak, Janusz; Witczak, Zbigniew J; Poplawski, Tomasz

2015-02-05

1,5-Anhydro-6-deoxy-methane-sulfamido-D-glucitol (FCP5) is a functionalized carbohydrate containing functional groups that render it potentially therapeutically useful. According to our concept of 'functional carb-pharmacophores' (FCPs) incorporation of the methanesulfonamido pharmacophore to 1,5 glucitol could create a therapeutically useful compound. Our previous studies revealed that FCP5 was cytotoxic to cancer cells. Therefore, in this work we assessed the cytotoxic mechanisms of FCP5 in four cancer cell lines - HeLa, LoVo, A549 and MCF-7, with particular focus on DNA damage and repair. A broad spectrum of methods, including comet assay with modifications, DNA repair enzyme assay, plasmid relaxation assay, and DNA fragmentation assay, were used. We also checked the potential for FCP5 to induce apoptosis. The results show that FCP5 can induce DNA strand breaks as well as oxidative modifications of DNA bases. DNA lesions induced by FCP5 were not entirely repaired in HeLa cells and DNA repair kinetics differs from other cell lines. Results from molecular docking and plasmid relaxation assay suggest that FCP5 binds to the major groove of DNA with a preference for adenosine-thymine base pair sequences and directly induces DNA strand breaks. Thus, FCP5 may represent a novel lead for the design of new major groove-binding compounds. The results also confirmed the validity of functional carb-pharmacophores as a new source of innovative drugs. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Radio- and photosensitization of DNA with compounds containing platinum and bromine atoms

NASA Astrophysics Data System (ADS)

Śmiałek, Małgorzata A.; Ptasińska, Sylwia; Gow, Jason; Vrønning Hoffmann, Søren; Mason, Nigel J.

2015-05-01

Irradiations of plasmid DNA by both X-rays and UV light in the presence and absence of compounds containing platinum and bromine atoms were performed in order to asses the sensitization potential of these compounds. Plasmid DNA pBR322 was incubated with platinum (II) bromide, hydrogen hexabromoplatinate (IV), hydrogen hexahydroxyplatinate (IV) and sodium hexahydroxyplatinate (IV). Incubation was followed by X-ray or UV irradiations. It was found that amongst the sensitizers tested, during irradiations carried out in the presence of platinum (II) bromide, the highest levels of double strand breaks formation upon X-ray treatment were recorded. In contrast much less damage was induced by UV light. Data presented here suggests that this compound may be a promising radiosensitizer for cancer treatment. Contribution to the Topical Issue "COST Action Nano-IBCT: Nano-scale Processes Behind Ion-Beam Cancer Therapy", edited by Andrey Solov'yov, Nigel Mason, Gustavo García, Eugene Surdutovich.

Synthesis, structure elucidation, DNA-PK and PI3K and anti-cancer activity of 8- and 6-aryl-substituted-1-3-benzoxazines.

PubMed

Morrison, Rick; Al-Rawi, Jasim M A; Jennings, Ian G; Thompson, Philip E; Angove, Michael J

2016-03-03

The synthesis of 6-aryl, 8- aryl, and 8-aryl-6-chloro-2-morpholino-1,3-benzoxazines with potent activity against PI3K and DNA-PK is described. Synthesis of thirty one analogues was facilitated by an improved synthesis of 3-bromo-2-hydroxybenzoic acid 13 by de-sulphonation of 3-bromo-2-hydroxy-5-sulfobenzoic acid 12 en route to 2-methylthio-substituted-benzoxazine intermediates 17-19. From this series, compound 20k (LTURM34) (dibenzo[b,d]thiophen-4-yl) (IC50 = 0.034 μM) was identified as a specific DNA-PK inhibitor, 170 fold more selective for DNA-PK activity compared to PI3K activity. Other compounds of the series show markedly altered selectivity for various PI3K isoforms including compound 20i (8-(naphthalen-1-yl) a potent and quite selective PI3Kδ inhibitor (IC50 = 0.64 μM). Finally, nine compounds were evaluated and showed antiproliferative activity against an NCI panel of cancer cell lines. Compound 20i (8-(naphthalen-1-yl) showed strong anti-proliferative activity against A498 renal cancer cells that warrants further investigation. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Pharmacological reduction of ER stress protects against TDP-43 neuronal toxicity in vivo.

PubMed

Vaccaro, Alexandra; Patten, Shunmoogum A; Aggad, Dina; Julien, Carl; Maios, Claudia; Kabashi, Edor; Drapeau, Pierre; Parker, J Alex

2013-07-01

C. elegans and D. rerio expressing mutant TAR DNA Binding Protein 43 (TDP-43) are powerful in vivo animal models for the genetics and pharmacology of amyotrophic lateral sclerosis (ALS). Using these small-animal models of ALS, we previously identified methylene blue (MB) as a potent suppressor of TDP-43 toxicity. Consequently here we investigated how MB might exert its neuroprotective properties and found that it acts through reduction of the endoplasmic reticulum (ER) stress response. We tested other compounds known to be active in the ER unfolded protein response in worms and zebrafish expressing mutant human TDP-43 (mTDP-43). We identified three compounds: salubrinal, guanabenz and a new structurally related compound phenazine, which also reduced paralysis, neurodegeneration and oxidative stress in our mTDP-43 models. Using C. elegans genetics, we showed that all four compounds act as potent suppressors of mTDP-43 toxicity through reduction of the ER stress response. Interestingly, these compounds operate through different branches of the ER unfolded protein pathway to achieve a common neuroprotective action. Our results indicate that protein-folding homeostasis in the ER is an important target for therapeutic development in ALS and other TDP-43-related neurodegenerative diseases. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Novel selection methods for DNA-encoded chemical libraries.

PubMed

Chan, Alix I; McGregor, Lynn M; Liu, David R

2015-06-01

Driven by the need for new compounds to serve as biological probes and leads for therapeutic development and the growing accessibility of DNA technologies including high-throughput sequencing, many academic and industrial groups have begun to use DNA-encoded chemical libraries as a source of bioactive small molecules. In this review, we describe the technologies that have enabled the selection of compounds with desired activities from these libraries. These methods exploit the sensitivity of in vitro selection coupled with DNA amplification to overcome some of the limitations and costs associated with conventional screening methods. In addition, we highlight newer techniques with the potential to be applied to the high-throughput evaluation of DNA-encoded chemical libraries. Copyright © 2015 Elsevier Ltd. All rights reserved.

Heteroplasmy and evidence for recombination in the mitochondrial control region of the flatfish Platichthys flesus.

PubMed

Hoarau, Galice; Holla, Suzanne; Lescasse, Rachel; Stam, Wytze T; Olsen, Jeanine L

2002-12-01

The general assumption that mitochondrial DNA (mtDNA) does not undergo recombination has been challenged recently in invertebrates. Here we present the first direct evidence for recombination in the mtDNA of a vertebrate, the flounder Platichthys flesus. The control region in the mtDNA of this flatfish is characterized by the presence of a variable number of tandem repeats and a high level of heteroplasmy. Two types of repeats were recognized, differing by two C-T point mutations. Most individuals carry a pure "C" or a pure "T" array, but one individual showed a compound "CT" array. Such a compound array is evidence for recombination in the mtDNA control region from the flounder.

Supramolecular approach to enantioselective DNA recognition using enantiomerically resolved cationic 4-amino-1,8-naphthalimide-based Tröger's bases.

PubMed

Banerjee, Swagata; Bright, Sandra A; Smith, Jayden A; Burgeat, Jeremy; Martinez-Calvo, Miguel; Williams, D Clive; Kelly, John M; Gunnlaugsson, Thorfinnur

2014-10-03

The synthesis and photophysical studies of two cationic Tröger's base (TB)-derived bis-naphthalimides 1 and 2 and the TB derivative 6, characterized by X-ray crystallography, are presented. The enantiomers of 1 and 2 are separated by cation-exchange chromatography on Sephadex C25 using sodium (-)-dibenzoyl-l-tartarate as the chiral mobile phase. The binding of enantiomers with salmon testes (st)-DNA and synthetic polynucleotides are studied by a variety of spectroscopic methods including UV/vis absorbance, circular dichroism, linear dichroism, and ethidium bromide displacement assays, which demonstrated binding of these compounds to the DNA grooves with very high affinity (K ∼ 10(6) M(-1)) and preferential binding of (-)-enantiomer. In all cases, binding to DNA resulted in a significant stabilization of the double-helical structure of DNA against thermal denaturation. Compound (±)-2 and its enantiomers possessed significantly higher binding affinity for double-stranded DNA compared to 1, possibly due to the presence of the methyl group, which allows favorable hydrophobic and van der Waals interactions with DNA. The TB derivatives exhibited marked preference for AT rich sequences, where the binding affinities follow the order (-)-enantiomer > (±) > (+)-enantiomer. The compounds exhibited significant photocleavage of plasmid DNA upon visible light irradiation and are rapidly internalized into malignant cell lines.

Protein targets for anticancer gold compounds: mechanistic inferences.

PubMed

Gabbiani, Chiara; Messori, Luigi

2011-12-01

Gold compounds form an interesting class of antiproliferative agents of potential pharmacological use in cancer treatment. Indeed, a number of gold compounds, either gold(III) or gold(I), were recently described and characterised that manifested remarkable cytotoxic properties in vitro against cultured cancer cells; for some of them encouraging in vivo results were also reported toward a few relevant animal models of cancer. The molecular mechanisms through which gold compounds exert their biological effects are still largely unknown and the subject of intense investigations. Recent studies point out that the modes of action of cytotoxic gold compounds are essentially DNA-independent and cisplatin-unrelated, relying -most likely- on gold interactions with a variety of protein targets. Notably, a few cellular proteins playing relevant functional roles were proposed to represent effective targets for cytotoxic gold compounds but these hypotheses need adequate validation. The state of the art of this research area and the perspectives for future studies are herein critically analysed and discussed.

Biological Characterization of Novel Inhibitors of the Gram-Positive DNA Polymerase IIIC Enzyme

PubMed Central

Kuhl, Alexander; Svenstrup, Niels; Ladel, Christoph; Otteneder, Michael; Binas, Annegret; Schiffer, Guido; Brands, Michael; Lampe, Thomas; Ziegelbauer, Karl; Rübsamen-Waigmann, Helga; Haebich, Dieter; Ehlert, Kerstin

2005-01-01

Novel N-3-alkylated 6-anilinouracils have been identified as potent and selective inhibitors of bacterial DNA polymerase IIIC, the enzyme essential for the replication of chromosomal DNA in gram-positive bacteria. A nonradioactive assay measuring the enzymatic activity of the DNA polymerase IIIC in gram-positive bacteria has been assembled. The 6-anilinouracils described inhibited the polymerase IIIC enzyme at concentrations in the nanomolar range in this assay and displayed good in vitro activity (according to their MICs) against staphylococci, streptococci, and enterococci. The MICs of the most potent derivatives were about 4 μg/ml for this panel of bacteria. The 50% effective dose of the best compound (6-[(3-ethyl-4-methylphenyl)amino]-3-{[1-(isoxazol-5-ylcarbonyl)piperidin-4-yl]methyl}uracil) was 10 mg/kg of body weight after intravenous application in a staphylococcal sepsis model in mice, from which in vivo pharmacokinetic data were also acquired. PMID:15728893

Epigenetic effects of green tea polyphenols in cancer

PubMed Central

Henning, Susanne M; Wang, Piwen; Carpenter, Catherine L; Heber, David

2014-01-01

Epigenetics describes heritable alterations of gene expression and chromatin organization without changes in DNA sequence. Both hypermethylation and hypomethylation of DNA can affect gene expression and the multistep process of carcinogenesis. Epigenetic changes are reversible and may be targeted by dietary interventions. Bioactive compounds from green tea (GT) such as (–)-epigallocatechin gallate have been shown to alter DNA methyltransferase activity in studies of esophageal, oral, skin, Tregs, lung, breast and prostate cancer cells, which may contribute to the chemopreventive effect of GT. Three out of four mouse model studies have confirmed the inhibitory effect of (–)-epigallocatechin gallate on DNA methylation. A human study demonstrated that decreased methylation of CDX2 and BMP-2 in gastric carcinoma was associated with higher GT consumption. It is the goal of this review to summarize our current knowledge of the potential of GT to alter epigenetic processes, which may be useful in chemoprevention. PMID:24283885

Anthocyanin Interactions with DNA: Intercalation, Topoisomerase I Inhibition and Oxidative Reactions

PubMed Central

Webb, Michael R.; Min, Kyungmi; Ebeler, Susan E.

2009-01-01

Anthocyanins and their aglycone anthocyanidins are pigmented flavonoids found in significant amounts in many commonly consumed foods. They exhibit a complex chemistry in aqueous solution, which makes it difficult to study their chemistry under physiological conditions. Here we used a gel electrophoresis assay employing supercoiled DNA plasmid to examine the ability of these compounds (1) to intercalate DNA, (2) to inhibit human topoisomerase I through both inhibition of plasmid relaxation activity (catalytic inhibition) and stabilization of the cleavable DNA-topoisomerase complex (poisoning), and (3) to inhibit or enhance oxidative single-strand DNA nicking. We found no evidence of DNA intercalation by anthocyan(id)ins in the physiological pH range for any of the compounds used in this study—cyanidin chloride, cyanidin 3-O-glucoside, cyanidin 3,5-O-diglucoside, malvidin 3-O-glucoside and luteolinidin chloride. The anthocyanins inhibited topoisomerase relaxation activity only at high concentrations (> 50 μM) and we could find no evidence of topoisomerase I cleavable complex stabilization by these compounds. However, we observed that all of the anthocyan(id)ins used in this study were capable of inducing significant oxidative DNA strand cleavage (nicking) in the presence of 1 mM DTT (dithiothreitol), while the free radical scavenger, DMSO, at concentrations typically used in similar studies, completely inhibited DNA nicking. Finally, we propose a mechanism to explain the anthocyan(id)in induced oxidative DNA cleavage observed under our experimental conditions. PMID:19924259

New potential antitumoral fluorescent tetracyclic thieno[3,2- b]pyridine derivatives: interaction with DNA and nanosized liposomes

NASA Astrophysics Data System (ADS)

Castanheira, Elisabete Ms; Carvalho, Maria Solange D.; Rodrigues, Ana Rita O.; Calhelha, Ricardo C.; Queiroz, Maria-João Rp

2011-05-01

Fluorescence properties of two new potential antitumoral tetracyclic thieno[3,2- b]pyridine derivatives were studied in solution and in liposomes of DPPC (dipalmitoyl phosphatidylcholine), egg lecithin (phosphatidylcholine from egg yolk; Egg-PC) and DODAB (dioctadecyldimethylammonium bromide). Compound 1, pyrido[2',3':3,2]thieno[4,5- d]pyrido[1,2- a]pyrimidin-6-one, exhibits reasonably high fluorescence quantum yields in all solvents studied (0.20 ≤ ΦF ≤ 0.30), while for compound 2, 3-[( p-methoxyphenyl)ethynyl]pyrido[2',3':3,2]thieno[4,5- d]pyrido[1,2- a]pyrimidin-6-one, the values are much lower (0.01 ≤ ΦF ≤ 0.05). The interaction of these compounds with salmon sperm DNA was studied using spectroscopic methods, allowing the determination of intrinsic binding constants, K i = (8.7 ± 0.9) × 103 M-1 for compound 1 and K i = (5.9 ± 0.6) × 103 M-1 for 2, and binding site sizes of n = 11 ± 3 and n = 7 ± 2 base pairs, respectively. Compound 2 is the most intercalative compound in salmon sperm DNA (35%), while for compound 1 only 11% of the molecules are intercalated. Studies of incorporation of both compounds in liposomes of DPPC, Egg-PC and DODAB revealed that compound 2 is mainly located in the hydrophobic region of the lipid bilayer, while compound 1 prefers a hydrated and fluid environment.

Measurement of pyrimidine (6-4) photoproducts in DNA by a mild acidic hydrolysis-HPLC fluorescence detection assay.

PubMed

Douki, T; Voituriez, L; Cadet, J

1995-03-01

Pyrimidine (6-4) pyrimidone photoproducts constitute one of the major classes of DNA lesions induced by far-UV irradiation. However, their biological role remains difficult to assess partly because of the lack of a specific and sensitive assay for monitoring their formation in DNA. Here is presented a measurement method based on the release of the (6-4) base adducts from DNA followed by an HPLC separation associated with a sensitive and specific fluorescence detection. The quantitative and mechanistic aspects of the chemical hydrolysis, based on the use of hydrogen fluoride stabilized in pyridine, were investigated, using dinucleoside monophosphate (6-4) photoproducts as model compounds. The final hydrolysis products were isolated and characterized by UV, fluorescence, mass, and 1H NMR spectroscopies. Application of the assay to far-UV irradiated calf thymus DNA provided information on the sequence effect on the rate of formation of three of the four possible bipyrimidine (6-4) photoproducts.

Systems and methods for modeling and analyzing networks

DOEpatents

Hill, Colin C; Church, Bruce W; McDonagh, Paul D; Khalil, Iya G; Neyarapally, Thomas A; Pitluk, Zachary W

2013-10-29

The systems and methods described herein utilize a probabilistic modeling framework for reverse engineering an ensemble of causal models, from data and then forward simulating the ensemble of models to analyze and predict the behavior of the network. In certain embodiments, the systems and methods described herein include data-driven techniques for developing causal models for biological networks. Causal network models include computational representations of the causal relationships between independent variables such as a compound of interest and dependent variables such as measured DNA alterations, changes in mRNA, protein, and metabolites to phenotypic readouts of efficacy and toxicity.

Effects of intermediates between vitamins K(2) and K(3) on mammalian DNA polymerase inhibition and anti-inflammatory activity.

PubMed

Mizushina, Yoshiyuki; Maeda, Jun; Irino, Yasuhiro; Nishida, Masayuki; Nishiumi, Shin; Kondo, Yasuyuki; Nishio, Kazuyuki; Kuramochi, Kouji; Tsubaki, Kazunori; Kuriyama, Isoko; Azuma, Takeshi; Yoshida, Hiromi; Yoshida, Masaru

2011-02-10

Previously, we reported that vitamin K(3) (VK(3)), but not VK(1) or VK(2) (=MK-4), inhibits the activity of human DNA polymerase γ (pol γ). In this study, we chemically synthesized three intermediate compounds between VK(2) and VK(3), namely MK-3, MK-2 and MK-1, and investigated the inhibitory effects of all five compounds on the activity of mammalian pols. Among these compounds, MK-2 was the strongest inhibitor of mammalian pols α, κ and λ, which belong to the B, Y and X families of pols, respectively; whereas VK(3) was the strongest inhibitor of human pol γ, an A-family pol. MK-2 potently inhibited the activity of all animal species of pol tested, and its inhibitory effect on pol λ activity was the strongest with an IC(50) value of 24.6 μM. However, MK-2 did not affect the activity of plant or prokaryotic pols, or that of other DNA metabolic enzymes such as primase of pol α, RNA polymerase, polynucleotide kinase or deoxyribonuclease I. Because we previously found a positive relationship between pol λ inhibition and anti-inflammatory action, we examined whether these compounds could inhibit inflammatory responses. Among the five compounds tested, MK-2 caused the greatest reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute inflammation in mouse ear. In addition, in a cell culture system using mouse macrophages, MK-2 displayed the strongest suppression of the production of tumor necrosis factor (TNF)-α induced by lipopolysaccharide (LPS). Moreover, MK-2 was found to inhibit the action of nuclear factor (NF)-κB. In an in vivo mouse model of LPS-evoked acute inflammation, intraperitoneal injection of MK-2 in mice led to suppression of TNF-α production in serum. In conclusion, this study has identified VK(2) and VK(3) intermediates, such as MK-2, that are promising anti-inflammatory candidates.

Targeting radiosensitizers to DNA by attachment of an intercalating group: Nitroimidazole-linked phenanthridines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cowan, D.S.; Panicucci, R.; McClelland, R.A.

The nitroimidazole-linked phenanthridine series of compounds (NLP-1, 2, and 3) were synthesized under the assumption that it should be possible to enhance the molar efficiency of 2-nitroimidazoles as hypoxic cell radiosensitizers and cytotoxins by targeting them to their likely site of action, DNA. The targeting group chosen was the phenanthridine moiety, the major component of the classical DNA intercalating compound, ethidium bromide. The sole difference between the compounds is the length of the hydrocarbon chain linking the nitroimidazole to the phenanthridine. The phenanthridine group with a three-carbon side chain, P-1, was also synthesized to allow studies on the effect ofmore » the targeting group by itself. The ability of the compounds to bind to DNA is inversely proportional to their linker chain length with binding constant values ranging from approximately 1 {times} 10(5) mol-1 for NLP-2 to 6 {times} 10(5) mol-1 for NLP-3. The NLP compounds show selective toxicity to hypoxic cells at 37 degrees C at external drug concentrations 10-40 times lower than would be required for untargeted 2-nitroimidazoles such as misonidazole in vitro. Toxicity to both hypoxic and aerobic cells is dependent on the linker chain: the shorter the chain, the greater the toxicity. In addition, the NLP compounds radiosensitize hypoxic cells at external drug concentrations as low as 0.05 mM with almost the full oxygen effect being observed at a concentration of 0.5 mM. These concentrations are 10-100 times lower than would be required for similar radiosensitization using misonidazole. Radiosensitizing ability is independent of linker chain length. The present compounds represent prototypes for further studies of the efficacy and mechanism of action of 2-nitroimidazoles targeted to DNA by linkage to an intercalating group.« less

A Benzothiazole Derivative (5g) Induces DNA Damage And Potent G2/M Arrest In Cancer Cells.

PubMed

Hegde, Mahesh; Vartak, Supriya V; Kavitha, Chandagirikoppal V; Ananda, Hanumappa; Prasanna, Doddakunche S; Gopalakrishnan, Vidya; Choudhary, Bibha; Rangappa, Kanchugarakoppal S; Raghavan, Sathees C

2017-05-31

Chemically synthesized small molecules play important role in anticancer therapy. Several chemical compounds have been reported to damage the DNA, either directly or indirectly slowing down the cancer cell progression by causing a cell cycle arrest. Direct or indirect reactive oxygen species formation causes DNA damage leading to cell cycle arrest and subsequent cell death. Therefore, identification of chemically synthesized compounds with anticancer potential is important. Here we investigate the effect of benzothiazole derivative (5g) for its ability to inhibit cell proliferation in different cancer models. Interestingly, 5g interfered with cell proliferation in both, cell lines and tumor cells leading to significant G2/M arrest. 5g treatment resulted in elevated levels of ROS and subsequently, DNA double-strand breaks (DSBs) explaining observed G2/M arrest. Consistently, we observed deregulation of many cell cycle associated proteins such as CDK1, BCL2 and their phosphorylated form, CyclinB1, CDC25c etc. Besides, 5g treatment led to decreased levels of mitochondrial membrane potential and activation of apoptosis. Interestingly, 5g administration inhibited tumor growth in mice without significant side effects. Thus, our study identifies 5g as a potent biochemical inhibitor to induce G2/M phase arrest of the cell cycle, and demonstrates its anticancer properties both ex vivo and in vivo.

Radiation-Induced Damage to Nucleic Acid Constituents

NASA Astrophysics Data System (ADS)

Kim, Heasook

The objective of this research was to identify the primary free radical species produced by ionizing radiation in DNA. The ultimate goal would be to use these data obtained from model compounds to analyze radiation-induced damage in DNA itself. The different single crystals were studied in detail. The first was the sodium salt of guanosine-3 ^':5^' -cyclic monophosphate (cyclic GMP). The results of studies on crystals irradiated at 4.2^ circK distinguished two species. One of these species exhibited a non-exchangeable proton coupling that was characterized by ENDOR spectroscopy and shown to be sigma proton. The spin density on C8 was deduced from the ENDOR hyperfine coupling tensor and found to be 0.15. The second species also exhibited a non-exchangeable sigma proton coupling and a beta proton coupling. The spin densities on C8 and N9 were deduced from ENDOR measurements to be 0.09 and 0.36. The former is attributed to the oxidation product and the latter to the primary reduction product. These products are respectively the guanine cation and anion. The second single crystal studied was a sodium salt of 2^'-deoxyguanosine -5^'-monophosphate tetrahydrate. The ESR and ENDOR spectra obtained from this crystal after x-irradiation at 4.2^circK were complex and the paramagnetic species were tentatively identified as ionic species. The third DNA model compound studied was thymidine. Single crystal of thymidine were irradiated at 1.6^ circK and at 4.2^circ K. The lower temperature preserved a more primitive stage of the radiation damage process. ENDOR measurements distinguished three paramagnetic species. The most interesting component of the paramagnetic absorption in crystals irradiated at 1.6^circK is attributed to trapped electron. These electrons are stabilized by the electrostatic fields generated by hydroxy dipoles. The hyperfine couplings between the trapped electron and the proton of these polar groups were deduced from ENDOR measurements. The ESR and ENDOR measurements described in this report were carried out DNA model compounds x-irradiated and measured at lower temperatures than reported previously. The experiments have demonstrated that an earlier stage of radiation damage can sometimes be stabilized and characterized in single crystals by maintaining the sample at 1.4 ^circK. (Abstract shortened with permission of author.).

4-Hydroxy-2-pyridones: Discovery and evaluation of a novel class of antibacterial agents targeting DNA synthesis.

PubMed

Arnold, Michael A; Gerasyuto, Aleksey I; Wang, Jiashi; Du, Wu; Gorske, Yi Jin Kim; Arasu, Tamil; Baird, John; Almstead, Neil G; Narasimhan, Jana; Peddi, Srinivasa; Ginzburg, Olya; Lue, Stanley W; Hedrick, Jean; Sheedy, Josephine; Lagaud, Guy; Branstrom, Arthur A; Weetall, Marla; Prasad, J V N Vara; Karp, Gary M

2017-11-15

The continued emergence of bacteria resistant to current standard of care antibiotics presents a rapidly growing threat to public health. New chemical entities (NCEs) to treat these serious infections are desperately needed. Herein we report the discovery, synthesis, SAR and in vivo efficacy of a novel series of 4-hydroxy-2-pyridones exhibiting activity against Gram-negative pathogens. Compound 1c, derived from the N-debenzylation of 1b, preferentially inhibits bacterial DNA synthesis as determined by standard macromolecular synthesis assays. The structural features of the 4-hydroxy-2-pyridone scaffold required for antibacterial activity were explored and compound 6q, identified through further optimization of the series, had an MIC 90 value of 8 μg/mL against a panel of highly resistant strains of E. coli. In a murine septicemia model, compound 6q exhibited a PD 50 of 8 mg/kg in mice infected with a lethal dose of E. coli. This novel series of 4-hydroxy-2-pyridones serves as an excellent starting point for the identification of NCEs treating Gram-negative infections. Copyright © 2017 Elsevier Ltd. All rights reserved.

Maternal exposure to anti-androgenic compounds, vinclozolin, flutamide and procymidone, has no effects on spermatogenesis and DNA methylation in male rats of subsequent generations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inawaka, Kunifumi; Kawabe, Mayumi; DIMS Institute of Medical Science, Inc., Ichinomiya

To verify whether anti-androgens cause transgenerational effects on spermatogenesis and DNA methylation in rats, gravid Crl:CD(SD) female rats (4 or 5/group, gestational day (GD) 0 = day sperm detected) were intraperitoneally treated with anti-androgenic compounds, such as vinclozolin (100 mg/kg/day), procymidone (100 mg/kg/day), or flutamide (10 mg/kg/day), from GD 8 to GD 15. Testes were collected from F1 male pups at postnatal day (PND) 6 for DNA methylation analysis of the region (210 bp including 7 CpG sites) within the lysophospholipase gene by bisulfite DNA sequencing method. F0 and F1 males underwent the sperm analysis (count, motility and morphology), followedmore » by DNA methylation analysis of the sperm. Remaining F1 males were cohabited with untreated-females to obtain F2 male pups for subsequent DNA methylation analysis of the testes at PND 6. These analyses showed no effects on spermatogenesis and fertility in F1 males of any treatment group. DNA methylation status in testes (F1 and F2 pups at PND 6) or sperms (F1 males at 13 weeks old) of the treatment groups were comparable to the control at all observation points, although DNA methylation rates in testes were slightly lower than those in sperm. In F0 males, no abnormalities in the spermatogenesis, fertility and DNA methylation status of sperm were observed. No transgenerational abnormalities of spermatogenesis and DNA methylation status caused by anti-androgenic compounds were observed.« less

Maternal exposure to anti-androgenic compounds, vinclozolin, flutamide and procymidone, has no effects on spermatogenesis and DNA methylation in male rats of subsequent generations.

PubMed

Inawaka, Kunifumi; Kawabe, Mayumi; Takahashi, Satoru; Doi, Yuko; Tomigahara, Yoshitaka; Tarui, Hirokazu; Abe, Jun; Kawamura, Satoshi; Shirai, Tomoyuki

2009-06-01

To verify whether anti-androgens cause transgenerational effects on spermatogenesis and DNA methylation in rats, gravid Crl:CD(SD) female rats (4 or 5/group, gestational day (GD) 0=day sperm detected) were intraperitoneally treated with anti-androgenic compounds, such as vinclozolin (100 mg/kg/day), procymidone (100 mg/kg/day), or flutamide (10 mg/kg/day), from GD 8 to GD 15. Testes were collected from F1 male pups at postnatal day (PND) 6 for DNA methylation analysis of the region (210 bp including 7 CpG sites) within the lysophospholipase gene by bisulfite DNA sequencing method. F0 and F1 males underwent the sperm analysis (count, motility and morphology), followed by DNA methylation analysis of the sperm. Remaining F1 males were cohabited with untreated-females to obtain F2 male pups for subsequent DNA methylation analysis of the testes at PND 6. These analyses showed no effects on spermatogenesis and fertility in F1 males of any treatment group. DNA methylation status in testes (F1 and F2 pups at PND 6) or sperms (F1 males at 13 weeks old) of the treatment groups were comparable to the control at all observation points, although DNA methylation rates in testes were slightly lower than those in sperm. In F0 males, no abnormalities in the spermatogenesis, fertility and DNA methylation status of sperm were observed. No transgenerational abnormalities of spermatogenesis and DNA methylation status caused by anti-androgenic compounds were observed.

Monitoring carcinogen actions on DNA by 32P-postlabeling.

PubMed

Randerath, K; Randerath, E

1990-01-01

Among several recently developed analytical methods, 32P-postlabeling analysis is a highly sensitive method for the detection and measurement of covalent carcinogen-DNA adducts and other DNA modifications. Since the method does not require radioactive carcinogens, it is suitable for DNA of humans exposed to environmental or occupational genotoxicants. The basic procedure entails the enzymatic incorporation of 32P-label into monomeric or dimeric hydrolysis products of DNA, followed by chromatographic mapping and autoradiography of the 32P-labeled digestion products and quantitation by scintillation spectrometry. Microgram amounts of DNA are analyzed; thus the assay is well suited for limited amounts of cells or tissue. Various versions of the assay afford different sensitivities of adduct detection. Under optimal conditions, one aromatic or bulky/hydrophobic adduct in 10(8)-10(10) nucleotides can be detected and measured (corresponding to 0.3-30 amol adduct/microgram DNA or 0.1-10 nmol adduct/mol DNA-P). The assay has been successfully applied to a variety of mutagenic (genotoxic) as well as non-mutagenic carcinogens. In humans, the 32P-postlabeling assay has been applied to DNA specimens from cigarette smokers, iron foundry workers, and coke oven workers. Estimation of total aromatic adduct levels in exposed individuals gave values of 1 adduct in 10(6)-10(8) DNA nucleotides. These values are similar to the total levels of persistent adducts in tissues of animals after exposure to initiating or carcinogenic doses of authentic aromatic genotoxicants. Among the non-mutagenic carcinogens investigated are estrogens, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), choline-devoid diet, carbon tetrachloride, and peroxisome proliferators. In addition, age-dependent DNA modifications (I-compounds) are being detected by 32P-postlabeling in animals that have not been knowingly exposed to mutagens/carcinogens. I-compound profiles and levels are dependent on species, tissue, sex, and diet. Reduced levels of I-compounds have been consistently noted in the target organ of carcinogen-exposed animals and in resulting neoplasms, suggesting that I-compound loss may play a role in carcinogenesis.

DNA interactions of non-chelating tinidazole-based coordination compounds and their structural, redox and cytotoxic properties.

PubMed

Castro-Ramírez, Rodrigo; Ortiz-Pastrana, Naytzé; Caballero, Ana B; Zimmerman, Matthew T; Stadelman, Bradley S; Gaertner, Andrea A E; Brumaghim, Julia L; Korrodi-Gregório, Luís; Pérez-Tomás, Ricardo; Gamez, Patrick; Barba-Behrens, Norah

2018-05-23

Novel tinidazole (tnz) coordination compounds of different geometries were synthesised, whose respective solid-state packing appears to be driven by inter- and intramolecular lone pairπ interactions. The copper(ii) compounds exhibit interesting redox properties originating from both the tnz and the metal ions. These complexes interact with DNA through two distinct ways, namely via electrostatic interactions or/and groove binding, and they can mediate the generation of ROS that damage the biomolecule. Cytotoxic studies revealed an interesting activity of the dinuclear compound [Cu(tnz)2(μ-Cl)Cl]2 7, which is further more efficient towards cancer cells, compared with normal cells.

From in vitro to in vivo: Integration of the virtual cell based assay with physiologically based kinetic modelling.

PubMed

Paini, Alicia; Sala Benito, Jose Vicente; Bessems, Jos; Worth, Andrew P

2017-12-01

Physiologically based kinetic (PBK) models and the virtual cell based assay can be linked to form so called physiologically based dynamic (PBD) models. This study illustrates the development and application of a PBK model for prediction of estragole-induced DNA adduct formation and hepatotoxicity in humans. To address the hepatotoxicity, HepaRG cells were used as a surrogate for liver cells, with cell viability being used as the in vitro toxicological endpoint. Information on DNA adduct formation was taken from the literature. Since estragole induced cell damage is not directly caused by the parent compound, but by a reactive metabolite, information on the metabolic pathway was incorporated into the model. In addition, a user-friendly tool was developed by implementing the PBK/D model into a KNIME workflow. This workflow can be used to perform in vitro to in vivo extrapolation and forward as backward dosimetry in support of chemical risk assessment. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Effects of methyl and inorganic mercury exposure on genome homeostasis and mitochondrial function in Caenorhabditis elegans.

PubMed

Wyatt, Lauren H; Luz, Anthony L; Cao, Xiou; Maurer, Laura L; Blawas, Ashley M; Aballay, Alejandro; Pan, William K Y; Meyer, Joel N

2017-04-01

Mercury toxicity mechanisms have the potential to induce DNA damage and disrupt cellular processes, like mitochondrial function. Proper mitochondrial function is important for cellular bioenergetics and immune signaling and function. Reported impacts of mercury on the nuclear genome (nDNA) are conflicting and inconclusive, and mitochondrial DNA (mtDNA) impacts are relatively unknown. In this study, we assessed genotoxic (mtDNA and nDNA), metabolic, and innate immune impacts of inorganic and organic mercury exposure in Caenorhabditis elegans. Genotoxic outcomes measured included DNA damage, DNA damage repair (nucleotide excision repair, NER; base excision repair, BER), and genomic copy number following MeHg and HgCl 2 exposure alone and in combination with known DNA damage-inducing agents ultraviolet C radiation (UVC) and hydrogen peroxide (H 2 O 2 ), which cause bulky DNA lesions and oxidative DNA damage, respectively. Following exposure to both MeHg and HgCl 2 , low-level DNA damage (∼0.25 lesions/10kb mtDNA and nDNA) was observed. Unexpectedly, a higher MeHg concentration reduced damage in both genomes compared to controls. However, this observation was likely the result of developmental delay. In co-exposure treatments, both mercury compounds increased initial DNA damage (mtDNA and nDNA) in combination with H 2 O 2 exposure, but had no impact in combination with UVC exposure. Mercury exposure both increased and decreased DNA damage removal via BER. DNA repair after H 2 O 2 exposure in mercury-exposed nematodes resulted in damage levels lower than measured in controls. Impacts to NER were not detected. mtDNA copy number was significantly decreased in the MeHg-UVC and MeHg-H 2 O 2 co-exposure treatments. Mercury exposure had metabolic impacts (steady-state ATP levels) that differed between the compounds; HgCl 2 exposure decreased these levels, while MeHg slightly increased levels or had no impact. Both mercury species reduced mRNA levels for immune signaling-related genes, but had mild or no effects on survival on pathogenic bacteria. Overall, mercury exposure disrupted mitochondrial endpoints in a mercury-compound dependent fashion. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of methyl and inorganic mercury exposure on genome homeostasis and mitochondrial function in Caenorhabditis elegans

PubMed Central

Wyatt, Lauren H.; Luz, Anthony L.; Cao, Xiou; Maurer, Laura L.; Blawas, Ashley M.; Aballay, Alejandro; Pan, William K.; Meyer, Joel N.

2017-01-01

Mercury toxicity mechanisms have the potential to induce DNA damage and disrupt cellular processes, like mitochondrial function. Proper mitochondrial function is important for cellular bioenergetics and immune signaling and function. Impacts of mercury on the nuclear genome (nDNA) are conflicting and inconclusive, and mitochondrial DNA (mtDNA) impacts are relatively unknown. In this study, we assessed genotoxic (mtDNA and nDNA), metabolic, and innate immune impacts of inorganic and organic mercury exposure in Caenorhabditis elegans. Genotoxic outcomes measured included DNA damage, DNA damage repair (nucleotide excision repair, NER; base excision repair, BER), and genomic copy number following MeHg and HgCl2 exposure alone and in combination with known DNA damage-inducing agents ultraviolet C radiation (UVC) and hydrogen peroxide (H2O2), which cause bulky DNA lesions and oxidative DNA damage, respectively. Following exposure to both MeHg and HgCl2, low-level DNA damage (~0.25 lesions/10 kb mtDNA and nDNA) was observed. Unexpectedly, a higher MeHg concentration reduced damage in both genomes compared to controls. However, this observation was likely the result of developmental delay. In co-exposure treatments, both mercury compounds increased initial DNA damage (mtDNA and nDNA) in combination with H2O2 exposure, but had no impact in combination with UVC exposure. Mercury exposure both increased and decreased DNA damage removal via BER. DNA repair after H2O2 exposure in mercury-exposed nematodes resulted in damage levels lower than measured in controls. Impacts to NER were not detected. mtDNA copy number was significantly decreased in the MeHg-UVC and MeHg-H2O2 co-exposure treatments. Mercury exposure had metabolic impacts (steady-state ATP levels) that differed between the compounds; HgCl2 exposure decreased these levels, while MeHg slightly increased levels or had no impact. Both mercury species reduced mRNA levels for immune signaling-related genes, but had mild or no effects on survival on pathogenic bacteria. Overall, mercury exposure disrupted mitochondrial endpoints in a mercury-compound dependent fashion. PMID:28242054

Repeated oral dosing of TAS-102 confers high trifluridine incorporation into DNA and sustained antitumor activity in mouse models

PubMed Central

TANAKA, NOZOMU; SAKAMOTO, KAZUKI; OKABE, HIROYUKI; FUJIOKA, AKIO; YAMAMURA, KEISUKE; NAKAGAWA, FUMIO; NAGASE, HIDEKI; YOKOGAWA, TATSUSHI; OGUCHI, KEI; ISHIDA, KEIJI; OSADA, AKIKO; KAZUNO, HIROMI; YAMADA, YUKARI; MATSUO, KENICHI

2014-01-01

TAS-102 is a novel oral nucleoside antitumor agent containing trifluridine (FTD) and tipiracil hydrochloride (TPI). The compound improves overall survival of colorectal cancer (CRC) patients who are insensitive to standard chemotherapies. FTD possesses direct antitumor activity since it inhibits thymidylate synthase (TS) and is itself incorporated into DNA. However, the precise mechanisms underlying the incorporation into DNA and the inhibition of TS remain unclear. We found that FTD-dependent inhibition of TS was similar to that elicited by fluorodeoxyuridine (FdUrd), another clinically used nucleoside analog. However, washout experiments revealed that FTD-dependent inhibition of TS declined rapidly, whereas FdUrd activity persisted. The incorporation of FTD into DNA was significantly higher than that of other antitumor nucleosides. Additionally, orally administered FTD had increased antitumor activity and was incorporated into DNA more effectively than continuously infused FTD. When TAS-102 was administered, FTD gradually accumulated in tumor cell DNA, in a TPI-independent manner, and significantly delayed tumor growth and prolonged survival, compared to treatment with 5-FU derivatives. TAS-102 reduced the Ki-67-positive cell fraction, and swollen nuclei were observed in treated tumor tissue. The amount of FTD incorporation in DNA and the antitumor activity of TAS-102 in xenograft models were positively and significantly correlated. These results suggest that TAS-102 exerts its antitumor activity predominantly due to its DNA incorporation, rather than as a result of TS inhibition. The persistence of FTD in the DNA of tumor cells treated with TAS-102 may underlie its ability to prolong survival in cancer patients. PMID:25230742

Identification of ATR-Chk1 pathway inhibitors that selectively target p53-deficient cells without directly suppressing ATR catalytic activity

PubMed Central

Kawasumi, Masaoki; Bradner, James E.; Tolliday, Nicola; Thibodeau, Renee; Sloan, Heather; Brummond, Kay M.; Nghiem, Paul

2014-01-01

Resistance to DNA-damaging chemotherapy is a barrier to effective treatment that appears to be augmented by p53 functional deficiency in many cancers. In p53-deficient cells where the G1/S checkpoint is compromised, cell viability after DNA damage relies upon intact intra-S and G2/M checkpoints mediated by the ATR and Chk1 kinases. Thus, a logical rationale to sensitize p53-deficient cancers to DNA-damaging chemotherapy is through the use of ATP-competitive inhibitors of ATR or Chk1. To discover small molecules that may act on uncharacterized components of the ATR pathway, we performed a phenotype-based screen of 9,195 compounds for their ability to inhibit hydroxyurea-induced phosphorylation of Ser345 on Chk1, known to be a critical ATR substrate. This effort led to the identification of four small-molecule compounds, three of which were derived from known bioactive library (anthothecol, dihydrocelastryl, and erysolin) and one of which was a novel synthetic compound termed MARPIN. These compounds all inhibited ATR-selective phosphorylation and sensitized p53-deficient cancer cells to DNA-damaging agents in vitro and in vivo. Notably, these compounds did not inhibit ATR catalytic activity in vitro, unlike typical ATP-competitive inhibitors, but acted in a mechanistically distinct manner to disable ATR-Chk1 function. Our results highlight a set of novel molecular probes to further elucidate druggable mechanisms to improve cancer therapeutic responses produced by DNA-damaging drugs. PMID:25336189

Identification of ATR-Chk1 pathway inhibitors that selectively target p53-deficient cells without directly suppressing ATR catalytic activity.

PubMed

Kawasumi, Masaoki; Bradner, James E; Tolliday, Nicola; Thibodeau, Renee; Sloan, Heather; Brummond, Kay M; Nghiem, Paul

2014-12-15

Resistance to DNA-damaging chemotherapy is a barrier to effective treatment that appears to be augmented by p53 functional deficiency in many cancers. In p53-deficient cells in which the G1-S checkpoint is compromised, cell viability after DNA damage relies upon intact intra-S and G2-M checkpoints mediated by the ATR (ataxia telangiectasia and Rad3 related) and Chk1 kinases. Thus, a logical rationale to sensitize p53-deficient cancers to DNA-damaging chemotherapy is through the use of ATP-competitive inhibitors of ATR or Chk1. To discover small molecules that may act on uncharacterized components of the ATR pathway, we performed a phenotype-based screen of 9,195 compounds for their ability to inhibit hydroxyurea-induced phosphorylation of Ser345 on Chk1, known to be a critical ATR substrate. This effort led to the identification of four small-molecule compounds, three of which were derived from known bioactive library (anthothecol, dihydrocelastryl, and erysolin) and one of which was a novel synthetic compound termed MARPIN. These compounds all inhibited ATR-selective phosphorylation and sensitized p53-deficient cancer cells to DNA-damaging agents in vitro and in vivo. Notably, these compounds did not inhibit ATR catalytic activity in vitro, unlike typical ATP-competitive inhibitors, but acted in a mechanistically distinct manner to disable ATR-Chk1 function. Our results highlight a set of novel molecular probes to further elucidate druggable mechanisms to improve cancer therapeutic responses produced by DNA-damaging drugs. ©2014 American Association for Cancer Research.

SOD activity and DNA binding properties of a new symmetric porphyrin Schiff base ligand and its metal complexes.

PubMed

Çay, Sevim; Köse, Muhammet; Tümer, Ferhan; Gölcü, Ayşegül; Tümer, Mehmet

2015-12-05

4-Methoxy-2,6-bis(hydroxymethyl)phenol (1) was prepared from the reaction of 4-methoxyphenol and formaldehyde. The compound (1) was then oxidized to the 4-methoxy-2,6-diformylphenol (2) compound. Molecular structure of compound (2) was determined by X-ray diffraction method. A new symmetric porphyrin Schiff base ligand 4-methoxy-2,6-bis[5-(4-iminophenyl)-10,15,20-triphenylporphyrin]phenol (L) was prepared from the reaction of the 5-(4-aminophenyl)-10,15,20-triphenylporphyrin (TTP-NH2) and the compound (2) in the toluene solution. The metal complexes (Cu(II), Fe(III), Mn(III), Pt(II) and Zn(II)) of the ligand (L) were synthesized and characterized by the spectroscopic and analytical methods. The DNA (fish sperm FSdsDNA) binding studies of the ligand and its complexes were performed using UV-vis spectroscopy. Additionally, superoxide dismutase activities of the porphyrin Schiff base metal complexes were investigated. Additionally, electrochemical, photoluminescence and thermal properties of the compounds were investigated. Copyright © 2015 Elsevier B.V. All rights reserved.

New pyridinium-based fluorescent dyes: A comparison of symmetry and side-group effects on G-Quadruplex DNA binding selectivity and application in live cell imaging.

PubMed

Lu, Yu-Jing; Hu, Dong-Ping; Zhang, Kun; Wong, Wing-Leung; Chow, Cheuk-Fai

2016-07-15

A series of C1-, C2-and C3-symmetric pyridinium conjugates with different styrene-like side groups were synthesized and were utilized as G-quadruplex selective fluorescent probes. The new compounds were well-characterized. Their selectivity, sensitivity, and stability towards G-quadruplex were studied by fluorescence titration, native PAGE experiments, FRET and circular dichroism (CD) analyses. These new compounds investigated in the fluorescence assays were preferentially bound with G-quadruplex DNA compared with other type of nucleic acids and it is fascinating to realize the effects of molecular symmetry and associated side groups showing unexpectedly great influence on the fluorescent signal enhancement for the discrimination of G-quadruplexes DNA from other nucleic acids. This may correlate with the pocket symmetry and shape of the G-quadruplex DNA inherently. Among the compounds, a C2-symmetric dye (2,6-bis-((E)-2-(1H-indol-3-yl)-vinyl)-1-methylpyridin-1-ium iodide) with indolyl-groups substituted was screened out from the series giving the best selectivity and sensitivity towards G-quadruplexes DNA, particularly telo21, due to its high equilibrium binding constant (K=2.17×10(5)M(-1)). In addition, the limit of detection (LOD) of the dye to determine telo21 DNA in bioassays was found as low as 33nM. The results of the study give insight and certain crucial factors, such as molecular symmetry and the associated side groups, on developing of effective fluorescent dyes for G-quadruplex DNA applications including G-quadruplex structure stabilization, biosensing and clinical applications. The compound was also demonstrated as a very selective G-quadruplex fluorescent agent for living cell staining and imaging. Copyright © 2016 Elsevier B.V. All rights reserved.

3-Acetyl-8-methoxy-2[H]-chromen-2-one derived Schiff bases as potent antiproliferative agents: Insight into the influence of 4(N)-substituents on the in vitro biological activity

NASA Astrophysics Data System (ADS)

Kalaiarasi, G.; Rex Jeya Rajkumar, S.; Aswini, G.; Dharani, S.; Fronczek, Frank R.; Prabhakaran, R.

2018-07-01

A series of 3-acetyl-8-methoxycoumarin appended thiosemicarbazones (1-4) was prepared from the reaction of 3-acetyl-8-methoxycoumarin with 4(N)-substituted thiosemicarbazides in a view of ascertaining their biological properties with the change of N-terminal substitution in the thiosemicarbazide moiety. Comprehensive characterization was brought about by various spectral and analytical methods. The molecular structures of all the compounds were determined by single crystal X-ray diffraction analysis. Binding studies with Calf thymus DNA (CT-DNA) and proteins such as Bovine Serum Albumin (BSA) and Human Serum Albumin (HSA) indicated an intercalative mode of binding with DNA and static quenching mechanism with proteins. The compounds cleaved plasmid DNA (pBR322) and acted well as free radical scavengers. A good spectrum of antimicrobial activity was observed against four bacterial and five fungal pathogens. The compounds exhibited profound antiproliferative activity on MCF-7 (human breast cancer) and A549 (human lung carcinoma) cell lines. Assay on human normal keratinocyte cell line HaCaT showed that the compounds were non-toxic to normal cells.

Synthesis, characterization, DNA-Binding, enzyme inhibition and antioxidant studies of new N-methylated derivatives of pyridinium amine

NASA Astrophysics Data System (ADS)

Zafar, Muhammad Naveed; Perveen, Fouzia; Nazar, Muhammad Faizan; Mughal, Ehsan Ullah; Rafique, Humera; Tahir, Muhammad Nawaz; Akbar, Muhammad Sharif; Zahra, Sabeen

2017-06-01

A series of novel N-methylated derivatives of pyridinium amine, [L1][Tf]-[L5][Tf], were synthesized and characterized by FTIR, NMR, MS and XRD analyses. Preliminary biological screening of these compounds including antioxidant, enzyme inhibition and DNA (salmon sperm) interaction studies were also carried out. The targeted compounds were synthesized by a melt reaction between 4-chloro-N-methyl pyridinium triflate and corresponding amines (1-naphthyl amine, o-ansidine, 2-nitroaniline, p-ansidine and cyclohexyl amine) at temperature of 230 °C. The DPPH radical antioxidant scavenging activities of these compounds at maximum concentration of 50 μg/mL were observed in the range of 60-70%. Acetylcholine esterase (AChE) and Butylcholine esterase (BChE) inhibitory activities of synthesized compounds at 2 mM concentration were also measured to be at maximum of 79 and 71% respectively. The spectral behavior of ligand-DNA obtained from photo-luminescent measurements showed that all ligands bind with DNA via non-covalent interactions. The binding constant values were determined by UV-visible and fluorescence spectroscopy and were quite close to that obtained from molecular docking studies.

Toxicity, mutagenicity and transport in Saccharomyces cerevisiae of three popular DNA intercalating fluorescent dyes.

PubMed

Sayas, Enric; García-López, Federico; Serrano, Ramón

2015-09-01

We have compared the toxicity, mutagenicity and transport in Saccharomyces cerevisiae of three DNA-intercalating fluorescent dyes widely used to stain DNA in gels. Safety data about ethidium bromide (EtBr) are contradictory, and two compounds of undisclosed structure (Redsafe and Gelred) have been proposed as safe alternatives. Our results indicate that all three compounds inhibit yeast growth, with Gelred being the most inhibitory and also the only one causing cell death. EtBr and Gelred, but not Redsafe, induce massive formation of petite (non-respiratory) mutants, but only EtBr induces massive loss of mitochondrial DNA. All three compounds increase reversion of a chromosomal point mutation (lys2-801(amber) ), with Gelred being the most mutagenic and Redsafe the least. These dyes are all cationic and are probably taken by cells through non-selective cation channels. We could measure the glucose-energized transport of EtBr and Gelred inside the cells, while uptake of Redsafe was below our detection limit. We conclude that although all three compounds are toxic and mutagenic in the yeast system, Redsafe is the safest for yeast, probably because of very limited uptake by these cells. Copyright © 2015 John Wiley & Sons, Ltd.

Identification of active compounds from Aurantii Immatri Pericarpium attenuating brain injury in a rat model of ischemia-reperfusion.

PubMed

Yang, Eun-Ju; Lim, Sun Ha; Song, Kyung-Sik; Han, Hyung Soo; Lee, Jongwon

2013-05-01

Ischemic stroke is caused by brain injury due to prolonged ischemia by occlusion of cerebral arteries. In this study, we isolated active compounds from an ethanol extract of Aurantii Immatri Pericarpium (HY5356). We first showed by DNA fragmentation assay that HY5356 improved human hepatocellular carcinoma cells (HepG2) under hypoxic conditions by inhibiting apoptosis. When HY5356 was fractionated with dichloromethane (MC), ethyl acetate (EA) and n-butanol (BU), the MC fraction improved cell viability at the lowest concentration (100 μg/ml). Intraperitoneal injection of HY5356 (200 mg/kg) or the MC fraction (200 mg/kg) to rats prior to occlusion attenuated brain injury significantly in a rat model of ischemia-reperfusion. Adopting cell viability under hypoxic conditions as an activity screening system, we isolated nobiletin and tangeretin as active compounds. The results suggest that intake of Aurantii Immatri Pericarpium containing nobiletin and tangeretin as active compounds might be beneficial for preventing ischemic stroke. Copyright © 2012 Elsevier Ltd. All rights reserved.

DNA damage protection against free radicals of two antioxidant neolignan glucosides from sugarcane molasses.

PubMed

Asikin, Yonathan; Takahashi, Makoto; Mizu, Masami; Takara, Kensaku; Oku, Hirosuke; Wada, Koji

2016-03-15

Sugarcane molasses is a potential by-product of the sugarcane manufacturing industry that is rich in antioxidant materials. The present study aimed to obtain antioxidative compounds from sugarcane molasses and to evaluate their ability to protect DNA from oxidative damage. Two neolignan glucosides were isolated from sugarcane molasses using bioassay and UV spectra monitoring-guided fractionation. The compounds were elucidated as (7R,8S)-dehydrodiconiferyl alcohol-4-O-β-d-glucoside (1) and (7S,8R)-simulanol-9'-O-β-d-glucoside (2). Neolignan glucoside 2 protected against DNA damage caused by free radicals more effectively than did neolignan glucoside 1 (13.62 and 9.08 µmol L(-1) for peroxyl and hydroxyl radicals, respectively, compared to 48.07 and 14.42 µmol L(-1) ). Additionally, neolignan glucoside 2 exhibited superior DNA protection against free radicals compared with various known antioxidative compounds, including p-coumaric acid, ferulic acid, vanillic acid and epigallocatechin gallate. The isolated neolignan glucosides from sugarcane molasses are able to protect DNA from oxidative damage caused by free radicals. This is the first identification of these two compounds in sugarcane molasses. The sugarcane molasses can therefore be used as potential nutraceutical preventative agents, and the findings may foster the utilization of this by-product as a bioresource-based product. © 2015 Society of Chemical Industry. Copyright © 2015 Society of Chemical Industry.

Screening and investigation of triplex DNA binders from Stephania tetrandra S. Moore by a combination of peak area-fading UHPLC with orbitrap MS and optical spectroscopies.

PubMed

Yang, Hongmei; Wang, Yihan; Yu, Wenjing; Shi, Lei; Wang, Hongfeng; Su, Rui; Chen, Changbao; Liu, Shuying

2018-05-15

The identification and screening of triplex DNA binders are important because these compounds, in many cases, are potential anticancer agents as well as promising drug candidates. Therefore, the ability to screen for these compounds in a high-throughput mode could dramatically improve the drug screening process. A method involving a combination of 96-well plate format and peak area-fading ultra high performance liquid chromatography coupled with Orbitrap mass spectrometry was employed for screening bioactive compounds binding to the triplex DNA from the extracts of Stephania tetrandra S. Moore. Two compounds were screened out and identified as fangchinoline and tetrandrine, based on the comparison of retention time and MS 2 data with those of standards. The binding mechanisms of fangchinoline and tetrandrine at the molecular level were explored using MS 2 , fluorescence spectroscopy, ultraviolet-visible spectroscopy, and circular dichroism. Collision-induced dissociation experiments showed that the complexes with fangchinoline and tetrandrine were dissociated by ligand elimination. According to these measurements, an intercalating binding is the most appropriate binding mode of these two alkaloids to the triplex DNA. The current work provides not only deep insight into alkaloid-triplex DNA complexes but also useful guidelines for the design of efficient anticancer agents. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Discovery of 2-aminothiazolyl berberine derivatives as effectively antibacterial agents toward clinically drug-resistant Gram-negative Acinetobacter baumanii.

PubMed

Gao, Wei-Wei; Gopala, Lavanya; Bheemanaboina, Rammohan R Yadav; Zhang, Guo-Biao; Li, Shuo; Zhou, Cheng-He

2018-02-25

Aminothiazolyl berberine derivatives as potentially antimicrobial agents were designed and synthesized in an effort to overcome drug resistance. The antimicrobial assay revealed that some target compounds exhibited significantly inhibitory efficiencies toward bacteria and fungi including drug-resistant pathogens, and the aminothiazole and Schiff base moieties were helpful structural fragments for aqueous solubility and antibacterial activity. Especially, aminothiazolyl 9-hexyl berberine 9c and 2,4-dichlorobenzyl derivative 18a exhibited good activities (MIC = 2 nmol/mL) against clinically drug-resistant Gram-negative Acinetobacter baumanii with low cytotoxicity to hepatocyte LO2 cells, rapidly bactericidal effects and quite slow development of bacterial resistance toward A. baumanii. Molecular modeling indicated that compounds 9c and 18a could bind with GLY-102, ARG-136 and/or ALA-100 residues of DNA gyrase through hydrogen bonds. It was found that compounds 9c and 18a were able to disturb the drug-resistant A. baumanii membrane effectively, and molecule 9c could not only intercalate but also cleave bacterial DNA isolated from resistant A. baumanii, which might be the preliminary antibacterial action mechanism of inhibiting the growth of A. baumanii strain. In particular, the combination use of compound 9c with norfloxacin could enhance the antibacterial activity, broaden antibacterial spectrum and overcome the drug resistance. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Compounds that target host cell proteins prevent varicella-zoster virus replication in culture, ex vivo, and in SCID-Hu mice.

PubMed

Rowe, Jenny; Greenblatt, Rebecca J; Liu, Dongmei; Moffat, Jennifer F

2010-06-01

Varicella-zoster virus (VZV) replicates in quiescent T cells, neurons, and skin cells. In cultured fibroblasts (HFFs), VZV induces host cyclin expression and cyclin-dependent kinase (CDK) activity without causing cell cycle progression. CDK1/cyclin B1 phosphorylates the major viral transactivator, and the CDK inhibitor roscovitine prevents VZV mRNA transcription. We investigated the antiviral effects of additional compounds that target CDKs or other cell cycle enzymes in culture, ex vivo, and in vivo. Cytotoxicity and cell growth arrest doses were determined by Neutral Red assay. Antiviral effects were evaluated in HFFs by plaque assay, genome copy number, and bioluminescence. Positive controls were acyclovir (400 microM) and phosphonoacetic acid (PAA, 1 mM). Test compounds were roscovitine, aloisine A, and purvalanol A (CDK inhibitors), aphidicolin (inhibits human and herpesvirus DNA polymerase), l-mimosine (indirectly inhibits human DNA polymerase), and DRB (inhibits casein kinase 2). All had antiviral effects below the concentrations required for cell growth arrest. Compounds were tested in skin organ culture at EC(99) doses; all prevented VZV replication in skin, except for aloisine A and purvalanol A. In SCID mice with skin xenografts, roscovitine (0.7 mg/kg/day) was as effective as PAA (36 mg/kg/day). The screening systems described here are useful models for evaluating novel antiviral drugs for VZV. Copyright 2010 Elsevier B.V. All rights reserved.

Novel Platinum (Pt)-Vandetanib Hybrid Compounds: Design, Synthesis and Investigation of Anti-cancer Activity and Mechanism of Action

NASA Astrophysics Data System (ADS)

Fei, Rong

Purpose: Lung cancer is one of the most common cancers and non-small cell lung cancer (NSCLC) accounts for 80-85% of lung cancers. 70% of individuals with NSCLC harboring somatic mutations in exons of the epidermal growth factor receptor (EGFR) gene that encode tyrosine kinase domain. EGFR tyrosine kinase inhibitors (TKIs) are promising molecular targeted therapy for NSCLC with sensitizing EGFR mutations. However, secondary mutation of EGFR after treatment of TKIs develops resistance. Vandetanib is introduced to overcome erlotinib resistance as a multi-targeted TKI. However, its anticancer effect is still compromised by EGFR T790M mutation. Therefore, new molecular anticancer strategies are necessarily needed. In this study, vandetanib is incorporated with Pt-based anticancer agents as hybrid compounds, aiming to circumvent TKI resistance. Furthermore, hybrid compounds are investigated in cisplatin resistant problem to expect to overcome resistance by introduction of vandetanib. Methods: Three novel Pt-vandetanib hybrid compounds were synthesized and its physicochemical properties were characterized. Anticancer activity and cytotoxicity were evaluated by sulforhodamine B assay and lactate dehydrogenase release. Docking simulation was performed to investigate the interaction of compounds with EGFR harboring different mutations. Inhibition efficacy of hybrids to kinases was evaluated by kinase inhibition profiling service and cell-free kinase inhibition assay. Mechanistic studies on cytotoxicity activity of the hybrid compounds were carried out. DNA damage response of hybrid compounds was further investigated in KB cells. The cytotoxicity of hybrids was tested in cisplatin resistant KB CP20 cells. Mechanistic of anticancer activity was studied to test inhibition on oncoprotein CIP2Aand DNA damage. Results: Platinum-vandetanib hybrid compounds were synthesized and test to be stable under extracellular condition. Hybrids reacted with 5'-GMP2- and glutathione, and both of them formed mono-dentate adducts. Moreover, hybrid compounds exhibited low toxicity in human normal kidney cells. Compounds maintained the inhibition selectivity towards EGFR from the results of kinase inhibition profiling and cell-free kinase inhibition assay. Hybrids formed strong H-bond at D800 on EGFR. Pt-vandetanib hybrids were highly effective against HCC827 cells harboring sensitizing EGFR mutation. Importantly, relative resistant rate of hybrids were much smaller than vandetanib in H1975 cells. Western blot analysis results revealed that the hybrid compounds could efficiently inhibit EGFR phosphorylation in a dose dependent manner in HCC827. While, inhibition of p-EGFR was not as good as the original TKI in H1975 cells. However, the hybrid compounds induced DNA damage and caused apoptosis of the NSCLC cells. Both of the two pathways were contributed to cancer cell death and overcome vandetanib resistance. Pt-vandetanib hybrids showed little resistance in cisplatin resistant cell line KB-CP20. Drug accumulation evaluation revealed that cisplatin accumulation in CP20 cells decreased to one eighth of that in the parental KB3.1 cells. While hybrids maintained similar drug accumulation extent in both cells lines. Mechanistic study showed that hybrid compounds could induce DNA damage and cause apoptosis, whereas cisplatin failed to cause DNA damage in KB-CP20 cells. Oncoprotein CIP2A was overexpressed in CP20 cell and was ascribed to CDDP resistance. The hybrids inhibited CIP2A expression and downstream AKT phosphorylation. It was hypothesized that downregulation of CIP2A contributed to circumvention platinum resistance. Conclusion: Novel Pt-vandetanib hybrid compounds were able to overcome vandetanib resistance in H1975 cells by maintaining inhibition to the EGFR and inducing DNA damage and apoptosis. Moreover, Pt-vandetanib hybrid compounds behaved low toxicity and overcome cisplatin resistance by being "non-substrate" to efflux transporter and successfully causing DNA damage. Hybrids were found to downregulate oncogene CIP2A expression level. The novel Pt-vandetanib hybrid compounds are potent for further development.

Polarizable Force Field for DNA Based on the Classical Drude Oscillator: I. Refinement Using Quantum Mechanical Base Stacking and Conformational Energetics.

PubMed

Lemkul, Justin A; MacKerell, Alexander D

2017-05-09

Empirical force fields seek to relate the configuration of a set of atoms to its energy, thus yielding the forces governing its dynamics, using classical physics rather than more expensive quantum mechanical calculations that are computationally intractable for large systems. Most force fields used to simulate biomolecular systems use fixed atomic partial charges, neglecting the influence of electronic polarization, instead making use of a mean-field approximation that may not be transferable across environments. Recent hardware and software developments make polarizable simulations feasible, and to this end, polarizable force fields represent the next generation of molecular dynamics simulation technology. In this work, we describe the refinement of a polarizable force field for DNA based on the classical Drude oscillator model by targeting quantum mechanical interaction energies and conformational energy profiles of model compounds necessary to build a complete DNA force field. The parametrization strategy employed in the present work seeks to correct weak base stacking in A- and B-DNA and the unwinding of Z-DNA observed in the previous version of the force field, called Drude-2013. Refinement of base nonbonded terms and reparametrization of dihedral terms in the glycosidic linkage, deoxyribofuranose rings, and important backbone torsions resulted in improved agreement with quantum mechanical potential energy surfaces. Notably, we expand on previous efforts by explicitly including Z-DNA conformational energetics in the refinement.

Synthesis, spectroscopic characterization, crystal structure, DNA interaction study and invitro biological screenings of 4-(5-chloro-2-hydroxyphenylamino)-4-oxobut-2-enoic acid

NASA Astrophysics Data System (ADS)

Sirajuddin, Muhammad; Nooruddin; Ali, Saqib; McKee, Vickie; Khan, Shahan Zeb; Malook, Khan

2015-01-01

The titled compound, 4-(5-chloro-2-hydroxyphenylamino)-4-oxobut-2-enoic acid was synthesized and characterized by various techniques like elemental analyses, FT-IR, NMR (1H, and 13C) and single crystal X-ray structural analysis. The appearance of the OH peak of the carboxylic acid in the FT-IR and NMR spectra conform the formation of the compound. A good agreement was found between the calculated values of C, H, N and found values in elemental analysis that show the purity of the compound. Protons H2 and H3 are in cis conformation with each other as conformed both from 1H NMR as well as from single crystal X-ray analysis. The molecular structure of the title compound, C10H10NO3Cl, is stabilized by short intramolecular Osbnd H- - -O hydrogen bonds within the molecule. In the crystal structure, intermolecular Nsbnd H- - -O hydrogen bonds link molecules into zigzag chains resulting in a dendrimer like structure. The title compound was screened for biological activities like interaction with DNA, cytotoxicity, antitumor and antioxidant activities. DNA interaction study reveals that the binding mode of interaction of the compound with SS-DNA is intercalative as it results in hypochromism along with significant red shift of 5 nm. It was also found to be effective antioxidant of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and show almost comparable antioxidant activity to that of the standard and known antioxidant, ascorbic acid, at higher concentration. The antitumor activity data of the compound shows that it can be used as potent antitumor agent.

Vanadium distribution in rats and DNA cleavage by vanadyl complex: implication for vanadium toxicity and biological effects.

PubMed Central

Sakurai, H

1994-01-01

Vanadium ion is toxic to animals. However, vanadium is also an agent used for chemoprotection against cancers in animals. To understand both the toxic and beneficial effects we studied vanadium distribution in rats. Accumulation of vanadium in the liver nuclei of rats given low doses of compounds in the +4 or +5 oxidation state was greater than in the liver nuclei of rats given high doses of vanadium compounds or the vanadate (+5 oxidation state) compound. Vanadium was incorporated exclusively in the vanadyl (+4 oxidation state) form. We also investigated the reactions of vanadyl ion and found that incubation of DNA with vanadyl ion and hydrogen peroxide (H2O2) led to intense DNA cleavage. ESR spin trapping demonstrated that hydroxyl radicals are generated during the reactions of vanadyl ion and H2O2. Thus, we propose that the mechanism for vanadium-dependent toxicity and antineoplastic action is due to DNA cleavage by hydroxyl radicals generated in living systems. PMID:7843133

Application of encoded library technology (ELT) to a protein-protein interaction target: discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists.

PubMed

Kollmann, Christopher S; Bai, Xiaopeng; Tsai, Ching-Hsuan; Yang, Hongfang; Lind, Kenneth E; Skinner, Steven R; Zhu, Zhengrong; Israel, David I; Cuozzo, John W; Morgan, Barry A; Yuki, Koichi; Xie, Can; Springer, Timothy A; Shimaoka, Motomu; Evindar, Ghotas

2014-04-01

The inhibition of protein-protein interactions remains a challenge for traditional small molecule drug discovery. Here we describe the use of DNA-encoded library technology for the discovery of small molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen 1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of 4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function-associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the target protein. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparison of DNA-Reactive Metabolites from Nitrosamine and Styrene Using Voltammetric DNA/Microsomes Sensors

PubMed Central

Krishnan, Sadagopan; Bajrami, Besnik; Mani, Vigneshwaran; Pan, Shenmin; Rusling, James F.

2012-01-01

Voltammetric sensors made with films of polyions, double-stranded DNA and liver microsomes adsorbed layer-by-layer onto pyrolytic graphite electrodes were evaluated for reactive metabolite screening. This approach features simple, inexpensive screening without enzyme purification for applications in drug or environmental chemical development. Cytochrome P450 enzymes (CYPs) in the liver microsomes were activated by an NADPH regenerating system or by electrolysis to metabolize model carcinogenic compounds nitrosamine and styrene. Reactive metabolites formed in the films were trapped as adducts with nucleobases on DNA. The DNA damage was detected by square-wave voltammetry (SWV) using Ru(bpy)32+ as a DNA-oxidation catalyst. These sensors showed a larger rate of increase in signal vs. reaction time for a highly toxic nitrosamine than for the moderately toxic styrene due to more rapid reactive metabolite-DNA adduct formation. Results were consistent with reported in vivo TD50 data for the formation of liver tumors in rats. Analogous polyion/ liver microsome films prepared on 500 nm silica nanoparticles (nanoreactors) and reacted with nitrosamine or styrene, provided LC-MS or GC analyses of metabolite formation rates that correlated well with sensor response. PMID:23100998

Pulsatilla saponin A, an active molecule from Pulsatilla chinensis, induces cancer cell death and inhibits tumor growth in mouse xenograft models.

PubMed

Liu, Qiang; Chen, Weichang; Jiao, Yang; Hou, Jianquan; Wu, Qingyu; Liu, Yanli; Qi, Xiaofei

2014-05-15

Many natural compounds possess antitumor growth activities. Pulsatilla chinensis is an herb used in traditional Chinese medicine to treat infectious diseases. More recently, extracts from P chinensis have been shown to contain antitumor activities. In this study, we isolated Pulsatilla saponin A as an active compound from P chinensis extracts and tested its anticancer activity in vitro and in vivo. In cell culture, Pulsatilla saponin A significantly inhibited the growth of human hepatocellular carcinoma SMCC-7721 cells and pancreatic BXPC3 and SW1990 cancer cells. Similar inhibitory activities were observed when the compound was tested in mouse xenograft tumor models using human hepatocellular carcinoma Bel-7402 and pancreatic cancer SW1990 cells. In Comet assay and flow cytometric analysis of cell cycle distribution and annexin V expression, DNA damage, G2 arrest, and apoptosis were identified in Pulsatilla saponin A-treated cancer cells. Based on the results of Western blotting, p53 and cyclin B protein levels were higher, whereas Bcl-2 protein levels were lower in Pulsatilla saponin A-treated cancer cells than in vehicle-treated cells. Pulsatilla saponin A may exert its antitumor effect by inducing DNA damage and causing G2 arrest and apoptosis in cancer cells. Pulsatilla saponin A and its derivatives may be developed as a new class of anticancer agents. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

Specific and highly efficient condensation of GC and IC DNA by polyaza pyridinophane derivatives.

PubMed

Stojković, Marijana Radić; Gonzalez-Garcia, Jorge; Šupljika, Filip; Galiana-Rosello, Cristina; Guijarro, Lluis; Gazze, Salvatore A; Francis, Lewis W; Piantanida, Ivo; Garcia-Espana, Enrique

2018-04-01

Two bis-polyaza pyridinophane derivatives and their monomeric reference compounds revealed strong interactions with ds-DNA and RNA. The bis-derivatives show a specific condensation of GC- and IC-DNA, which is almost two orders of magnitude more efficient than the well-known condensation agent spermine. The type of condensed DNA was identified as ψ-DNA, characterized by the exceptionally strong CD signals. At variance to the almost silent AT(U) polynucleotides, these strong CD signals allow the determination of GC-condensates at nanomolar nucleobase concentrations. Detailed thermodynamic characterisation by ITC reveals significant differences between the DNA binding of the bis-derivative compounds (enthalpy driven) and that of spermine and of their monomeric counterparts (entropy driven). Atomic force microscopy confirmed GC-DNA compaction by the bis-derivatives and the formation of toroid- and rod-like structures responsible for the ψ-type pattern in the CD spectra. Copyright © 2017 Elsevier B.V. All rights reserved.

Novel 2-step synthetic indole compound 1,1,3-tri(3-indolyl)cyclohexane inhibits cancer cell growth in lung cancer cells and xenograft models.

PubMed

Lee, Ching-Hsiao; Yao, Ching-Fa; Huang, Sin-Ming; Ko, Shengkai; Tan, Yi-Hung; Lee-Chen, Guey-Jen; Wang, Yi-Ching

2008-08-15

The clinical responses to chemotherapy in lung cancer patients are unsatisfactory. Thus, the development of more effective anticancer drugs for lung cancer is urgently needed. A 2-step novel synthetic compound, referred to as 1,1,3-tri(3-indolyl)cyclohexane (3-indole), was generated in high purity and yield. 3-Indole was tested for its biologic activity in A549, H1299, H1435, CL1-1, and H1437 lung cancer cells. Animal studies were also performed. The data indicate that 3-indole induced apoptosis in various lung cancer cells. Increased cytochrome-c release from mitochondria to cytosol, decreased expression of antiapoptotic Bcl-2, and increased expression of proapoptotic Bax were observed. In addition, 3-indole stimulated caspases-3, -9, and to a lesser extent caspase-8 activities in cancer cells, suggesting that the intrinsic mitochondria pathway was the potential mechanism involved in 3-indole-induced apoptosis. 3-Indole-induced a concentration-dependent mitochondrial membrane potential dissipation and an increase in reactive oxygen species (ROS) production. Activation of c-Jun N-terminal kinase (JNK) and triggering of DNA damage were also apparent. Note that 3-indole-induced JNK activation and DNA damage can be partially suppressed by an ROS inhibitor. Apoptosis induced by 3-indole could be abrogated by ROS or JNK inhibitors, suggesting the importance of ROS and JNK stress-related pathways in 3-indole-induced apoptosis. Moreover, 3-indole showed in vivo antitumor activities against human xenografts in murine models. On the basis of its potent anticancer activity in cell and animal models, the data suggest that this 2-step synthetic 3-indole compound of high purity and yield is a potential candidate to be tested as a lead pharmaceutical compound for cancer treatment. 2008 American Cancer Society

New arylated benzo[h]quinolines induce anti-cancer activity by oxidative stress-mediated DNA damage.

PubMed

Yadav, Dharmendra K; Rai, Reeta; Kumar, Naresh; Singh, Surjeet; Misra, Sanjeev; Sharma, Praveen; Shaw, Priyanka; Pérez-Sánchez, Horacio; Mancera, Ricardo L; Choi, Eun Ha; Kim, Mi-Hyun; Pratap, Ramendra

2016-12-06

The anti-cancer activity of the benzo[h]quinolines was evaluated on cultured human skin cancer (G361), lung cancer (H460), breast cancer (MCF7) and colon cancer (HCT116) cell lines. The inhibitory effect of these compounds on the cell growth was determined by the MTT assay. The compounds 3e, 3f, 3h and 3j showed potential cytotoxicity against these human cancer cell lines. Effect of active compounds on DNA oxidation and expression of apoptosis related gene was studied. We also developed a quantitative method to measure the activity of cyclin-dependent kinases-2 (CDK2) by western blotting in the presence of active compound. In addition, molecular docking revealed that benzo[h]quinolines can correctly dock into the hydrophobic pocket of the targets receptor protein aromatase and CDK2, while their bioavailability/drug-likeness was predicted to be acceptable but requires future optimization. These findings reveal that benzo[h]quinolines act as anti-cancer agents by inducing oxidative stress-mediated DNA damage.

Bioactivation of carboxylic acid compounds by UDP-Glucuronosyltransferases to DNA-damaging intermediates: role of glycoxidation and oxidative stress in genotoxicity.

PubMed

Sallustio, Benedetta C; Degraaf, Yvette C; Weekley, Josephine S; Burcham, Philip C

2006-05-01

Nonenzymatic modification of proteins by acyl glucuronides is well documented; however, little is known about their potential to damage DNA. We have previously reported that clofibric acid undergoes glucuronidation-dependent bioactivation to DNA-damaging species in cultured mouse hepatocytes. The aim of this study was to investigate the mechanisms underlying such DNA damage, and to screen chemically diverse carboxylic acid drugs for their DNA-damaging potential in glucuronidation proficient murine hepatocytes. Cells were incubated with each aglycone for 18 h, followed by assessment of compound cytotoxicity using the MTT assay and evaluation of DNA damage using the Comet assay. Relative cytotoxic potencies were ketoprofen > diclofenac, benoxaprofen, nafenopin > gemfibrozil, probenecid > bezafibrate > clofibric acid. At a noncytotoxic (0.1 mM) concentration, only benoxaprofen, nafenopin, clofibric acid, and probenecid significantly increased Comet moments (P < 0.05 Kruskal-Wallis). Clofibric acid and probenecid exhibited the greatest DNA-damaging potency, producing significant DNA damage at 0.01 mM concentrations. The two drugs produced maximal increases in Comet moment of 4.51 x and 2.57 x control, respectively. The glucuronidation inhibitor borneol (1 mM) abolished the induction of DNA damage by 0.5 mM concentrations of clofibric acid and probenecid. In an in vitro cell-free system, clofibric acid glucuronide was 10 x more potent than glucuronic acid in causing DNA strand-nicking, although both compounds showed similar rates of autoxidation to generate hydroxyl radicals. In cultured hepatocytes, the glycation inhibitor, aminoguanidine, and the iron chelator, desferrioxamine mesylate, inhibited DNA damage by clofibric acid, whereas the free radical scavengers Trolox and butylated hydroxytoluene, and the superoxide dismutase mimetic bis-3,5-diisopropylsalicylate had no effect. In conclusion, clinically relevant concentrations of two structurally unrelated carboxylic acids, probenecid and clofibric acid, induced DNA damage in isolated hepatocytes via glucuronidation- dependent pathways. These findings suggest acyl glucuronides are able to access and damage nuclear DNA via iron-catalyzed glycation/glycoxidative processes.

Chemical form of selenium differentially influences DNA repair pathways following exposure to lead nitrate.

PubMed

McKelvey, Shauna M; Horgan, Karina A; Murphy, Richard A

2015-01-01

Lead, an environmental toxin is known to induce a broad range of physiological and biochemical dysfunctions in humans through a number of mechanisms including the deactivation of antioxidants thus leading to generation of reactive oxygen species (ROS) and subsequent DNA damage. Selenium on the other hand has been proven to play an important role in the protection of cells from free radical damage and oxidative stress, though its effects are thought to be form and dose dependent. As the liver is the primary organ required for metabolite detoxification, HepG2 cells were chosen to assess the protective effects of various selenium compounds following exposure to the genotoxic agent lead nitrate. Initially DNA damage was quantified using a comet assay, gene expression patterns associated with DNA damage and signalling were also examined using PCR arrays and the biological pathways which were most significantly affected by selenium were identified. Interestingly, the organic type selenium compounds (selenium yeast and selenomethionine) conferred protection against lead induced DNA damage in HepG2 cells; this is evident by reduction in the quantity of DNA present in the comet tail of cells cultured in their presence with lead. This trend also followed through the gene expression changes noted in DNA damage pathways analysed. These results were in contrast with those of inorganic sodium selenite which promoted lead induced DNA damage evident in both the comet assay results and the gene expression analysis. Over all this study provided valuable insights into the effects which various selenium compounds had on the DNA damage and signalling pathway indicating the potential for using organic forms of selenium such as selenium enriched yeast to protect against DNA damaging agents. Copyright © 2014 Elsevier GmbH. All rights reserved.

Use of a combined effect model approach for discriminating between ABCB1- and ABCC1-type efflux activities in native bivalve gill tissue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faria, Melissa; CESAM & Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro; Pavlichenko, Vasiliy

Aquatic organisms, such as bivalves, employ ATP binding cassette (ABC) transporters for efflux of potentially toxic chemicals. Anthropogenic water contaminants can, as chemosensitizers, disrupt efflux transporter function enabling other, putatively toxic compounds to enter the organism. Applying rapid amplification of cDNA ends (RACE) PCR we identified complete cDNAs encoding ABCB1- and ABCC1-type transporter homologs from zebra mussel providing the molecular basis for expression of both transporter types in zebra mussel gills. Further, efflux activities of both transporter types in gills were indicated with dye accumulation assays where efflux of the dye calcein-am was sensitive to both ABCB1- (reversin 205, verapamil)more » and ABCC1- (MK571) type specific inhibitors. The assumption that different inhibitors targeted different efflux pump types was confirmed when comparing measured effects of binary inhibitor compound mixtures in dye accumulation assays with predictions from mixture effect models. Effects by the MK571/reversin 205 mixture corresponded better with independent action, whereas reversin 205/verapamil joint effects were better predicted by the concentration addition model indicating different and equal targets, respectively. The binary mixture approach was further applied to identify the efflux pump type targeted by environmentally relevant chemosensitizing compounds. Pentachlorophenol and musk ketone, which were selected after a pre-screen of twelve compounds that previously had been identified as chemosensitizers, showed mixture effects that corresponded better with concentration addition when combined with reversine 205 but with independent action predictions when combined with MK571 indicating targeting of an ABCB1-type efflux pump by these compounds. - Highlights: • Sequences and function of ABC efflux transporters in bivalve gills were explored. • Full length Dreissena polymorpha abcb1 and abcc1 cDNA sequences were identified. • A mixture effect design with inhibitors was applied in transporter activity assays. • ABCB1- and ABCC-type efflux activities were distinguished in native gill tissue. • Inhibitory action of environmental chemicals targeted ABCB1-type efflux activity.« less

Model based on GRID-derived descriptors for estimating CYP3A4 enzyme stability of potential drug candidates

NASA Astrophysics Data System (ADS)

Crivori, Patrizia; Zamora, Ismael; Speed, Bill; Orrenius, Christian; Poggesi, Italo

2004-03-01

A number of computational approaches are being proposed for an early optimization of ADME (absorption, distribution, metabolism and excretion) properties to increase the success rate in drug discovery. The present study describes the development of an in silico model able to estimate, from the three-dimensional structure of a molecule, the stability of a compound with respect to the human cytochrome P450 (CYP) 3A4 enzyme activity. Stability data were obtained by measuring the amount of unchanged compound remaining after a standardized incubation with human cDNA-expressed CYP3A4. The computational method transforms the three-dimensional molecular interaction fields (MIFs) generated from the molecular structure into descriptors (VolSurf and Almond procedures). The descriptors were correlated to the experimental metabolic stability classes by a partial least squares discriminant procedure. The model was trained using a set of 1800 compounds from the Pharmacia collection and was validated using two test sets: the first one including 825 compounds from the Pharmacia collection and the second one consisting of 20 known drugs. This model correctly predicted 75% of the first and 85% of the second test set and showed a precision above 86% to correctly select metabolically stable compounds. The model appears a valuable tool in the design of virtual libraries to bias the selection toward more stable compounds. Abbreviations: ADME - absorption, distribution, metabolism and excretion; CYP - cytochrome P450; MIFs - molecular interaction fields; HTS - high throughput screening; DDI - drug-drug interactions; 3D - three-dimensional; PCA - principal components analysis; CPCA - consensus principal components analysis; PLS - partial least squares; PLSD - partial least squares discriminant; GRIND - grid independent descriptors; GRID - software originally created and developed by Professor Peter Goodford.

Synthesis and Biological Evaluation of Ru(II) and Pt(II) Complexes Bearing Carboxyl Groups as Potential Anticancer Targeted Drugs.

PubMed

Martínez, Ma Ángeles; Carranza, M Pilar; Massaguer, Anna; Santos, Lucia; Organero, Juan A; Aliende, Cristina; de Llorens, Rafael; Ng-Choi, Iteng; Feliu, Lidia; Planas, Marta; Rodríguez, Ana M; Manzano, Blanca R; Espino, Gustavo; Jalón, Félix A

2017-11-20

The synthesis and characterization of Pt(II) (1 and 2) and Ru(II) arene (3 and 4) or polypyridine (5 and 6) complexes is described. With the aim of having a functional group to form bioconjugates, one uncoordinated carboxyl group has been introduced in all complexes. Some of the complexes were selected for their potential in photodynamic therapy (PDT). The molecular structures of complexes 2 and 5, as well as that of the sodium salt of the 4'-(4-carboxyphenyl)-2,2':6',2″-terpyridine ligand (cptpy), were determined by X-ray diffraction. Different techniques were used to evaluate the binding capacity to model DNA molecules, and MTT cytotoxicity assays were performed against four cell lines. Compounds 3, 4, and 5 showed little tendency to bind to DNA and exhibited poor biological activity. Compound 2 behaves as bonded to DNA probably through a covalent interaction, although its cytotoxicity was very low. Compound 1 and possibly 6, both of which contain a cptpy ligand, were able to intercalate with DNA, but toxicity was not observed for 6. However, compound 1 was active in all cell lines tested. Clonogenic assays and apoptosis induction studies were also performed on the PC-3 line for 1. The photodynamic behavior for complexes 1, 5, and 6 indicated that their nuclease activity was enhanced after irradiation at λ = 447 nm. The cell viability was significantly reduced only in the case of 5. The different behavior in the absence or presence of light makes complex 5 a potential prodrug of interest in PDT. Molecular docking studies followed by molecular dynamics simulations for 1 and the counterpart without the carboxyl group confirmed the experimental data that pointed to an intercalation mechanism. The cytotoxicity of 1 and the potential of 5 in PDT make them good candidates for subsequent conjugation, through the carboxyl group, to "selected peptides" which could facilitate the selective vectorization of the complex toward receptors that are overexpressed in neoplastic cell lines.

Dinuclear RuII(bipy)2 Derivatives: Structural, Biological, and in Vivo Zebrafish Toxicity Evaluation.

PubMed

Lenis-Rojas, Oscar A; Roma-Rodrigues, Catarina; Fernandes, Alexandra R; Marques, Fernanda; Pérez-Fernández, David; Guerra-Varela, Jorge; Sánchez, Laura; Vázquez-García, Digna; López-Torres, Margarita; Fernández, Alberto; Fernández, Jesús J

2017-06-19

Ruthenium-based drugs exhibit interesting properties as potential anticancer pharmaceuticals. We herein present the synthesis and characterization of a new family of ruthenium complexes with formulas [{Ru(bipy) 2 } 2 (μ-L)][CF 3 SO 3 ] 4 (L = bptz, 1a) and [{Ru(bipy) 2 } 2 (μ-L)][CF 3 SO 3 ] 2 (L = arphos, 2a; dppb, 3a; dppf, 4a), which were synthesized from the Ru(II) precursor compound cis-Ru(bipy) 2 Cl 2 . The complexes were characterized by elemental analysis, mass spectrometry, 1 H and 31 P{ 1 H} NMR, IR spectroscopy, and conductivity measurements. The molecular structures for three Ru(II) compounds were determined by single-crystal X-ray diffraction. The newly developed compounds interact with CT-DNA by intercalation, in particular, 2a, 3a, and 4a, which also seemed to induce some extent of DNA degradation. This effect seemed to be related with the formation of reactive oxygen species. The cytotoxic activity was evaluated against A2780, MCF7, and MDAMB231 human tumor cells. Compounds 2a and 4a were the most cytotoxic with activity compared to cisplatin (∼2 μM, 72 h) in the A2780 cisplatin sensitive cells. All the compounds induced A2780 cell death by apoptosis, however, to a lesser extent for compounds 4a and 2a. For these compounds, the mechanism of cell death in addition to apoptosis seemed to involve autophagy. In vivo toxicity was evaluated using the zebrafish embryo model. LC 50 estimates varied from 5.397 (3a) to 39.404 (1a) mg/L. Considering the in vivo toxicity in zebrafish embryos and the in vitro cytotoxicity in cancer cells, compound 1a seems to be the safest having no effect on dechirionation and presenting a good antiproliferative activity against ovarian carcinoma cells.

Fluorescence-based recombination assay for sensitive and specific detection of genotoxic carcinogens in human cells.

PubMed

Ireno, Ivanildce C; Baumann, Cindy; Stöber, Regina; Hengstler, Jan G; Wiesmüller, Lisa

2014-05-01

In vitro genotoxicity tests are known to suffer from several shortcomings, mammalian cell-based assays, in particular, from low specificities. Following a novel concept of genotoxicity detection, we developed a fluorescence-based method in living human cells. The assay quantifies DNA recombination events triggered by DNA double-strand breaks and damage-induced replication fork stalling predicted to detect a broad spectrum of genotoxic modes of action. To maximize sensitivities, we engineered a DNA substrate encompassing a chemoresponsive element from the human genome. Using this substrate, we screened various human tumor and non-transformed cell types differing in the DNA damage response, which revealed that detection of genotoxic carcinogens was independent of the p53 status but abrogated by apoptosis. Cell types enabling robust and sensitive genotoxicity detection were selected for the generation of reporter clones with chromosomally integrated DNA recombination substrate. Reporter cell lines were scrutinized with 21 compounds, stratified into five sets according to the established categories for identification of carcinogenic compounds: genotoxic carcinogens ("true positives"), non-genotoxic carcinogens, compounds without genotoxic or carcinogenic effect ("true negatives") and non-carcinogenic compounds, which have been reported to induce chromosomal aberrations or mutations in mammalian cell-based assays ("false positives"). Our results document detection of genotoxic carcinogens in independent cell clones and at levels of cellular toxicities <60 % with a sensitivity of >85 %, specificity of ≥90 % and detection of false-positive compounds <17 %. Importantly, through testing cyclophosphamide in combination with primary hepatocyte cultures, we additionally provide proof-of-concept for the identification of carcinogens requiring metabolic activation using this novel assay system.

Ferrocene-based guanidine derivatives: in vitro antimicrobial, DNA binding and docking supported urease inhibition studies.

PubMed

Gul, Rukhsana; Rauf, Muhammad Khawar; Badshah, Amin; Azam, Syed Sikander; Tahir, Muhammad Nawaz; Khan, Azim

2014-10-06

Some novel ferrocenyl guanidines 1-8 were synthesized and characterized by different spectroscopic methods, elemental analysis and single crystal X-rays diffraction techniques. The crystallographic studies revealed that the existence of the strong non-bonding interactions facilitate these molecules to interact with biological macro-molecules like DNA that described to inherit good biological activities. The DNA interaction studies carried out by cyclic voltammetry (CV) and UV-visible spectroscopy are in close agreement with the binding constants (K) (0.79-5.4) × 10(5) (CV) and (0.72-5.1) × 10(5) (UV-vis). The shift in peak potential, current and absorption maxima of the studied ferrocenyl guanidines in the presence of DNA revealed that CV coupled with UV-vis spectroscopy could provide an opportune to characterize metal-based compounds-DNA interaction mechanism, a prerequisite for the design of new anticancer agents and understanding the molecular basis of their action. The compounds 1-8 have been screened for their antibacterial, antifungal and urease inhibition potency. A concurrent in silico study has also been applied on ferrocene moiety impregnated guanidines 1-8 to identify most active compounds having for inhibiting the activity of urease (pdb id 3LA4). Most of the compounds were found as potent inhibitors of urease and the compound 1 was found to be the most active with an IC50 of 16.83 ± 0.03 μM. The docking scores are in close agreement with the in vitro obtained IC50 values of inhibitors 1-8. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

A compact two-wave dichrometer of an optical biosensor analytical system for medicine

NASA Astrophysics Data System (ADS)

Chulkov, D. P.; Gusev, V. M.; Kompanets, O. N.; Vereschagin, F. V.; Skuridin, S. G.; Yevdokimov, Yu. M.

2017-01-01

An experimental model has been developed of a compact two-wave dichrometer on the base of LEDs that is well-suited to work with "liquid" DNA nanoconstructions as biosensing units. The mobile and inexpensive device is intended for use in a biosensor analytical system for rapid determination of biologically active compounds in liquids to solve practical problems of clinic medicine and pharmacology.

Synthesis, spectral and quantum chemical studies and use of (E)-3-[(3,5-bis(trifluoromethyl)phenylimino)methyl]benzene-1,2-diol and its Ni(II) and Cu(II) complexes as an anion sensor, DNA binding, DNA cleavage, anti-microbial, anti-mutagenic and anti-cancer agent

NASA Astrophysics Data System (ADS)

Ünver, Hüseyin; Boyacıoğlu, Bahadır; Zeyrek, Celal Tuğrul; Yıldız, Mustafa; Demir, Neslihan; Yıldırım, Nuray; Karaosmanoğlu, Oğuzhan; Sivas, Hülya; Elmalı, Ayhan

2016-12-01

We report the synthesis of a novel Schiff base (E)-3-[(3,5-bis(trifluoromethyl) phenylimino)methyl] benzene-1,2-diol from the reaction of 2,3-dihydroxybenzaldehyde with 3,5-bis(trifluoromethyl)aniline, and its Ni(II) and Cu(II) complexes. The molecular structure of the Schiff base was experimentally determined using X-ray single-crystal data and was compared to the structure predicted by theoretical calculations using density functional theory (DFT), Hartree-Fock (HF) and Möller-Plesset second-order perturbation (MP2). In addition, nonlinear optical (NLO) effects of the compound was predicted using DFT. The antimicrobial activities of the compounds were investigated for their minimum inhibitory concentration. UV-Vis spectroscopy studies of the interactions between the compounds and calf thymus DNA (CT-DNA) showed that the compounds interacts with CT-DNA via intercalative binding. A DNA cleavage study showed that the Cu(II) complex cleaved DNA without any external agents. The compounds inhibited the base pair mutation in the absence of S9 with high inhibition rate. In addition, in vitro cytotoxicity of the Ni(II) complex towards HepG2 cell line was assayed by the MTT method. Also, the colorimetric response of the Schiff base in DMSO to the addition of equivalent amount of anions (F-, Br-, I-, CN-, SCN-, ClO4-, HSO4-, AcO-, H2PO4-, N3- and OH-) was investigated. In this regard, while the addition of F-, CN-, AcO- and OH- anions into the solution containing Schiff base resulted in a significant color change, the addition of Br-, I-, SCN-, ClO4-, HSO4-, H2PO4- and N3- anions resulted in no color change. The most discernable color change in the Schiff base was caused by CN-, which demonstrated that the ligand can be used to selectively detect CN-.

The formation of DNA sugar radicals from photoexcitation of guanine cation radicals.

PubMed

Shukla, Lata I; Pazdro, Robert; Huang, James; DeVreugd, Christopher; Becker, David; Sevilla, Michael D

2004-05-01

In this investigation of radical formation and reaction in gamma- irradiated DNA and model compounds, we report the conversion of the guanine cation radical (one-electron oxidized guanine, G(.+)) to the C1' sugar radical and another sugar radical at the C3' or C4' position (designated C3'(.)/C4'(.)) by visible and UV photolysis. Electron spin resonance (ESR) spectroscopic investigations were performed on salmon testes DNA as well as 5'-dGMP, 3'-dGMP, 2'-deoxyguanosine and other nucleosides/nucleotides as model systems. DNA samples (25- 150 mg/ml D(2)O) were prepared with Tl(3+) or Fe(CN)(3-)(6) as electron scavengers. Upon gamma irradiation of such samples at 77 K, the electron-gain path in the DNA is strongly suppressed and predominantly G(.+) is found; after UV or visible photolysis, the fraction of the C1' sugar radical increases with a concomitant reduction in the fraction of G(.+). In model systems, 3'- dGMP(+.) and 5'-dGMP(+.) were produced by attack of Cl(.-)(2) on the parent nucleotide in 7 M LiCl glass. Subsequent visible photolysis of the 3'-dGMP(+.) (77 K) results predominantly in formation of C1'(.) whereas photolysis of 5'-dGMP(+.) results predominantly in formation of C3'(.)/C4'(.). We propose that sugar radical formation is a result of delocalization of the hole in the electronically excited base cation radical into the sugar ring, followed by deprotonation at specific sites on the sugar.

DNA tests for strawberry: ¿-decalactone "fruity" aroma - qFaFAD1

USDA-ARS?s Scientific Manuscript database

Strawberry fruit produce hundreds of volatile compounds that contribute to the characteristic flavor of the fruit that many enjoy. One important compound is gamma-decalactone. This compound has been described as “peachy” and is indeed a very valuable compound contributing to the characteristic flavo...

Metabolic interrogation as a tool to optimize chemotherapeutic regimens.

PubMed

Sandulache, Vlad C; Chen, Yunyun; Feng, Lei; William, William N; Skinner, Heath D; Myers, Jeffrey N; Meyn, Raymond E; Li, Jinzhong; Mijiti, Ainiwaer; Bankson, James A; Fuller, Clifton D; Konopleva, Marina Y; Lai, Stephen Y

2017-03-14

Platinum-based (Pt) chemotherapy is broadly utilized in the treatment of cancer. Development of more effective, personalized treatment strategies require identification of novel biomarkers of treatment response. Since Pt compounds are inactivated through cellular metabolic activity, we hypothesized that metabolic interrogation can predict the effectiveness of Pt chemotherapy in a pre-clinical model of head and neck squamous cell carcinoma (HNSCC).We tested the effects of cisplatin (CDDP) and carboplatin (CBP) on DNA damage, activation of cellular death cascades and tumor cell metabolism, specifically lactate production. Pt compounds induced an acute dose-dependent, transient drop in lactate generation in vitro, which correlated with effects on DNA damage and cell death. Neutralization of free radical stress abrogated these effects. The magnitude of this effect on lactate production correlated with the differential sensitivity of HNSCC cells to Pt compounds (CDDP vs CBP) and p53-driven Pt chemotherapy resistance. Using dual flank xenograft tumors, we demonstrated that Pt-driven effects on lactate levels correlate with effects on tumor growth delay in a dose-dependent manner and that lactate levels can define the temporal profile of Pt chemotherapy-induced metabolic stress. Lactate interrogation also predicted doxorubicin effects on cell death in both solid tumor (HNSCC) and acute myelogenous leukemia (AML) cell lines.Real-time metabolic interrogation of acute changes in cell and tumor lactate levels reflects chemotherapy effects on DNA damage, cell death and tumor growth delay. We have identified a real-time biomarker of chemotherapy effectiveness which can be used to develop adaptive, iterative and personalized treatment regimens against a variety of solid and hematopoietic malignancies.

Metabolic interrogation as a tool to optimize chemotherapeutic regimens

PubMed Central

Feng, Lei; William, William N.; Skinner, Heath D.; Myers, Jeffrey N.; Meyn, Raymond E.; Li, Jinzhong; Mijiti, Ainiwaer; Bankson, James A.; Fuller, Clifton D.; Konopleva, Marina Y.; Lai, Stephen Y.

2017-01-01

Platinum-based (Pt) chemotherapy is broadly utilized in the treatment of cancer. Development of more effective, personalized treatment strategies require identification of novel biomarkers of treatment response. Since Pt compounds are inactivated through cellular metabolic activity, we hypothesized that metabolic interrogation can predict the effectiveness of Pt chemotherapy in a pre-clinical model of head and neck squamous cell carcinoma (HNSCC). We tested the effects of cisplatin (CDDP) and carboplatin (CBP) on DNA damage, activation of cellular death cascades and tumor cell metabolism, specifically lactate production. Pt compounds induced an acute dose-dependent, transient drop in lactate generation in vitro, which correlated with effects on DNA damage and cell death. Neutralization of free radical stress abrogated these effects. The magnitude of this effect on lactate production correlated with the differential sensitivity of HNSCC cells to Pt compounds (CDDP vs CBP) and p53-driven Pt chemotherapy resistance. Using dual flank xenograft tumors, we demonstrated that Pt-driven effects on lactate levels correlate with effects on tumor growth delay in a dose-dependent manner and that lactate levels can define the temporal profile of Pt chemotherapy-induced metabolic stress. Lactate interrogation also predicted doxorubicin effects on cell death in both solid tumor (HNSCC) and acute myelogenous leukemia (AML) cell lines. Real-time metabolic interrogation of acute changes in cell and tumor lactate levels reflects chemotherapy effects on DNA damage, cell death and tumor growth delay. We have identified a real-time biomarker of chemotherapy effectiveness which can be used to develop adaptive, iterative and personalized treatment regimens against a variety of solid and hematopoietic malignancies. PMID:28184025

Sulfur and selenium antioxidants: challenging radical scavenging mechanisms and developing structure-activity relationships based on metal binding.

PubMed

Zimmerman, Matthew T; Bayse, Craig A; Ramoutar, Ria R; Brumaghim, Julia L

2015-04-01

Because sulfur and selenium antioxidants can prevent oxidative damage, numerous animal and clinical trials have investigated the ability of these compounds to prevent the oxidative stress that is an underlying cause of cardiovascular disease, Alzheimer's disease, and cancer, among others. One of the most common sources of oxidative damage is metal-generated hydroxyl radical; however, very little research has focused on determining the metal-binding abilities and structural attributes that affect oxidative damage prevention by sulfur and selenium compounds. In this review, we describe our ongoing investigations into sulfur and selenium antioxidant prevention of iron- and copper-mediated oxidative DNA damage. We determined that many sulfur and selenium compounds inhibit Cu(I)-mediated DNA damage and that DNA damage prevention varies dramatically when Fe(II) is used in place of Cu(I) to generate hydroxyl radical. Oxidation potentials of the sulfur or selenium compounds do not correlate with their ability to prevent DNA damage, highlighting the importance of metal coordination rather than reactive oxygen species scavenging as an antioxidant mechanism. Additional gel electrophoresis, mass spectrometry, and UV-visible studies confirmed sulfur and selenium antioxidant binding to Cu(I) and Fe(II). Ultimately, our studies established that both the hydroxyl-radical-generating metal ion and the chemical environment of the sulfur or selenium significantly affect DNA damage prevention and that metal coordination is an essential mechanism for these antioxidants. Copyright © 2015 Elsevier Inc. All rights reserved.

Indicine N-oxide binds to tubulin at a distinct site and inhibits the assembly of microtubules: a mechanism for its cytotoxic activity.

PubMed

Appadurai, Prakash; Rathinasamy, Krishnan

2014-02-10

Indicine N-oxide, a pyrrolizidine alkaloid present in the plant Heliotropium indicum had shown promising cytotoxic activity in various tumor models. The compound exhibited severe toxicity to hepatocytes and bone marrow cells. The present work was aimed to evaluate the molecular mechanism of the toxicity of indicine N-oxide. We found that indicine N-oxide inhibited the proliferation of various cancer cell lines in a concentration dependent manner with IC50 ranging from 46 to 100 μM. At the half maximal inhibitory concentration it blocked the cell cycle progression at mitosis without significantly altering the organization of the spindle and interphase microtubules. The toxicities of the compound at higher concentrations are attributed to its severe depolymerizing effect on both the interphase and spindle microtubules. Binding studies using purified goat brain tubulin indicated that indicine N-oxide binds to tubulin at a distinct site not shared by colchicine or taxol. It decreased the polymer mass of both purified tubulin and MAP-rich tubulin. It was found to induce cleavage of DNA using pUC18 plasmid. The interactions of indicine N-oxide on DNA were also confirmed by computational analysis; which predicted its binding site at the minor groove of DNA. These studies bring to light that the toxicities of indicine N-oxide were due to its DNA damaging effects and depolymerization of microtubules. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

DNA-encoded chemical libraries: advancing beyond conventional small-molecule libraries.

PubMed

Franzini, Raphael M; Neri, Dario; Scheuermann, Jörg

2014-04-15

DNA-encoded chemical libraries (DECLs) represent a promising tool in drug discovery. DECL technology allows the synthesis and screening of chemical libraries of unprecedented size at moderate costs. In analogy to phage-display technology, where large antibody libraries are displayed on the surface of filamentous phage and are genetically encoded in the phage genome, DECLs feature the display of individual small organic chemical moieties on DNA fragments serving as amplifiable identification barcodes. The DNA-tag facilitates the synthesis and allows the simultaneous screening of very large sets of compounds (up to billions of molecules), because the hit compounds can easily be identified and quantified by PCR-amplification of the DNA-barcode followed by high-throughput DNA sequencing. Several approaches have been used to generate DECLs, differing both in the methods used for library encoding and for the combinatorial assembly of chemical moieties. For example, DECLs can be used for fragment-based drug discovery, displaying a single molecule on DNA or two chemical moieties at the extremities of complementary DNA strands. DECLs can vary substantially in the chemical structures and the library size. While ultralarge libraries containing billions of compounds have been reported containing four or more sets of building blocks, also smaller libraries have been shown to be efficient for ligand discovery. In general, it has been found that the overall library size is a poor predictor for library performance and that the number and diversity of the building blocks are rather important indicators. Smaller libraries consisting of two to three sets of building blocks better fulfill the criteria of drug-likeness and often have higher quality. In this Account, we present advances in the DECL field from proof-of-principle studies to practical applications for drug discovery, both in industry and in academia. DECL technology can yield specific binders to a variety of target proteins and is likely to become a standard tool for pharmaceutical hit discovery, lead expansion, and Chemical Biology research. The introduction of new methodologies for library encoding and for compound synthesis in the presence of DNA is an exciting research field and will crucially contribute to the performance and the propagation of the technology.

The role of arsenic in the hydrolysis and DNA metalation processes in an arsenous acid-platinum(ii) anticancer complex.

PubMed

Marino, T; Parise, A; Russo, N

2017-01-04

Platinum(ii)-based molecules are the most commonly used anticancer drugs in the chemotherapeutic treatment of tumours but possess serious side effects and some cancer types exhibit resistance with respect to these compounds (e.g. cisplatin). For these reasons, the research of new compounds that can bypass this limitation is in continuous development. Recently, mixed Pt(ii)-As(iii) systems have been synthesized and tested as potential anticancer agents. The mechanism of action of these kinds of drugs is unclear. Since in other platinum(ii) containing drugs, hydrolysis plays an important role in the activation of the compound before it reaches DNA, we have explored the aquation process using density functional theory (DFT), focusing our attention on the arsenoplatin complex, [Pt(μ-NHC(CH 3 )O) 2 ClAs(OH) 2 ]. As DNA is believed to be the cellular target for Pt anticancer drugs, the metalation mechanism of DNA purine bases has been also investigated. Also for this new drug it appears that guanine is the preferred site with respect to adenine as with other platinum-containing compounds. A comparison with cisplatin is performed in order to highlight the contribution of arsenic in the anticancer activity of this new proposed anticancer agent.

Immunomodulatory Effect of Agave tequilana Evaluated on an Autoimmunity Like-SLE Model Induced in Balb/c Mice with Pristane.

PubMed

Gutiérrez Nava, Zúlima Jannette; Jiménez-Aparicio, Antonio Ruperto; Herrera-Ruiz, Maribel Lucila; Jiménez-Ferrer, Enrique

2017-05-25

In this work, the immunomodulatory activity of the acetone extract and the fructans obtained from Agave tequilana were evaluated, on the systemic autoimmunity type-SLE model generated by the administration of 2,6,10,14-tetramethylpentadecane (TMPD, also known as pristane) on Balb/c female mice. The systemic autoimmunity type-SLE was observed seven months after the application of TMPD, in which the animals from the negative control group (animals with damage and without any other treatment) developed articular inflammation, proteinuria, an increment of the antinuclear antibody titters and tissue pro-inflammatory cytokines levels (IL-1β, IL-6, TNF-α e IFN-γ) as well as the anti-inflammatory cytokine IL-10. The administration of the different treatments and the extracts of A. tequilana , provoked the decrease of: articular inflammation, the development of proteinuria, ssDNA/dsDNA antinuclear antibody titters and cytokines IL-1β, IL-6, TNF-α, IFN-γ and IL-10. The phytochemical analysis of the acetone extract identified the presence of the following compounds: β-sitosterol glycoside; 3,7,11,15-tetramethyl-2-hexadecen-1-ol (phytol); octadecadienoic acid-2,3-dihydroxypropyl ester; stigmasta-3,5-dien-7-one; cycloartenone and cycloartenol. Therefore, A. tequilana contains active compounds with the capacity to modify the evolution of the systemic autoimmunity type-SLE on a murine model.

Differential Targeting of Unpaired Bases within Duplex DNA by the Natural Compound Clerocidin: A Valuable Tool to Dissect DNA Secondary Structure

PubMed Central

Nadai, Matteo; Palù, Giorgio; Palumbo, Manlio; Richter, Sara N.

2012-01-01

Non-canonical DNA structures have been postulated to mediate protein-nucleic acid interactions and to function as intermediates in the generation of frame-shift mutations when errors in DNA replication occur, which result in a variety of diseases and cancers. Compounds capable of binding to non-canonical DNA conformations may thus have significant diagnostic and therapeutic potential. Clerocidin is a natural diterpenoid which has been shown to selectively react with single-stranded bases without targeting the double helix. Here we performed a comprehensive analysis on several non-canonical DNA secondary structures, namely mismatches, nicks, bulges, hairpins, with sequence variations in both the single-stranded region and the double-stranded flanking segment. By analysis of clerocidin reactivity, we were able to identify the exposed reactive residues which provided information on both the secondary structure and the accessibility of the non-paired sites. Mismatches longer than 1 base were necessary to be reached by clerocidin reactive groups, while 1-base nicks were promptly targeted by clerocidin; in hairpins, clerocidin reactivity increased with the length of the hairpin loop, while, interestingly, reactivity towards bulges reached a maximum in 3-base-long bulges and declined in longer bulges. Electrophoretic mobility shift analysis demonstrated that bulges longer than 3 bases (i.e. 5- and 7-bases) folded or stacked on the duplex region therefore being less accessible by the compound. Clerocidin thus represents a new valuable diagnostic tool to dissect DNA secondary structures. PMID:23285245

Differential targeting of unpaired bases within duplex DNA by the natural compound clerocidin: a valuable tool to dissect DNA secondary structure.

PubMed

Nadai, Matteo; Palù, Giorgio; Palumbo, Manlio; Richter, Sara N

2012-01-01

Non-canonical DNA structures have been postulated to mediate protein-nucleic acid interactions and to function as intermediates in the generation of frame-shift mutations when errors in DNA replication occur, which result in a variety of diseases and cancers. Compounds capable of binding to non-canonical DNA conformations may thus have significant diagnostic and therapeutic potential. Clerocidin is a natural diterpenoid which has been shown to selectively react with single-stranded bases without targeting the double helix. Here we performed a comprehensive analysis on several non-canonical DNA secondary structures, namely mismatches, nicks, bulges, hairpins, with sequence variations in both the single-stranded region and the double-stranded flanking segment. By analysis of clerocidin reactivity, we were able to identify the exposed reactive residues which provided information on both the secondary structure and the accessibility of the non-paired sites. Mismatches longer than 1 base were necessary to be reached by clerocidin reactive groups, while 1-base nicks were promptly targeted by clerocidin; in hairpins, clerocidin reactivity increased with the length of the hairpin loop, while, interestingly, reactivity towards bulges reached a maximum in 3-base-long bulges and declined in longer bulges. Electrophoretic mobility shift analysis demonstrated that bulges longer than 3 bases (i.e. 5- and 7-bases) folded or stacked on the duplex region therefore being less accessible by the compound. Clerocidin thus represents a new valuable diagnostic tool to dissect DNA secondary structures.

Unexpected DNA affinity and sequence selectivity through core rigidity in guanidinium-based minor groove binders.

PubMed

Nagle, Padraic S; McKeever, Caitriona; Rodriguez, Fernando; Nguyen, Binh; Wilson, W David; Rozas, Isabel

2014-09-25

In this paper we report the design and biophysical evaluation of novel rigid-core symmetric and asymmetric dicationic DNA binders containing 9H-fluorene and 9,10-dihydroanthracene cores as well as the synthesis of one of these fluorene derivatives. First, the affinity toward particular DNA sequences of these compounds and flexible core derivatives was evaluated by means of surface plasmon resonance and thermal denaturation experiments finding that the position of the cations significantly influence the binding strength. Then their affinity and mode of binding were further studied by performing circular dichroism and UV studies and the results obtained were rationalized by means of DFT calculations. We found that the fluorene derivatives prepared have the ability to bind to the minor groove of certain DNA sequences and intercalate to others, whereas the dihydroanthracene compounds bind via intercalation to all the DNA sequences studied here.

Determination of the DNA-binding characteristics of ethidium bromide, proflavine, and cisplatin by flow injection analysis: usefulness in studies on antitumor drugs.

PubMed

Alonso, A; Almendral, M J; Curto, Y; Criado, J J; Rodríguez, E; Manzano, J L

2006-08-15

Flow injection analysis was used to study the reactions occurring between DNA and certain compounds that bind to its double helix, deforming this and even breaking it, such that some of them (e.g., cisplatin) are endowed with antitumoral activity. Use of this technique in the merging zones and stopped-flow modes afforded data on the binding parameters and the kinetic characteristics of the process. The first compound studied was ethidium bromide (EtdBr), used as a fluorescent marker because its fluorescence is enhanced when it binds to DNA. The DNA-EtdBr binding parameters, the apparent intrinsic binding constant (0.31+/-0.02 microM(-1)), and the maximum number of binding sites per nucleotide (0.327+/-0.009) were determined. The modification introduced in these parameters by the presence of proflavine (Prf), a classic competitive inhibitor of the binding of EtdBr to the DNA double helix, was also studied, determining the value of the intrinsic binding constant of Prf (K(Prf) = 0.119+/-9x10(-3) microM(-1)). Finally, we determined the binding parameters between DNA and EtdBr in the presence of the antitumor agent cisplatin, a noncompetitive inhibitor of such binding. This provided information about the binding mechanism as well as the duration and activity of the binding of the compound in its pharmacological use.

Evaluation of various glyphosate concentrations on DNA damage in human Raji cells and its impact on cytotoxicity.

PubMed

Townsend, Michelle; Peck, Connor; Meng, Wei; Heaton, Matthew; Robison, Richard; O'Neill, Kim

2017-04-01

Glyphosate is a highly used active compound in agriculturally based pesticides. The literature regarding the toxicity of glyphosate to human cells has been highly inconsistent. We studied the resulting DNA damage and cytotoxicity of various glyphosate concentrations on human cells to evaluate DNA damaging potential. Utilizing human Raji cells, DNA damage was quantified using the comet assay, while cytotoxicity was further analyzed using MTT viability assays. Several glyphosate concentrations were assessed, ranging from 15 mM to 0.1 μM. We found that glyphosate treatment is lethal to Raji cells at concentrations above 10 mM, yet has no cytotoxic effects at concentrations at or below 100 μM. Treatment concentrations of 1 mM and 5 mM induce statistically significant DNA damage to Raji cells following 30-60 min of treatment, however, cells show a slow recovery from initial damage and cell viability is unaffected after 2 h. At these same concentrations, cells treated with additional compound did not recover and maintained high levels of DNA damage. While the cytotoxicity of glyphosate appears to be minimal for physiologically relevant concentrations, the compound has a definitive cytotoxic nature in human cells at high concentrations. Our data also suggests a mammalian metabolic pathway for the degradation of glyphosate may be present. Copyright © 2017 Elsevier Inc. All rights reserved.

Genotoxic chemical carcinogens target inducible genes in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamilton, J.W.; McCaffrey, J.; Caron, R.M.

1994-12-31

Our laboratory is interested in whether carcinogen-induced DNA damage is distributed nonrandomly in the genome - that is, {open_quotes}targeted{close_quotes} to specific genes or gene regions in vivo. As an indirect measure of whether targeting occurs at the gene level, we have examined whether carcinogens differentially alter the expression of individual genes. We have compared the effects of model genotoxic carcinogens that principally induce either strand breaks, simple alkylations, bulky lesions, or DNA cross-links on the expression of several constitutive and inducible genes in a simple in vivo system, the chick embryo. Each agent was examined for its effects on genemore » expression over a 24 hour period corresponding to the period of maximal DNA damage and repair induced by each compound. The doses used in these studies represented the maximum doses that caused no overt toxicity over a 96 hour period but that induced significant levels of DNA damage. Our results demonstrate that inducible genes are targeted by chemical carcinogens. We hypothesize that such effects may be a result of DNA damage specifically altering DNA-protein interactions within the promoters of inducible genes.« less

Genotoxic activity of 4,4',5'-trimethylazapsoralen on plasmid DNA.

PubMed

Lagatolla, C; Dolzani, L; Granzotto, M; Monti-Bragadin, C

1998-01-01

The genotoxic activities of 8-methoxypsoralen (8-MOP) and 4,4',5'-trimethylazapsoralen (4,4',5'-TMAP) on plasmid DNA have been compared. In a previous work, 4,4',5'-TMAP, a methyl derivative of a psoralen isoster, had shown potential photochemotherapeutic activity. The mutagenic activity of mono- and bifunctional lesions caused by these compounds was evaluated both after UVA irradiation, which causes the formation of both kinds of lesions, and after a two-step irradiation procedure of the psoralen-plasmid DNA complex, which allowed monoadducts and interstrand crosslinks to be studied separately. Furthermore, we used a procedure that allowed us to evaluate both the mutagenic and recombinogenic activity of the two compounds. Results indicate that the most important difference between 8-MOP and 4,4',5'-TMAP consists in their mode of photoreaction with DNA rather than in their mutagenic potential. In fact, in all of the experimental procedures, 4,4',5'-TMAP shows a lower ability than 8-MOP to generate interstrand crosslinks. However, when comparable toxicity levels are reached, the two compounds show the same mutagenic potentiality.

Novel trifluoromethylated 9-amino-3,4-dihydroacridin-1(2H)-ones act as covalent poisons of human topoisomerase IIα.

PubMed

Infante Lara, Lorena; Sledge, Alexis; Laradji, Amine; Okoro, Cosmas O; Osheroff, Neil

2017-02-01

A number of topoisomerase II-targeted anticancer drugs, including amsacrine, utilize an acridine or related aromatic core as a scaffold. Therefore, to further explore the potential of acridine-related compounds to act as topoisomerase II poisons, we synthesized a series of novel trifluoromethylated 9-amino-3,4-dihydroacridin-1(2H)-one derivatives and examined their ability to enhance DNA cleavage mediated by human topoisomerase IIα. Derivatives containing a H, Cl, F, and Br at C7 enhanced enzyme-mediated double-stranded DNA cleavage ∼5.5- to 8.5-fold over baseline, but were less potent than amsacrine. The inclusion of an amino group at C9 was critical for activity. The compounds lost their activity against topoisomerase IIα in the presence of a reducing agent, displayed no activity against the catalytic core of topoisomerase IIα, and inhibited DNA cleavage when incubated with the enzyme prior to the addition of DNA. These findings strongly suggest that the compounds act as covalent, rather than interfacial, topoisomerase II poisons. Published by Elsevier Ltd.

The PARP inhibitor PJ-34 sensitizes cells to UVA-induced phototoxicity by a PARP independent mechanism.

PubMed

Lakatos, Petra; Hegedűs, Csaba; Salazar Ayestarán, Nerea; Juarranz, Ángeles; Kövér, Katalin E; Szabó, Éva; Virág, László

2016-08-01

A combination of a photosensitizer with light of matching wavelength is a common treatment modality in various diseases including psoriasis, atopic dermatitis and tumors. DNA damage and production of reactive oxygen intermediates may impact pathological cellular functions and viability. Here we set out to investigate the role of the nuclear DNA nick sensor enzyme poly(ADP-ribose) polymerase 1 in photochemical treatment (PCT)-induced tumor cell killing. We found that silencing PARP-1 or inhibition of its enzymatic activity with Veliparib had no significant effect on the viability of A431 cells exposed to 8-methoxypsoralen (8-MOP) and UVA (2.5J/cm(2)) indicating that PARP-1 is not likely to be a key player in either cell survival or cell death of PCT-exposed cells. Interestingly, however, another commonly used PARP inhibitor PJ-34 proved to be a photosensitizer with potency equal to 8-MOP. Irradiation of PJ-34 with UVA caused changes both in the UV absorption and in the 1H NMR spectra of the compound with the latter suggesting UVA-induced formation of tautomeric forms of the compound. Characterization of the photosensitizing effect revealed that PJ-34+UVA triggers overproduction of reactive oxygen species, induces DNA damage, activation of caspase 3 and caspase 8 and internucleosomal DNA fragmentation. Cell death in this model could not be prevented by antioxidants (ascorbic acid, trolox, glutathione, gallotannin or cell permeable superoxide dismutase or catalase) but could be suppressed by inhibitors of caspase-3 and -8. In conclusion, PJ-34 is a photosensitizer and PJ-34+UVA causes DNA damage and caspase-mediated cell death independently of PARP-1 inhibition. Copyright © 2016 Elsevier B.V. All rights reserved.

High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion.

PubMed

Shiratori, Hiromi; Feinweber, Carmen; Knothe, Claudia; Lötsch, Jörn; Thomas, Dominique; Geisslinger, Gerd; Parnham, Michael J; Resch, Eduard

2016-01-01

DNA methylation is a major regulatory process of gene transcription, and aberrant DNA methylation is associated with various diseases including cancer. Many compounds have been reported to modify DNA methylation states. Despite increasing interest in the clinical application of drugs with epigenetic effects, and the use of diagnostic markers for genome-wide hypomethylation in cancer, large-scale screening systems to measure the effects of drugs on DNA methylation are limited. In this study, we improved the previously established fluorescence polarization-based global DNA methylation assay so that it is more suitable for application to human genomic DNA. Our methyl-sensitive fluorescence polarization (MSFP) assay was highly repeatable (inter-assay coefficient of variation = 1.5%) and accurate (r2 = 0.99). According to signal linearity, only 50-80 ng human genomic DNA per reaction was necessary for the 384-well format. MSFP is a simple, rapid approach as all biochemical reactions and final detection can be performed in one well in a 384-well plate without purification steps in less than 3.5 hours. Furthermore, we demonstrated a significant correlation between MSFP and the LINE-1 pyrosequencing assay, a widely used global DNA methylation assay. MSFP can be applied for the pre-screening of compounds that influence global DNA methylation states and also for the diagnosis of certain types of cancer.

Human DNA adduct measurements: State of the art

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poirier, M.C.; Weston, A.

1996-10-01

Human DNA adduct formation (covalent modification of DNA with chemical carcinogens) is a promising biomarker for elucidating the molecular epidemiology of cancer. Classes of compounds for which human DNA adducts have been observed include polycyclic aromatic hydrocarbons (PAHs), nitrosamines, mycotoxins, aromatic amines, heterocyclic amines, ultraviolet light, and alkylating cancer chemotherapeutic agents. Most human DNA adduct exposure monitoring has been performed with either {sup 32}P-postlabeling or immunoassays, neither of which is able to chemically characterize specific DNA adducts. Recently developed combinations of methods with chemical and physical end points have allowed identification of specific adducts in human tissues. Studies are presentedmore » that demonstrate that high ambient levels of benzo[a]pyrene are associated with high levels of DNA adducts in human blood cell DNA and that the same DNA adduct levels drop when the ambient PAH levels decrease significantly. DNA adduct dosimetry, which has been achieved with some dietary carcinogens and cancer chemotherapeutic agents, is described, as well as studies correlating DNA adducts with other biomarkers. It is likely that some toxic, noncarcinogenic compounds may have genotoxic effects, including oxidative damage, and that adverse health outcomes other than cancer may be correlated with DNA adduct formation. The studies presented here may serve as useful prototypes for exploration of other toxicological end points. 156 refs., 1 fig., 3 tabs.« less

Preventive Role of Specific Dietary Factors and Natural Compounds Against DNA Damage and Oxidative Stress.

DTIC Science & Technology

1998-08-01

vitamins (C and E) and natural compounds (caffeic acid phenethyl ester [CAPE] and epigallocatechin gallate [ EGCG ] may be protective against mammary...caffeic acid phenethyl ester (CAPE), and epigallocatechin gallate ( EGCG ) in inhibiting DNA damage. These antioxidants are found in natural products such...as fruits and vegetables (vitamins C and E), the popular medicine honeybee propolis (CAPE), or green tea ( EGCG ). Studies carried out to date suggest

Genotoxicity of Tri- and Hexavalent Chromium Compounds In Vivo and Their Modes of Action on DNA Damage In Vitro

PubMed Central

Fang, Zhijia; Zhao, Min; Zhen, Hong; Chen, Lifeng; Shi, Ping; Huang, Zhiwei

2014-01-01

Chromium occurs mostly in tri- and hexavalent states in the environment. Hexavalent chromium [Cr(VI)] compounds are extensively used in diverse industries, and trivalent chromium [Cr(III)] salts are used as micronutrients and dietary supplements. In the present work, we report that they both induce genetic mutations in yeast cells. They both also cause DNA damage in both yeast and Jurkat cells and the effect of Cr(III) is greater than that of Cr(VI). We further show that Cr(III) and Cr(VI) cause DNA damage through different mechanisms. Cr(VI) intercalates DNA and Cr(III) interferes base pair stacking. Based on our results, we conclude that Cr(III) can directly cause genotoxicity in vivo. PMID:25111056

Activity of cationically substituted bis-benzimidazoles against experimental Pneumocystis carinii pneumonia.

PubMed Central

Tidwell, R R; Jones, S K; Naiman, N A; Berger, L C; Brake, W B; Dykstra, C C; Hall, J E

1993-01-01

On the basis of a previously observed correlation between the antimicrobial activity and DNA binding strength of dicationic molecules, a series of 10 dicationically substituted bis-benzimidazoles were tested for activity in the rat model of Pneumocystis carinii pneumonia. One of the compounds, 1,4-bis[5-(2-imidazolinyl)-2-benzimidazolyl]butane, was found to be more potent and less toxic than pentamidine. PMID:8215291

A more detailed picture of the interactions between virtual screening-derived hits and the DNA G-quadruplex: NMR, molecular modelling and ITC studies.

PubMed

Trotta, Roberta; De Tito, Stefano; Lauri, Ilaria; La Pietra, Valeria; Marinelli, Luciana; Cosconati, Sandro; Martino, Luigi; Conte, Maria R; Mayol, Luciano; Novellino, Ettore; Randazzo, Antonio

2011-08-01

The growing amount of literature about G-quadruplex DNA clearly demonstrates that such a structure is no longer viewed as just a biophysical strangeness but it is instead being considered as an important target for the treatment of various human disorders such as cancers or venous thrombosis. In this scenario, with the aim of finding brand new molecular scaffolds able to interact with the groove of the DNA quadruplex [d(TGGGGT)](4), we recently performed a successful structure-based virtual screening (VS) campaign. As a result, six molecules were found to be somehow groove binders. Herein, we report the results of novel NMR titration experiments of these VS-derived ligands with modified quadruplexes, namely [d(TGG(Br)GGT)](4) and [d(TGGGG(Br)T)](4). The novel NMR spectroscopy experiments combined with molecular modelling studies, allow for a more detailed picture of the interaction between each binder and the quadruplex DNA. Noteworthy, isothermal titration calorimetry (ITC) measurements on the above-mentioned compounds revealed that 2, 4, and 6 besides their relatively small dimensions bind the DNA quadruplex [d(TGGGGT)](4) with higher affinity than distamycin A, to the best of our knowledge, the most potent groove binder identified thus far. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

A reaction mechanism-based prediction of mutagenicity: α-halo carbonyl compounds adduct with DNA by SN2 reaction.

PubMed

Haranosono, Yu; Ueoka, Hiroki; Kito, Gakushi; Nemoto, Shingo; Kurata, Masaaki; Sakaki, Hideyuki

2018-01-01

Most of the α-halo carbonyl (AHC) compounds tend to be predicted as mutagenic by structure-activity relationship based on structural category only, because they have an alkyl halide structure as a structural alert of mutagenicity. However, some AHC compounds are not mutagenic. We hypothesized that AHC reacts with DNA by S N 2 reaction, and the reactivity relates to mutagenicity. As an index of S N 2 reactivity, we focused on molecular orbitals (MOs), as the direction and position of two molecules in collision are important in the S N 2 reaction. The MOs suitable for S N 2 reaction (SN2MOs) were selected by chemical-visual inspection based on the shape of the MO. We used the level gap and the energy gap between SN2MO and the lowest unoccupied molecular orbital as the descriptors of S N 2 reactivity. As the results, S N 2 reactivity related to mutagenicity and we were able to predict mutagenicity of 20 AHC compounds with 95.0% concordance. It was suggested that S N 2 reaction is a reaction mechanism of AHC compounds and DNA in the mutagenic process. The method allows for discrimination among structurally similar compounds by combination with quantitative structure-activity relationships. The combination approach is expected to be useful for the mutagenic assessment of pharmaceutical impurities.

Metabolite Profiling and Classification of DNA-Authenticated Licorice Botanicals

PubMed Central

Simmler, Charlotte; Anderson, Jeffrey R.; Gauthier, Laura; Lankin, David C.; McAlpine, James B.; Chen, Shao-Nong; Pauli, Guido F.

2015-01-01

Raw licorice roots represent heterogeneous materials obtained from mainly three Glycyrrhiza species. G. glabra, G. uralensis, and G. inflata exhibit marked metabolite differences in terms of flavanones (Fs), chalcones (Cs), and other phenolic constituents. The principal objective of this work was to develop complementary chemometric models for the metabolite profiling, classification, and quality control of authenticated licorice. A total of 51 commercial and macroscopically verified samples were DNA authenticated. Principal component analysis and canonical discriminant analysis were performed on 1H NMR spectra and area under the curve values obtained from UHPLC-UV chromatograms, respectively. The developed chemometric models enable the identification and classification of Glycyrrhiza species according to their composition in major Fs, Cs, and species specific phenolic compounds. Further key outcomes demonstrated that DNA authentication combined with chemometric analyses enabled the characterization of mixtures, hybrids, and species outliers. This study provides a new foundation for the botanical and chemical authentication, classification, and metabolomic characterization of crude licorice botanicals and derived materials. Collectively, the proposed methods offer a comprehensive approach for the quality control of licorice as one of the most widely used botanical dietary supplements. PMID:26244884

Chemical composition of the Lippia origanoides essential oils and their antigenotoxicity against bleomycin-induced DNA damage.

PubMed

Vicuña, Gloria Carolina; Stashenko, Elena E; Fuentes, Jorge Luis

2010-07-01

The present work evaluated the chemical composition of the essential oils (EO) obtained from Lippia origanoides and their DNA protective effect against bleomycin-induced genotoxicity. L. origanoides EO chemical composition was determined by gas chromatography-mass spectrometry (GC-MS). The major compounds of the L. origanoides EOs were thymol (34-58%) and carvacrol (26%). The antigenotoxic effects of the EOs, major compounds and standard compound (epigallocatechin gallate) were assayed in co-incubation procedures using the SOS chromotest in Escherichia coli. Both EOs and their major compounds protected bacterial cells against bleomycin-induced genotoxicity indicating that these two compounds were principally responsible for the antigenotoxicity detected in the oils. Thymol and carvacrol antigenotoxicity was lower than those observed with epigallocatechin gallate. The results were discussed in relation to the chemopreventive potential of L. origanoides EOs and their major components, carvacrol and thymol. Copyright 2009 Elsevier B.V. All rights reserved.

Cell death induced by flavonoid glycosides with and without copper.

PubMed

Hsu, Hsue-Yin; Tsang, Shih-Fang; Lin, Kai-Wei; Yang, Shyh-Chyun; Lin, Chun-Nan

2008-07-01

The ability of flavonoid glycosides isolated from several plants to induce DNA breakage was examined using supercoiled plasmid pBR322 DNA by agarose gel electrophoresis in the presence of Cu(II). Among all the compounds, 1, 4, and 6 could cause significant breakages of supercoiled plasmid pBR322 DNA in the presence of Cu(II). Cu(I) was not shown to be an essential intermediate in the process of pBR322 DNA breakage by using the Cu(I)-specific sequestering reagent neocuproine. A decreased cell viability was enhanced in gastric carcinoma SCM-1 cells treating with lower concentrations of 1 and 6 when cotreated with increased concentrations of Cu(II), respectively. Treatments of SCM-1 cells with 500 microM of 1 in the presence of 300 or 500 microM of Cu(II) inhibited the Cu(II)-induced apoptosis. Compound 1 (500 microM) could prevent cell death by inhibiting the 500 microM Cu(II)-induced apoptosis and necrosis, but did not have any effect on the mitochondrial membrane potential changed by 500 microM Cu(II). Both compounds 1 and 6 could inhibit the DNA breakages caused by O2- while 1 also revealed inhibitory effect on xanthine oxidase with an IC50 value of 22.7+/-6.9 microM. These results indicated that compound 1 with a higher concentration may probably mediate through the suppression of xanthine oxidase activity and reduce reactive oxygen species (ROS) induced by high concentration of Cu(II) (500 microM) and prevent the following cell death.

Deoxynybomycins inhibit mutant DNA gyrase and rescue mice infected with fluoroquinolone-resistant bacteria.

PubMed

Parkinson, Elizabeth I; Bair, Joseph S; Nakamura, Bradley A; Lee, Hyang Y; Kuttab, Hani I; Southgate, Emma H; Lezmi, Stéphane; Lau, Gee W; Hergenrother, Paul J

2015-04-24

Fluoroquinolones are one of the most commonly prescribed classes of antibiotics, but fluoroquinolone resistance (FQR) is widespread and increasing. Deoxynybomycin (DNM) is a natural-product antibiotic with an unusual mechanism of action, inhibiting the mutant DNA gyrase that confers FQR. Unfortunately, isolation of DNM is difficult and DNM is insoluble in aqueous solutions, making it a poor candidate for development. Here we describe a facile chemical route to produce DNM and its derivatives. These compounds possess excellent activity against FQR methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci clinical isolates and inhibit mutant DNA gyrase in-vitro. Bacteria that develop resistance to DNM are re-sensitized to fluoroquinolones, suggesting that resistance that emerges to DNM would be treatable. Using a DNM derivative, the first in-vivo efficacy of the nybomycin class is demonstrated in a mouse infection model. Overall, the data presented suggest the promise of DNM derivatives for the treatment of FQR infections.

Improved Force Fields for Peptide Nucleic Acids with Optimized Backbone Torsion Parameters.

PubMed

Jasiński, Maciej; Feig, Michael; Trylska, Joanna

2018-06-06

Peptide nucleic acids are promising nucleic acid analogs for antisense therapies as they can form stable duplex and triplex structures with DNA and RNA. Computational studies of PNA-containing duplexes and triplexes are an important component for guiding their design, yet existing force fields have not been well validated and parametrized with modern computational capabilities. We present updated CHARMM and Amber force fields for PNA that greatly improve the stability of simulated PNA-containing duplexes and triplexes in comparison with experimental structures and allow such systems to be studied on microsecond time scales. The force field modifications focus on reparametrized PNA backbone torsion angles to match high-level quantum mechanics reference energies for a model compound. The microsecond simulations of PNA-PNA, PNA-DNA, PNA-RNA, and PNA-DNA-PNA complexes also allowed a comprehensive analysis of hydration and ion interactions with such systems.

Synthesis, spectroscopic characterization and in vitro antimicrobial, anticancer and antileishmanial activities as well interaction with Salmon sperm DNA of newly synthesized carboxylic acid derivative, 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoic acid

NASA Astrophysics Data System (ADS)

Sirajuddin, Muhammad; Ali, Saqib; McKee, Vickie; Ullah, Hameed

2015-03-01

This paper stresses on the synthesis, characterization of novel carboxylic acid derivative and its application in pharmaceutics. Carboxylic acid derivatives have a growing importance in medicine, particularly in oncology. A novel carboxylic acid, 4-(4-methoxy-2-nitrophenylamino)-4-oxobutanoic acid, was synthesized and characterized by elemental analysis, FT-IR, NMR (1H, and 13C), mass spectrometry and single crystal X-ray structural analysis. The structure of the title compound, C11H12N2O6, shows the molecules dimerised by short intramolecular Osbnd H⋯O hydrogen bonds. The compound was screened for in vitro antimicrobial, anticancer, and antileishmanial activities as well as interaction with SS-DNA. The compound was also checked for in vitro anticancer activity against BHK-21, H-157 and HCEC cell lines, and showed significant anticancer activity. The compound was almost non-toxic towards human corneal epithelial cells (HCEC) and did not show more than 7.4% antiproliferative activity when used at the 2.0 μg/mL end concentration. It was also tested for antileishmanial activity against the promastigote form of leishmania major and obtained attractive result. DNA interaction study exposes that the binding mode of the compound with SS-DNA is an intercalative as it results in hypochromism along with minor red shift. A new and efficient strategy to identify pharmacophores sites in carboxylic acid derivative for antibacterial/antifungal activity using Petra, Osiris and Molinspiration (POM) analyses was also carried out.

Inhibition of HMGA2 binding to DNA by netropsin

PubMed Central

Miao, Yi; Cui, Tengjiao; Leng, Fenfei; Wilson, W. David

2008-01-01

The design of small synthetic molecules that can be used to affect gene expression is an area of active interest for development of agents in therapeutic and biotechnology applications. Many compounds that target the minor groove in AT sequences in DNA are well characterized and are promising reagents for use as modulators of protein-DNA complexes. The mammalian high mobility group transcriptional factor, HMGA2, also targets the DNA minor groove and plays critical roles in disease processes from cancer to obesity. Biosensor-surface plasmon resonance methods were used to monitor HMGA2 binding to target sites on immobilized DNA and a competition assay for inhibition of the HMGA2-DNA complex was designed. HMGA2 binds strongly to the DNA through AT hook domains with KD values of 20 - 30 nM depending on the DNA sequence. The well-characterized minor groove binder, netropsin, was used to develop and test the assay. The compound has two binding sites in the protein-DNA interaction sequence and this provides an advantage for inhibition. An equation for analysis of results when the inhibitor has two binding sites in the biopolymer recognition surface is presented with the results. The assay provides a platform for discovery of HMGA2 inhibitors. PMID:18023407

An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening

PubMed Central

2017-01-01

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing. PMID:28199790

An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening.

PubMed

MacConnell, Andrew B; Price, Alexander K; Paegel, Brian M

2017-03-13

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing.

Synthesis, antimicrobial evaluation and docking studies of some novel quinazolinone Schiff base derivatives

PubMed Central

Nasab, Rezvan Rezaee; Mansourian, Mahboubeh; Hassanzadeh, Farshid

2018-01-01

The quinazolin-4(3H)-one structural motif possesses a wide spectrum of biological activities. DNA gyrase play an important role in induction of bacterial death. It has been shown that many quinazolin-4(3H)-one derivatives have antibacterial effects through inhibition of DNA gyrase. Based on this information we decided to synthesize novel quinazolinone Schiff base derivatives in order to evaluate their antibacterial effects. A series of novel quinazolinone Schiff base derivatives were designed and synthesized from benzoic acid. The potential DNA gyrase inhibitory activity of these compounds was investigated using in silico molecular docking simulation. All new synthesized derivatives were screened for their antimicrobial activities against three species of Gram-negative bacteria including Escherichia coli, Pseudomonas aeruginosa, Salmonella entritidis and three species of Gram-positive bacteria comprising of Staphylococcus aurous, Bacillus subtilis, Listeria monocitogenes as well as for antifungal activities against Candida albicans using the conventional micro dilution method. Most of the compounds have shown good antibacterial activities, especially against E. coli at 128 µg/mL concentration while no remarkable antifungal activities were observed for these compounds. All the synthesized compounds exhibit dock score values between -5.96 and -8.58 kcal/mol. The highest dock score among them was -8.58 kcal/mol for compound 4c. PMID:29853931

New investigations into the genotoxicity of cobalt compounds and their impact on overall assessment of genotoxic risk.

PubMed

Kirkland, David; Brock, Tom; Haddouk, Hasnaà; Hargeaves, Victoria; Lloyd, Melvyn; Mc Garry, Sarah; Proudlock, Raymond; Sarlang, Séverine; Sewald, Katherina; Sire, Guillaume; Sokolowski, Andrea; Ziemann, Christina

2015-10-01

The genotoxicity of cobalt metal and cobalt compounds has been widely studied. Several publications show induction of chromosomal aberrations, micronuclei or DNA damage in mammalian cells in vitro in the absence of S9. Mixed results were seen in gene mutation studies in bacteria and mammalian cells in vitro, and in chromosomal aberration or micronucleus assays in vivo. To resolve these inconsistencies, new studies were performed with soluble and poorly soluble cobalt compounds according to OECD-recommended protocols. Induction of chromosomal damage was confirmed in vitro, but data suggest this may be due to oxidative stress. No biologically significant mutagenic responses were obtained in bacteria, Tk(+/-) or Hprt mutation tests. Negative results were also obtained for chromosomal aberrations (in bone marrow and spermatogonia) and micronuclei at maximum tolerated doses in vivo. Poorly soluble cobalt compounds do not appear to be genotoxic. Soluble compounds do induce some DNA and chromosomal damage in vitro, probably due to reactive oxygen. The absence of chromosome damage in robust GLP studies in vivo suggests that effective protective processes are sufficient to prevent oxidative DNA damage in whole mammals. Overall, there is no evidence of genetic toxicity with relevance for humans of cobalt substances and cobalt metal. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

The lignan glycosides lyoniside and saracoside poison the unusual type IB topoisomerase of Leishmania donovani and kill the parasite both in vitro and in vivo.

PubMed

Saha, Sourav; Mukherjee, Tulika; Chowdhury, Sayan; Mishra, Amartya; Chowdhury, Somenath Roy; Jaisankar, Parasuraman; Mukhopadhyay, Sibabrata; Majumder, Hemanta K

2013-12-15

Lignans are diphenyl propanoids with vast range of biological activities. The present study provides an important insight into the anti-leishmanial activities of two lignan glycosides, viz. lyoniside and saracoside. These compounds inhibit catalytic activities of topoisomerase IB (LdTopIB) of Leishmania donovani in non-competitive manner and stabilize the LdTopIB mediated cleavage complex formation both in vitro and in Leishmania promastigotes and subsequently inhibit the religation of cleaved strand. These two compounds not only poison LdTopIB but also can interact with the free enzyme LdTopIB. We have also shown that lyoniside and saracoside are cytotoxic to promastigotes and intracellular amastigotes. The protein-DNA complex formation leads to double strand breaks in DNA which ultimately triggers apoptosis-like cell death in the parasite. Along with their cytotoxicity towards sodium antimony gluconate (SAG) sensitive AG83 strain, their ability to kill SAG resistant GE1 strain makes these two compounds potential anti-leishmanial candidates. Not only they effectively kill L. donovani amastigotes inside macrophages in vitro, lyoniside and saracoside demonstrated strong anti-leishmanial efficacies in BALB/c mice model of leishmaniasis. Treatment with these lignan glycosides produce nitric oxide and reactive oxygen species which result in almost complete clearance of the liver and splenic parasite burden. These compounds do not inhibit human topoisomerase IB upto 200μM concentrations and had poor cytotoxic effect on uninfected cultured murine peritoneal macrophages upto 100μM concentrations. Taken together it can be concluded that these compounds can be developed into excellent therapeutic agent against deadly disease leishmaniasis. Copyright © 2013 Elsevier Inc. All rights reserved.

In Situ Analysis of Metabolic Characteristics Reveals the Key Yeast in the Spontaneous and Solid-State Fermentation Process of Chinese Light-Style Liquor

PubMed Central

Kong, Yu; Wu, Qun; Zhang, Yan

2014-01-01

The in situ metabolic characteristics of the yeasts involved in spontaneous fermentation process of Chinese light-style liquor are poorly understood. The covariation between metabolic profiles and yeast communities in Chinese light-style liquor was modeled using the partial least square (PLS) regression method. The diversity of yeast species was evaluated by sequence analysis of the 26S ribosomal DNA (rDNA) D1/D2 domains of cultivable yeasts, and the volatile compounds in fermented grains were analyzed by gas chromatography (GC)-mass spectrometry (MS). Eight yeast species and 58 volatile compounds were identified, respectively. The modulation of 16 of these volatile compounds was associated with variations in the yeast population (goodness of prediction [Q2] > 20%). The results showed that Pichia anomala was responsible for the characteristic aroma of Chinese liquor, through the regulation of several important volatile compounds, such as ethyl lactate, octanoic acid, and ethyl tetradecanoate. Correspondingly, almost all of the compounds associated with P. anomala were detected in a pure culture of this yeast. In contrast to the PLS regression results, however, ethyl lactate and ethyl isobutyrate were not detected in the same pure culture, which indicated that some metabolites could be generated by P. anomala only when it existed in a community with other yeast species. Furthermore, different yeast communities provided different volatile patterns in the fermented grains, which resulted in distinct flavor profiles in the resulting liquors. This study could help identify the key yeast species involved in spontaneous fermentation and provide a deeper understanding of the role of individual yeast species in the community. PMID:24727269

Molecular modeling of the AhR structure and interactions can shed light on ligand-dependent activation and transformation mechanisms.

PubMed

Bonati, Laura; Corrada, Dario; Tagliabue, Sara Giani; Motta, Stefano

2017-02-01

Molecular modeling has given important contributions to elucidation of the main stages in the AhR signal transduction pathway. Despite the lack of experimentally determined structures of the AhR functional domains, information derived from homologous systems has been exploited for modeling their structure and interactions. Homology models of the AhR PASB domain have provided information on the binding cavity and contributed to elucidate species-specific differences in ligand binding. Molecular Docking simulations of the ligand binding process have given insights into differences in binding of diverse agonists, antagonists, and selective AhR modulators, and their application to virtual screening of large databases of compounds have allowed identification of novel AhR ligands. Recently available structural information on protein-protein and protein-DNA complexes of other bHLH-PAS systems has opened the way for modeling the AhR:ARNT dimer structure and investigating the mechanisms of AhR transformation and DNA binding. Future research directions should include simulation of the protein dynamics to obtain a more reliable description of intermolecular interactions involved in signal transmission.

Nanogravimetric and voltammetric DNA-hybridization biosensors for studies of DNA damage by common toxicants and pollutants.

PubMed

Nowicka, Anna M; Kowalczyk, Agata; Stojek, Zbigniew; Hepel, Maria

2010-01-01

Electrochemical and nanogravimetric DNA-hybridization biosensors have been developed for sensing single mismatches in the probe-target ssDNA sequences. The voltammetric transduction was achieved by coupling ferrocene moiety to streptavidin linked to biotinylated tDNA. The mass-related frequency transduction was implemented by immobilizing the sensory pDNA on a gold-coated quartz crystal piezoresonators oscillating in the 10MHz band. The high sensitivity of these sensors enabled us to study DNA damage caused by representative toxicants and environmental pollutants, including Cr(VI) species, common pesticides and herbicides. We have found that the sensor responds rapidly to any damage caused by Cr(VI) species, with more severe DNA damage observed for Cr(2)O(7)(2-) and for CrO(4)(2-) in the presence of H(2)O(2) as compared to CrO(4)(2-) alone. All herbicides and pesticides examined caused DNA damage or structural alterations leading to the double-helix unwinding. Among these compounds, paraoxon-ethyl and atrazine caused the fastest and most severe damage to DNA. The physico-chemical mechanism of damaging interactions between toxicants and DNA has been proposed. The methodology of testing voltammetric and nanogravimetric DNA-hybridization biosensors developed in this work can be employed as a simple protocol to obtain rapid comparative data concerning DNA damage caused by herbicide, pesticides and other toxic pollutants. The DNA-hybridization biosensor can, therefore, be utilized as a rapid screening device for classifying environmental pollutants and to evaluate DNA damage induced by these compounds.

Functional lipids based on [12]aneN3 and naphthalimide as efficient non-viral gene vectors.

PubMed

Gao, Yong-Guang; Alam, Uzair; Tang, Quan; Shi, You-Di; Zhang, Ying; Wang, Ruibing; Lu, Zhong-Lin

2016-07-14

Small organic non-viral gene vectors with the structural combinations of (aliphatic chain)-naphthalimide-[12]aneN3 (11a, b) and naphthalimide-(aliphatic chain)-[12]aneN3 (12a-c) were synthesized and fully characterized. Agarose gel electrophoresis experiments indicated that the first type of compounds, 11a and 11b, could completely retard DNA at the concentration of 5 μM in the presence of DOPE. Within the second type of compounds, 12c with the decane chain showed a complete retardation of DNA at the concentration of 20 μM, whereas 12a and 12b with the ethyl and hexyl chains could not retard DNA effectively. Dynamic light scattering measurements indicated that compounds 11a, 11b and 12b, 12c condensed DNA into nanoparticles with the size in the range of 60-160 nm. Due to the strong fluorescence of 11a and 11b, the distribution of lipids/DNA complexes and the process of DNA release from the lipids were clearly observed via cellular uptake experiments. On the other hand, the non-fluorescent 12a-c enabled the EB exclusion assay to afford the binding constants of 4.88 × 10(6) M(-1) (12a), 4.18 × 10(6) M(-1) (12b) and 3.39 × 10(6) M(-1) (12c), respectively. The MTT assay revealed that both types of compounds have low cytotoxicity. Non-fluorescent 12c was successfully applied in the eGFP expression experiments in A549 cells and showed stronger green fluorescence emission than that of lipofectamine 2000. Quantitative transfection experiments through the luciferase assay further revealed that compounds 11a, 11b and 12c can act as non-viral gene vectors in different cell lines. Among them, 12c gave the highest transfection efficiency in HeLa cells, which was about 2 times that offered by lipofectamine 2000. This work clearly demonstrated that the right combination of different functional units and long aliphatic linkers will likely promote gene delivery and transfection efficiency.

Metabolic Engineering for the Production of Natural Products

PubMed Central

Pickens, Lauren B.; Tang, Yi; Chooi, Yit-Heng

2014-01-01

Natural products and natural product derived compounds play an important role in modern healthcare as frontline treatments for many diseases and as inspiration for chemically synthesized therapeutics. With advances in sequencing and recombinant DNA technology, many of the biosynthetic pathways responsible for the production of these chemically complex and pharmaceutically valuable compounds have been elucidated. With an ever expanding toolkit of biosynthetic components, metabolic engineering is an increasingly powerful method to improve natural product titers and generate novel compounds. Heterologous production platforms have enabled access to pathways from difficult to culture strains; systems biology and metabolic modeling tools have resulted in increasing predictive and analytic capabilities; advances in expression systems and regulation have enabled the fine-tuning of pathways for increased efficiency, and characterization of individual pathway components has facilitated the construction of hybrid pathways for the production of new compounds. These advances in the many aspects of metabolic engineering have not only yielded fascinating scientific discoveries but also make it an increasingly viable approach for the optimization of natural product biosynthesis. PMID:22432617

Zebrafish embryos as a screen for DNA methylation modifications after compound exposure.

PubMed

Bouwmeester, Manon C; Ruiter, Sander; Lommelaars, Tobias; Sippel, Josefine; Hodemaekers, Hennie M; van den Brandhof, Evert-Jan; Pennings, Jeroen L A; Kamstra, Jorke H; Jelinek, Jaroslav; Issa, Jean-Pierre J; Legler, Juliette; van der Ven, Leo T M

2016-01-15

Modified epigenetic programming early in life is proposed to underlie the development of an adverse adult phenotype, known as the Developmental Origins of Health and Disease (DOHaD) concept. Several environmental contaminants have been implicated as modifying factors of the developing epigenome. This underlines the need to investigate this newly recognized toxicological risk and systematically screen for the epigenome modifying potential of compounds. In this study, we examined the applicability of the zebrafish embryo as a screening model for DNA methylation modifications. Embryos were exposed from 0 to 72 h post fertilization (hpf) to bisphenol-A (BPA), diethylstilbestrol, 17α-ethynylestradiol, nickel, cadmium, tributyltin, arsenite, perfluoroctanoic acid, valproic acid, flusilazole, 5-azacytidine (5AC) in subtoxic concentrations. Both global and site-specific methylation was examined. Global methylation was only affected by 5AC. Genome wide locus-specific analysis was performed for BPA exposed embryos using Digital Restriction Enzyme Analysis of Methylation (DREAM), which showed minimal wide scale effects on the genome, whereas potential informative markers were not confirmed by pyrosequencing. Site-specific methylation was examined in the promoter regions of three selected genes vasa, vtg1 and cyp19a2, of which vasa (ddx4) was the most responsive. This analysis distinguished estrogenic compounds from metals by direction and sensitivity of the effect compared to embryotoxicity. In conclusion, the zebrafish embryo is a potential screening tool to examine DNA methylation modifications after xenobiotic exposure. The next step is to examine the adult phenotype of exposed embryos and to analyze molecular mechanisms that potentially link epigenetic effects and altered phenotypes, to support the DOHaD hypothesis. Copyright © 2015 Elsevier Inc. All rights reserved.

Anticancer activity of botanical alkyl hydroquinones attributed to topoisomerase II poisoning

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, C.-P.; Fang, W.-H.; Lin, L.-I.

2008-03-15

Cytotoxic alkyl hydroquinone compounds have been isolated from many plants. We previously isolated 3 structurally similar cytotoxic alkyl hydroquinone compounds from the sap of the lacquer tree Rhus succedanea L. belonging to the sumac family, which have a long history of medicinal use in Asia. Each has an unsaturated alkyl chain attached to the 2-position of a hydroquinone ring. One of these isolates, 10'(Z),13'(E),15'(E)-heptadecatrienylhydroquinone [HQ17(3)], being the most cytotoxic, was chosen for studying the anticancer mechanism of these compounds. We found that HQ17(3) was a topoisomerase (Topo) II poison. It irreversibly inhibited Topo II{alpha} activity through the accumulation of Topomore » II-DNA cleavable complexes. A cell-based assay showed that HQ17(3) inhibited the growth of leukemia HL-60 cells with an EC{sub 50} of 0.9 {mu}M, inhibited the topoisomerase-II-deficient cells HL-60/MX2 with an EC{sub 50} of 9.6 {mu}M, and exerted no effect on peripheral blood mononuclear cells at concentrations up to 50 {mu}M. These results suggest that Topo II is the cellular drug target. In HL-60 cells, HQ17(3) promptly inhibited DNA synthesis, induced chromosomal breakage, and led to cell death with an EC{sub 50} about one-tenth that of hydroquinone. Pretreatment of the cells with N-acetylcysteine could not attenuate the cytotoxicity and DNA damage induced by HQ17(3). However, N-acetylcysteine did significantly reduce the cytotoxicity of hydroquinone. In F344 rats, intraperitoneal injection of HQ17(3) for 28 days induced no clinical signs of toxicity. These results indicated that HQ17(3) is a potential anticancer agent, and its structural features could be a model for anticancer drug design.« less

Novel Design Strategy for Checkpoint Kinase 2 Inhibitors Using Pharmacophore Modeling, Combinatorial Fusion, and Virtual Screening

PubMed Central

Wang, Yen-Ling

2014-01-01

Checkpoint kinase 2 (Chk2) has a great effect on DNA-damage and plays an important role in response to DNA double-strand breaks and related lesions. In this study, we will concentrate on Chk2 and the purpose is to find the potential inhibitors by the pharmacophore hypotheses (PhModels), combinatorial fusion, and virtual screening techniques. Applying combinatorial fusion into PhModels and virtual screening techniques is a novel design strategy for drug design. We used combinatorial fusion to analyze the prediction results and then obtained the best correlation coefficient of the testing set (r test) with the value 0.816 by combining the BesttrainBesttest and FasttrainFasttest prediction results. The potential inhibitors were selected from NCI database by screening according to BesttrainBesttest + FasttrainFasttest prediction results and molecular docking with CDOCKER docking program. Finally, the selected compounds have high interaction energy between a ligand and a receptor. Through these approaches, 23 potential inhibitors for Chk2 are retrieved for further study. PMID:24864236

Azapsoralens: new potential photochemotherapeutic agents for psoriasis.

PubMed

Vedaldi, D; Caffieri, S; Miolo, G; Dall'Acqua, F; Baccichetti, F; Guiotto, A; Benetollo, F; Bombieri, G; Recchia, G; Cristofolini, M

1991-12-01

New bioisoters of psoralen, obtained by replacing carbon 8 of the central benzene ring with a nitrogen, were studied from the photochemical, photobiological and phototherapeutic points of view. In particular, 4,4'-, 4',5'-dimetyl, 4,4',5'-trimethyl and 3,4,4',5'-tetramethylazapsoralen were studied. The crystal and molecular structure of 4,4',5'-trimethylazapsoralen, obtained by X ray diffraction, was also reported. Like psoralen, these compounds form a molecular complex with DNA, undergoing intercalation inside the double helix of the macromolecule. When irridiated with long ultraviolet light (365 nm), the intercalated drug photoconjugates covalently to the macromolecule, forming mono- and diadducts. The photobinding rate show the following order of magnitude: 4,4',5'-trimetylazapsoralen (4,4',5'-TMAP) = 3,4,4',5'-tetramethylazapsoralen (3,4,4',5'-TMAP) greater than 4',5'-dimethylazapsoralen (4',5'-DMAP) = 4,4'-dimethylazapsoralen (4,4'-DMAP). The DNA photobinding rate of 8-methoxypsoralen (8-MOP), taken as reference compound, is similar to that of the two dimetylazapsoralens but lower than tri- and tetramethyl derivatives. The ability of azapsoralens to form cross-links in DNA is lower than that of 8-MOP. However, capacity to induce cross-links does not parallel the DNA photobinding rate; it is higher for trimethyl derivate and lower for tetramethylazapsoralen. Azapsoralens show evident antiproliferative activity. The trimethyl derivative is the most active, followed by tetrametyl, both these compounds showing activity slightly higher than that of 8-MOP. The two dimethylderivatives are less active. The mautagenic activity of azapsoralens on E. coli WP2 TM6 is lower than that of 8-MOP in the same conditions. The new compounds do not show any skin phototoxicity on guinea pig skin. On the basis of its DNA photobinding, antiproliferative activity, mutagenicity and lack of skin phototoxicity, 4,4',5'-TMAP was chosen for clinical evaluation. Clinical results obtained by topical treatment of psoriatic plaques reveal evident therapeutic effectiveness and clearing is between good and moderate, although 8-MOP, used as reference compound, is more effective.

Exocyclic Deoxyadenosine Adducts of 1,2,3,4-Diepoxybutane: Synthesis, Structural Elucidation, and Mechanistic Studies

PubMed Central

Seneviratne, Uthpala; Antsypovich, Sergey; Goggin, Melissa; Dorr, Danae Quirk; Guza, Rebecca; Moser, Adam; Thompson, Carrie; York, Darrin M.; Tretyakova, Natalia

2009-01-01

1,2,3,4-Diepoxybutane (DEB)1 is considered the ultimate carcinogenic metabolite of 1,3-butadiene, an important industrial chemical and environmental pollutant present in urban air. Although it preferentially modifies guanine within DNA, DEB induces a large number of A → T transversions, suggesting that it forms strongly mispairing lesions at adenine nucleobases. We now report the discovery of three potentially mispairing exocyclic adenine lesions of DEB: N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (compound 2), 1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (compound 3), and 1,N6-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2′-deoxyadenosine (compound 4). The structures and stereochemistry of the novel DEB-dA adducts were determined by a combination of UV and NMR spectroscopy, tandem mass spectrometry, and independent synthesis. We found that synthetic N6-(2-hydroxy-3,4-epoxybut-1-yl)-2′-deoxyadenosine (compound 1) representing the product of N6-adenine alkylation by DEB spontaneously cyclizes to form 3 under aqueous conditions or 2 under anhydrous conditions in the presence of organic base. Compound 3 can be interconverted with 4 by a reversible unimolecular pericyclic reaction favoring 4 as a more thermodynamically stable product. Both 3 and 4 are present in double stranded DNA treated with DEB in vitro and in liver DNA of laboratory mice exposed to 1,3-butadiene by inhalation. We propose that in DNA under physiological conditions, DEB alkylates the N-1 position of adenine in DNA to form N1-(2-hydroxy-3,4-epoxybut-1-yl)-adenine adducts, which undergo an SN2-type intramolecular nucleophilic substitution and rearrangement to give 3 (minor) and 4 (major). Formation of exocyclic DEB-adenine lesions following exposure to 1,3-butadiene provides a possible mechanism of mutagenesis at the A:T base pairs. PMID:19883087

Dimeric fluorescent energy transfer dyes comprising asymmetric cyanine azole-indolenine chromophores

DOEpatents

Glazer, Alexander N.; Benson, Scott C.

1998-01-01

Novel fluorescent heterodimeric DNA-staining energy transfer dyes are provided combining asymmetric cyanine azole-indolenine dyes, which provide for strong DNA affinity, large Stokes shifts and emission in the red region of the spectrum. The dyes find particular application in gel electrophoresis and for labels which may be bound to a variety of compositions in a variety of contexts. Kits and individual compounds are provided, where the kits find use for simultaneous detection of a variety of moieties, particularly using a single narrow wavelength irradiation source. The individual compounds are characterized by high donor quenching and high affinity to dsDNA as a result of optimizing the length of the linking group separating the two chromophores.

Influence of amine and thiol modifications at the 3' ends of single stranded DNA molecules on their adsorption on gold surface and the efficiency of their hybridization.

PubMed

Jaworska, Aleksandra; Jablonska, Anna; Wilanowski, Tomasz; Palys, Barbara; Sek, Slawomir; Kudelski, Andrzej

2018-05-24

Adsorption of molecules of DNA (deoxyribonucleic acid) or modified DNA on gold surfaces is often the first step in construction of many various biosensors, including biosensors for detection of DNA with a particular sequence. In this work we study the influence of amine and thiol modifications at the 3' ends of single stranded DNA (ssDNA) molecules on their adsorption on the surface of gold substrates and on the efficiency of hybridization of immobilized DNA with the complementary single stranded DNA. The characterization of formed layers has been carried out using infrared spectroscopy and atomic force microscopy. As model single stranded DNA we used DNA containing 20 adenine bases, whereas the complementary DNA contained 20 thymine bases. We found that the bands in polarization modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS) spectra of layers formed from thiol-modified DNA are significantly narrower and sharper, indicating their higher regularity in the orientation of DNA on gold surface when using thiol linker. Also, hybridization of the layer of thiol-modified DNA containing 20 adenine bases with the respective DNA containing thymine bases leads to formation of much more organized structures than in the case of unmodified DNA or DNA with the amine linker. We conclude that the thiol-modified ssDNA is more promising for the preparation of biosensors, in comparison with the amine-modified or unmodified ssDNA. We have also found that the above-mentioned modifications at the 3' end of ssDNA significantly influence the IR spectrum (and hence the structure) of polycrystalline films formed from such compounds, even though adsorbed fragments contain less than 5% of the DNA chain. This effect should be taken into account when comparing IR spectra of various polycrystalline films formed from modified and unmodified DNA. Copyright © 2018. Published by Elsevier B.V.

Shikonin, a natural product from the root of Lithospermum erythrorhizon, is a cytotoxic DNA-binding agent.

PubMed

Chen, Changmin; Shanmugasundaram, Kumaran; Rigby, Alan C; Kung, Andrew L

2013-04-11

To search for compounds that disrupt binding of the EWS-FLI1 fusion protein to its cognate targets, we developed a homogeneous high-throughput proximity assay and screened 5200 small molecule compounds. Many well-known DNA-binding chemotherapeutic agents, such as actinomycin D, cisplatin, doxorubicin, daunorubicin, and epirubicin scored in the assay and not surprising also disrupted the binding of other transcription factors. Unexpectedly, we found that Shikonin, a natural product from the root of Lithospermum erythrorhizon, similarly disrupted protein-DNA interactions. Mechanistic studies demonstrated that Shikonin displaces SYBR green from binding to the minor groove of DNA and is able to inhibit topoisomerase mediated DNA relaxation. In cells, Shikonin blocked the binding of EWS-FLI1 to the NR0B1 promoter, and attenuated gene expression. Shikonin rapidly induced G2/M arrest and apoptosis in Ewing sarcoma cells. These results demonstrate that contrary to other purported mechanisms of action, Shikonin is a DNA-binding cytotoxic agent. Copyright © 2013 Elsevier B.V. All rights reserved.

Evaluation of Novel Imidazotetrazine Analogues Designed to Overcome Temozolomide Resistance and Glioblastoma Regrowth

PubMed Central

Ramirez, Yulian P.; Mladek, Ann C.; Phillips, Roger M.; Gynther, Mikko; Rautio, Jarkko; Ross, Alonzo H.; Wheelhouse, Richard T.; Sakaria, Jann N.

2014-01-01

The cellular responses to two new temozolomide (TMZ) analogues, DP68 and DP86, acting against glioblastoma multiforme (GBM) cell lines and primary culture models are reported. Dose-response analysis of cultured GBM cells revealed that DP68 is more potent than DP86 and TMZ and that DP68 was effective even in cell lines resistant to TMZ. Based on a serial neurosphere assay, DP68 inhibits repopulation of these cultures at low concentrations. The efficacy of these compounds was independent of MGMT and MMR functions. DP68-induced interstrand DNA crosslinks were demonstrated with H2O2-treated cells. Furthermore, DP68 induced a distinct cell cycle arrest with accumulation of cells in S phase that is not observed for TMZ. Consistent with this biological response, DP68 induces a strong DNA damage response, including phosphorylation of ATM, Chk1 and Chk2 kinases, KAP1, and histone variant H2AX. Suppression of FANCD2 expression or ATR expression/kinase activity enhanced anti-glioblastoma effects of DP68. Initial pharmacokinetic analysis revealed rapid elimination of these drugs from serum. Collectively, these data demonstrate that DP68 is a novel and potent anti-glioblastoma compound that circumvents TMZ resistance, likely as a result of its independence from MGMT and mismatch repair and its capacity to crosslink strands of DNA. PMID:25351918

Virtual Screening for the Development of Dual-Inhibitors Targeting Topoisomerase IB and Tyrosyl-DNA Phosphodiesterase 1.

PubMed

Cardamone, Francesca; Pizzi, Simone; Iacovelli, Federico; Falconi, Mattia; Desideri, Alessandro

2017-01-01

Human topoisomerase IB is an important target in cancer therapy and drugs selectively stabilizing the topoisomerase IB-DNA covalent complex are in clinical use for several cancer types. Tyrosyl- DNA phosphodiesterase 1 is involved in the DNA repair resolving the topoisomerase IB-DNA covalent complex that is extremely dangerous for the survival of the cells since it produces an irreversible DNA damage. Given the close biological relationship between these two enzymes, the development of synergistic inhibitors, called dual-inhibitors, is an important challenge in cancer therapy and computer-aided drug design may help in the identification of the best compounds. In this review, an overview of the compounds inhibiting one of the two enzymes or acting as dual inhibitors is provided. Moreover, the general procedures of the virtual screening approach, providing a description of two widely used opensource programs, namely AutoDock4 and AutoDock Vina, are described. Finally, an application of the two programs on a selected number of dual inhibitors for tyrosyl-DNA phosphodiesterase 1 and topoisomerase IB and their performance is briefly discussed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A smart magnetic resonance imaging contrast agent responsive to adenosine based on a DNA aptamer-conjugated gadolinium complex.

PubMed

Xu, Weichen; Lu, Yi

2011-05-07

We report a general strategy for developing a smart MRI contrast agent for the sensing of small molecules such as adenosine based on a DNA aptamer that is conjugated to a Gd compound and a protein streptavidin. The binding of adenosine to its aptamer results in the dissociation of the Gd compound from the large protein, leading to decreases in the rotational correlation time and thus change of MRI contrast. © The Royal Society of Chemistry 2011

Application of Biocatalysis to on-DNA Carbohydrate Library Synthesis.

PubMed

Thomas, Baptiste; Lu, Xiaojie; Birmingham, William R; Huang, Kun; Both, Peter; Reyes Martinez, Juana Elizabeth; Young, Robert J; Davie, Christopher P; Flitsch, Sabine L

2017-05-04

DNA-encoded libraries are increasingly used for the discovery of bioactive lead compounds in high-throughput screening programs against specific biological targets. Although a number of libraries are now available, they cover limited chemical space due to bias in ease of synthesis and the lack of chemical reactions that are compatible with DNA tagging. For example, compound libraries rarely contain complex biomolecules such as carbohydrates with high levels of functionality, stereochemistry, and hydrophilicity. By using biocatalysis in combination with chemical methods, we aimed to significantly expand chemical space and generate generic libraries with potentially better biocompatibility. For DNA-encoded libraries, biocatalysis is particularly advantageous, as it is highly selective and can be performed in aqueous environments, which is an essential feature for this split-and-mix library technology. In this work, we demonstrated the application of biocatalysis for the on-DNA synthesis of carbohydrate-based libraries by using enzymatic oxidation and glycosylation in combination with traditional organic chemistry. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cypermethrin and lambda-cyhalothrin induced in vivo alterations in nucleic acids and protein contents in a freshwater catfish, Clarias batrachus (Linnaeus; Family-Clariidae).

PubMed

Kumar, Amit; Sharma, Bechan; Pandey, Ravi S

2009-08-01

The fresh water fish, Clarias batrachus, were exposed to sub-acute concentrations of cypermethrin and lambda-cyhalothrin for 96 h to assess their impact on the levels of nucleic acids and protein in different organs of fish. DNA content was found almost unchanged with a single exception of liver, which showed significant increment in the levels of DNA in response to the separate treatments of both compounds. Both RNA and protein contents declined in brain, liver, and muscle while sharp increase was observed in gills. However, in kidney, RNA contents depicted significant enhancement only at higher concentrations, with initial decline at lower concentrations. The trends of alterations in RNA/DNA and protein/DNA ratios were quite similar to the corresponding results explained above for RNA and protein. The results clearly indicated that both of these pyrethroids exerted their effects at transcriptional and translational levels while DNA synthesis was found to be unaffected by these compounds with an exception of liver.

Minor groove binding of a bis-quaternary ammonium compound: the crystal structure of SN 7167 bound to d(CGCGAATTCGCG)2.

PubMed Central

Squire, C J; Clark, G R; Denny, W A

1997-01-01

The X-ray crystal structure of the complex between the synthetic antitumour and antiviral DNA binding ligand SN 7167 and the DNA oligonucleotide d(CGCGAATTCGCG)2 has been determined to an R factor of 18.3% at 2.6 A resolution. The ligand is located within the minor groove and covers almost 6 bp with the 1-methylpyridinium ring extending as far as the C9-G16 base pair and the 1-methylquinolinium ring lying between the G4-C21 and A5-T20 base pairs. The ligand interacts only weakly with the DNA, as evidenced by long range contacts and shallow penetration into the groove. This structure is compared with that of the complex between the parent compound SN 6999 and the alkylated DNA sequence d(CGC[e6G]AATTCGCG)2. There are significant differences between the two structures in the extent of DNA bending, ligand conformation and groove binding. PMID:9321660

Human topoisomerase I poisoning: docking protoberberines into a structure-based binding site model

NASA Astrophysics Data System (ADS)

Kettmann, Viktor; Košt'álová, Daniela; Höltje, Hans-Dieter

2004-12-01

Using the X-ray crystal structure of the human topoisomerase I (top1) - DNA cleavable complex and the Sybyl software package, we have developed a general model for the ternary cleavable complex formed with four protoberberine alkaloids differing in the substitution on the terminal phenyl rings and covering a broad range of the top1-poisoning activities. This model has the drug intercalated with its planar chromophore between the -1 and +1 base pairs flanking the cleavage site, with the nonplanar portion pointing into the minor groove. The ternary complexes were geometry-optimized and relative interaction energies, computed by using the Tripos force field, were found to rank in correct order the biological potency of the compounds; in addition, the model is also consistent with the top1-poisoning inactivity of berberine, a major prototype of the protoberberine alkaloids. The model might serve as a rational basis for elaboration of the most active compound as a lead structure, in order to develop more potent top1 poisons as next generation anti-cancer drugs.

Quantitative comparison between in vivo DNA adduct formation from exposure to selected DNA-reactive carcinogens, natural background levels of DNA adduct formation and tumour incidence in rodent bioassays.

PubMed

Paini, Alicia; Scholz, Gabriele; Marin-Kuan, Maricel; Schilter, Benoît; O'Brien, John; van Bladeren, Peter J; Rietjens, Ivonne M C M

2011-09-01

This study aimed at quantitatively comparing the occurrence/formation of DNA adducts with the carcinogenicity induced by a selection of DNA-reactive genotoxic carcinogens. Contrary to previous efforts, we used a very uniform set of data, limited to in vivo rat liver studies in order to investigate whether a correlation can be obtained, using a benchmark dose (BMD) approach. Dose-response data on both carcinogenicity and in vivo DNA adduct formation were available for six compounds, i.e. 2-acetylaminofluorene, aflatoxin B1, methyleugenol, safrole, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and tamoxifen. BMD(10) values for liver carcinogenicity were calculated using the US Environmental Protection Agency BMD software. DNA adduct levels at this dose were extrapolated assuming linearity of the DNA adduct dose response. In addition, the levels of DNA adducts at the BMD(10) were compared to available data on endogenous background DNA damage in the target organ. Although for an individual carcinogen the tumour response increases when adduct levels increase, our results demonstrate that when comparing different carcinogens, no quantitative correlation exists between the level of DNA adduct formation and carcinogenicity. These data confirm that the quantity of DNA adducts formed by a DNA-reactive compound is not a carcinogenicity predictor but that other factors such as type of adduct and mutagenic potential may be equally relevant. Moreover, comparison to background DNA damage supports the notion that the mere occurrence of DNA adducts above or below the level of endogenous DNA damage is neither correlated to development of cancer. These data strongly emphasise the need to apply the mode of action framework to understand the contribution of other biological effect markers playing a role in carcinogenicity.

Effects of Caffeine and Chlorogenic Acid on Propidium Iodide Accessibility to DNA: Consequences on Genome Size Evaluation in Coffee Tree

PubMed Central

NOIROT, M.; BARRE, P.; DUPERRAY, C.; LOUARN, J.; HAMON, S.

2003-01-01

Estimates of genome size using flow cytometry can be biased by the presence of cytosolic compounds, leading to pseudo‐intraspecific variation in genome size. Two important compounds present in coffee trees—caffeine and chlorogenic acid—modify accessibility of the dye propidium iodide to Petunia DNA, a species used as internal standard in our genome size evaluation. These compounds could be responsible for intraspecific variation in genome size since their contents vary between trees. They could also be implicated in environmental variations in genome size, such as those revealed when comparing the results of evaluations carried out on different dates on several genotypes. PMID:12876189

Novel aminopyrimidinyl benzimidazoles as potentially antimicrobial agents: Design, synthesis and biological evaluation.

PubMed

Liu, Han-Bo; Gao, Wei-Wei; Tangadanchu, Vijai Kumar Reddy; Zhou, Cheng-He; Geng, Rong-Xia

2018-01-01

A series of novel aminopyrimidinyl benzimidazoles as potentially antimicrobial agents were designed, synthesized and characterized by IR, NMR and HRMS spectra. The biological evaluation in vitro revealed that some of the target compounds exerted good antibacterial and antifungal activity in comparison with the reference drugs. Noticeably, compound 7d could effectively inhibit the growth of A. flavus, E. coli DH52 and MRSA with MIC values of 1, 1 and 8 μg/mL, respectively. Further studies revealed that pyrimidine derivative 7d could exhibit bactericidal mode of action against both Gram positive (S. aureus and MRSA) and Gram negative (P. aeruginosa) bacteria. The active molecule 7d showed low cell toxicity and did not obviously trigger the development of resistance in bacteria even after 16 passages. Furthermore, compound 7d was able to beneficially regulate reactive oxygen species (ROS) generation for an excellent safety profile. Molecular docking study revealed that compound 7d could bind with DNA gyrase by the formation of hydrogen bonds. The preliminary exploration for antimicrobial mechanism disclosed that compound 7d could effectively intercalate into calf thymus DNA to form a steady supramolecular complex, which might further block DNA replication to exert the powerful bioactivities. The binding investigation of compound 7d with human serum albumins (HSA) revealed that this molecule could be effectively transported by HSA. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Melatonin and hydroxytyrosol-rich wines influence the generation of DNA oxidation catabolites linked to mutagenesis after the ingestion of three types of wine by healthy volunteers.

PubMed

Marhuenda, Javier; Medina, Sonia; Martínez-Hernández, Pedro; Arina, Simón; Zafrilla, Pilar; Mulero, Juana; Genieser, Hans-Gottfried; Ferreres, Federico; Gil-Izquierdo, Ángel

2016-12-07

The Mediterranean Diet (MD) has been proved to exert benefits with respect to the maintenance of the redox balance, and wine is a representative component. Bioactive compounds such as polyphenols, melatonin and hydroxytyrosol act as radical scavengers and regulate the oxidation status of organisms. Oxidative damage to DNA yields a large range of end products. The repair of oxidized DNA entails the removal of the useless bases and/or nucleotides as well as the release of circulating nucleotides and nucleosides. The current research aims to elucidate, for the first time, the DNA protection against oxidative stress provided by three types of red wine - relating it to the intake of bioactive compounds - after the intake of a serving of red wine/must by 18 healthy female volunteers during a short term double-blind, crossover and placebo-controlled study. The novelty of our work is to describe the importance of melatonin and hydroxytyrosol and its metabolites (from gut microflora) in comparison with polyphenols in a red wine matrix (excluding colon derivatives). The results show that the intake of red wine and must secondarily reduces oxidative stress and carcinogenesis due to their content of homovanillic acid, as measured by decreases in the plasmatic concentration of 8-hydroxy-2'deoxyguanosine, 8-hydroxyguanine, and 8-nitroguanosine. Moreover, the intake of wine appears to exert vasodilatory effects, mediated by the action of nitric oxide and increased plasma guanosine-3'-5'-cyclic monophosphate plasmatic levels, owing to the intake of wines higher in melatonin and homovanillic acid. Therefore, the results obtained in the present study revealed that polyphenols, despite being the major compounds in the red wine matrix, are not the most effective compounds protecting DNA from oxidative attack.

Microbial mutagenic effects of the DNA minor groove binder pibenzimol (Hoechst 33258) and a series of mustard analogues.

PubMed

Ferguson, L R; Denny, W A

1995-06-01

A series of aniline mustards and half-mustards targeted to DNA by linkage (through a polymethylene chain) to the bisbenzimidazole chromophore of pibenzimol (Hoechst 33258) have been evaluated for their mutagenic properties, as estimated in three strains of Salmonella typhimurium, and for their mitotic crossing-over and petite mutagenesis activities in Saccharomyces cerevisiae strain D5. Agarose gel electrophoresis studies showed that only the derivative with the longest linker chain cross-linked DNA, with the remaining compounds being monoalkylators. The parent (non-alkylator) minor groove binding ligand (Hoechst 33258) was inactive in the bacterial strains TA98 or TA100 but weakly mutagenic in TA102, and caused neither mitotic crossing-over nor 'petite' mutagenesis in yeast. Aniline half-mustard itself (monoalkylator) was an effective base-pair substitution mutagen (events in S. typhimurium strain TA100) with some frameshift mutagenesis activity in TA98, but showed only weak effects in the yeast assays, whereas aniline mustard (cross-linker) was inactive in these bacterial systems but caused substantial amounts of mitotic crossing-over in yeast. The composite molecules studied here showed effects more characteristic of the minor groove binding chromophore than of alkylating moieties. All showed weak mutagenic activity in TA102 and none in TA98. The only compound to show significant mitotic crossing-over ability was the long-chain derivative which cross-linked DNA. For most of the compounds, the mutagenicity data provided no supportive evidence for DNA alkylation. Since other evidence suggests this does occur readily, it is likely to have a different target to that seen with untargeted aniline mustards. The significant antitumor activity and low mutagenic potential shown by these compounds make them worthy of further study.

32P-postlabeling analysis of dibenz[a,j]acridine DNA adducts in mice: preliminary determination of initial genotoxic metabolites and their effect on biomarker levels.

PubMed

Roh, J; Schamer, M; Reilman, R; Xue, W; Warshawsky, D; Talaska, G

1993-01-01

N-Heterocyclic aromatics (NHA) are widely occurring environmental pollutants formed during the pyrolysis of nitrogen-containing organic chemicals. NHA are found in significant amounts in tobacco condensates, synthetic fuels, gasoline engine exhaust, and effluents from the heating of coal. Dibenz[a,j]acridine (DBA) is an example of NHA. The potency of many carcinogenic compounds is related, at least in part, to the efficiency of their biological activation. We undertook studies to determine which initial metabolites of DBA lead to the formation of high levels of carcinogen-DNA adducts in vivo. DBA and its metabolites, trans-DBA-1,2-dihydrodiol (DBA-1,2-DHD), trans-DBA-3,4-dihydrodiol (DBA-3,4-DHD), and trans-DBA-5,6-dihydrodiol (DBA-5,6-DHD), were applied to the skin of mice. DNA was isolated using enzyme-solvent extraction method. DNA was 32P-postlabeled under conditions of limiting [32P]ATP. In skin, DBA produced two distinct adducts. The same two adducts were seen when DBA-3,4-DHD was applied. In addition the total adduct level elicited by DBA-3,4-DHD was higher than that of parent compound. Two adducts were seen when DBA-5,6DHD was applied, but these were very different from adducts seen with DBA. These results suggested that activation of DBA to DNA-binding compounds in skin includes initial formation of DBA-3,4-DHD. The data support development of biomarkers for the exposure and effect of this compound, and also suggest that specific metabolic susceptibility markers might be able to predict populations at increased risk.

Dual targeting of GyrB and ParE by a novel aminobenzimidazole class of antibacterial compounds.

PubMed

Grossman, Trudy H; Bartels, Douglas J; Mullin, Steve; Gross, Christian H; Parsons, Jonathan D; Liao, Yusheng; Grillot, Anne-Laure; Stamos, Dean; Olson, Eric R; Charifson, Paul S; Mani, Nagraj

2007-02-01

A structure-guided drug design approach was used to optimize a novel series of aminobenzimidazoles that inhibit the essential ATPase activities of bacterial DNA gyrase and topoisomerase IV and that show potent activities against a variety of bacterial pathogens. Two such compounds, VRT-125853 and VRT-752586, were characterized for their target specificities and preferences in bacteria. In metabolite incorporation assays, VRT-125853 inhibited both DNA and RNA synthesis but had little effect on protein synthesis. Both compounds inhibited the maintenance of negative supercoils in plasmid DNA in Escherichia coli at the MIC. Sequencing of DNA corresponding to the GyrB and ParE ATP-binding regions in VRT-125853- and VRT-752586-resistant mutants revealed that their primary target in Staphylococcus aureus and Haemophilus influenzae was GyrB, whereas in Streptococcus pneumoniae it was ParE. In Enterococcus faecalis, the primary target of VRT-125853 was ParE, whereas for VRT-752586 it was GyrB. DNA transformation experiments with H. influenzae and S. aureus proved that the mutations observed in gyrB resulted in decreased susceptibilities to both compounds. Novobiocin resistance-conferring mutations in S. aureus, H. influenzae, and S. pneumoniae were found in gyrB, and these mutants showed little or no cross-resistance to VRT-125853 or VRT-752586 and vice versa. Furthermore, gyrB and parE double mutations increased the MICs of VRT-125853 and VRT-752586 significantly, providing evidence of dual targeting. Spontaneous frequencies of resistance to VRT-752586 were below detectable levels (<5.2x10(-10)) for wild-type E. faecalis but were significantly elevated for strains containing single and double target-based mutations, demonstrating that dual targeting confers low levels of resistance emergence and the maintenance of susceptibility in vitro.

Novobiocin: redesigning a DNA gyrase inhibitor for selective inhibition of hsp90.

PubMed

Burlison, Joseph A; Neckers, Len; Smith, Andrew B; Maxwell, Anthony; Blagg, Brian S J

2006-12-06

Novobiocin is a member of the coumermycin family of antibiotics and is a well-established inhibitor of DNA gyrase. Recent studies have shown that novobiocin binds to a previously unrecognized ATP-binding site at the C-terminus of Hsp90 and induces degradation of Hsp90-dependent client proteins at approximately 700 microM. In an effort to develop more efficacious inhibitors of the C-terminal binding site, a library of novobiocin analogues was prepared and initial structure-activity relationships revealed. These data suggested that the 4-hydroxy moiety of the coumarin ring and the 3'-carbamate of the noviose appendage were detrimental to Hsp90 inhibitory activity. In an effort to confirm these findings, 4-deshydroxy novobiocin (DHN1) and 3'-descarbamoyl-4-deshydroxynovobiocin (DHN2) were prepared and evaluated against Hsp90. Both compounds were significantly more potent than the natural product, and DHN2 proved to be more active than DHN1. In an effort to determine whether these moieties are important for DNA gyrase inhibition, these compounds were tested for their ability to inhibit DNA gyrase and found to exhibit significant reduction in gyrase activity. Thus, we have established the first set of compounds that clearly differentiate between the C-terminus of Hsp90 and DNA gyrase, converted a well-established gyrase inhibitor into a selective Hsp90 inhibitor, and confirmed essential structure-activity relationships for the coumermycin family of antibiotics.

Morphological and molecular identification of filamentous Aspergillus flavus and Aspergillus parasiticus isolated from compound feeds in South Africa.

PubMed

Iheanacho, Henry E; Njobeh, Patrick B; Dutton, Francis M; Steenkamp, Paul A; Steenkamp, Lucia; Mthombeni, Julian Q; Daru, Barnabas H; Makun, Anthony H

2014-12-01

Isolation of filamentous species of two Aspergillum genera from compound feeds produced in South Africa, and subsequent extraction of their individual DNA in this study, presents a simple but rapid molecular procedure for high through-put analysis of the individual morphological forms. DNA was successfully isolated from the Aspergillus spp. from agar cultures by use of a commercial kit. Agarose gel electrophoresis fractionation of the fungi DNA, showed distinct bands. The DNA extracted by this procedure appears to be relatively pure with a ratio absorbance at 260 and 280 nm. However, the overall morphological and molecular data indicated that 67.5 and 51.1% of feed samples were found to be contaminated with Aspergillus flavus and Aspergillus parasiticus, respectively, with poultry feed having the highest contamination mean level of 5.7 × 105 CFU/g when compared to cattle (mean: 4.0 × 106 CFU/g), pig (mean: 2.7 × 104 CFU/g) and horse (1.0 × 102 CFU) feed. This technique presents a readily achievable, easy to use method in the extraction of filamentous fungal DNA and it's identification. Hence serves as an important tool towards molecular study of these organisms for routine analysis check in monitoring and improving compound feed quality against fungal contamination. Copyright © 2014 Elsevier Ltd. All rights reserved.

Hydrogen Peroxide Activated Quinone Methide Precursors with Enhanced DNA Cross-Linking Capability and Cytotoxicity towards Cancer Cells

PubMed Central

Wang, Yibin; Fan, Heli; Balakrishnan, Kumudha; Lin, Zechao; Cao, Sheng; Chen, Wenbing; Fan, Yukai; Guthrie, Quibria A.; Sun, Huabing; Teske, Kelly A.; Gandhi, Varsha; Arnold, Leggy A.; Peng, Xiaohua

2017-01-01

Quinone methide (QM) formation induced by endogenously generated H2O2 is attractive for biological and biomedical applications. To overcome current limitations due to low biological activity of H2O2-activated QM precursors, we are introducing herein several new arylboronates with electron donating substituents at different positions of benzene ring and/or different neutral leaving groups. The reaction rate of the arylboronate esters with H2O2 and subsequent bisquinone methides formation and DNA cross-linking was accelerated with the application of Br as a leaving group instead of acetoxy groups. Additionally, a donating group placed meta to the nascent exo-methylene group of the quinone methide greatly improves H2O2-induced DNA interstrand cross-link formation as well as enhances the cellular activity. Multiple donating groups decrease the stability and DNA cross-linking capability, which lead to low cellular activity. A cell-based screen demonstrated that compounds 2a and 5a with a OMe or OH group dramatically inhibited the growth of various tissue-derived cancer cells while normal cells were less affected. Induction of H2AX phosphorylation by these compounds in CLL lymphocytes provide evidence for a correlation between cell death and DNA damage. The compounds presented herein showed potent anticancer activities and selectivity, which represent a novel scaffold for anticancer drug development. PMID:28388522

Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents

PubMed Central

Klapacz, Joanna; Pottenger, Lynn H.; Engelward, Bevin P.; Heinen, Christopher D.; Johnson, George E.; Clewell, Rebecca A.; Carmichael, Paul L.; Adeleye, Yeyejide; Andersen, Melvin E.

2016-01-01

From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations. PMID:27036068

Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents.

PubMed

Klapacz, Joanna; Pottenger, Lynn H; Engelward, Bevin P; Heinen, Christopher D; Johnson, George E; Clewell, Rebecca A; Carmichael, Paul L; Adeleye, Yeyejide; Andersen, Melvin E

2016-01-01

From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance of a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations. Copyright © 2015 Elsevier B.V. All rights reserved.

Key role of hydrogen peroxide in antimicrobial activity of spring, Honeydew maquis and chestnut grove Corsican honeys on Pseudomonas aeruginosa DNA.

PubMed

Poli, J-P; Guinoiseau, E; Luciani, A; Yang, Y; Battesti, M-J; Paolini, J; Costa, J; Quilichini, Y; Berti, L; Lorenzi, V

2018-05-01

In honeys, several molecules have been known for their antibacterial or wound healing properties. Corsican honeys just began to be tested for their antimicrobial activity with promising results on Pseudomonas aeruginosa. So, identification of active molecules and their mode of action was determined. Hydrogen peroxide concentrations were evaluated and, in parallel, the minimal inhibitory concentrations (MIC) values were performed with and without catalase. More, the quantity of phenolic compounds and ORAC assay were measured. Observation of antibacterial action was done using scanning electron microscopy (SEM) followed by plasmidic DNA extraction. MIC values of chestnut grove and honeydew maquis honeys vary between 7 and 8%, showing a strong antimicrobial capacity, associated with a plasmidic DNA degradation. When catalase is added, MIC values significatively increase (25%) without damaging DNA, proving the importance of H 2 O 2 . This hypothesis is confirmed by SEM micrographies which did not show any morphological damages but a depletion in bacterial population. Although, such low concentrations of H 2 O 2 (between 23 μmol l -1 and 54 μmol l -1 ) cannot explain antimicrobial activity and might be correlated with phenolic compounds concentration. Thus, Corsican honeys seem to induce DNA damage when H 2 O 2 and phenolic compounds act in synergy by a putative pro-oxidant effect. We started to determine the antibacterial efficiency of Corsican chestnut grove and honeydew maquis honeys on Pseudomonas aeruginosa. No morphological alteration of the bacterial surface was observed. Antimicrobial action seems to be related to the synergy between hydrogen peroxide and phenolic compounds. The exerted pro-oxidant activity leads to a degradation of P. aeruginosa plasmidic DNA. This is the first study that investigate the primary antibacterial mechanism of Corsican honeys. © 2018 The Society for Applied Microbiology.

c-MYC G-quadruplex binding by the RNA polymerase I inhibitor BMH-21 and analogues revealed by a combined NMR and biochemical Approach.

PubMed

Musso, Loana; Mazzini, Stefania; Rossini, Anna; Castagnoli, Lorenzo; Scaglioni, Leonardo; Artali, Roberto; Di Nicola, Massimo; Zunino, Franco; Dallavalle, Sabrina

2018-03-01

Pyridoquinazolinecarboxamides have been reported as RNA polymerase I inhibitors and represent a novel class of potential antitumor agents. BMH-21, was reported to intercalate with GC-rich rDNA, resulting in nucleolar stress as a primary mechanism of cytotoxicity. The interaction of BMH-21 and analogues with DNA G-quadruplex structures was studied by NMR and molecular modelling. The cellular response was investigated in a panel of human tumor cell lines and protein expression was examined by Western Blot analysis. We explored the ability of BMH-21 and its analogue 2 to bind to G-quadruplex present in the c-MYC promoter, by NMR and molecular modelling studies. We provide evidence that both compounds are not typical DNA intercalators but are effective binders of the tested G-quadruplex. The interaction with c-MYC G-quadruplex was reflected in down-regulation of c-Myc expression in human tumor cells. The inhibitory effect was almost complete in lymphoma cells SUDHL4 characterized by overexpression of c-Myc protein. This downregulation reflected an early and persistent modulation of cMyc mRNA. Given the relevance of c-MYC in regulation of ribosome biogenesis, it is conceivable that the inhibition of c-MYC contributes to the perturbation of nuclear functions and RNA polymerase I activity. Similar experiments with CX-5461, another RNA polymerase I transcription inhibitor, indicate the same behaviour in G-quadruplex stabilization. Our results support the hypothesis that BMH-21 and analogue compounds share the same mechanism, i.e. G-quadruplex binding as a primary event of a cascade leading to inhibition of RNA polymerase I and apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Mitochondrial encephalomyopathy and retinoblastoma explained by compound heterozygosity of SUCLA2 point mutation and 13q14 deletion

PubMed Central

Matilainen, Sanna; Isohanni, Pirjo; Euro, Liliya; Lönnqvist, Tuula; Pihko, Helena; Kivelä, Tero; Knuutila, Sakari; Suomalainen, Anu

2015-01-01

Mutations in SUCLA2, encoding the ß-subunit of succinyl-CoA synthetase of Krebs cycle, are one cause of mitochondrial DNA depletion syndrome. Patients have been reported to have severe progressive childhood-onset encephalomyopathy, and methylmalonic aciduria, often leading to death in childhood. We studied two families, with children manifesting with slowly progressive mitochondrial encephalomyopathy, hearing impairment and transient methylmalonic aciduria, without mtDNA depletion. The other family also showed dominant inheritance of bilateral retinoblastoma, which coexisted with mitochondrial encephalomyopathy in one patient. We found a variant in SUCLA2 leading to Asp333Gly change, homozygous in one patient and compound heterozygous in one. The latter patient also carried a deletion of 13q14 of the other allele, discovered with molecular karyotyping. The deletion spanned both SUCLA2 and RB1 gene regions, leading to manifestation of both mitochondrial disease and retinoblastoma. We made a homology model for human succinyl-CoA synthetase and used it for structure–function analysis of all reported pathogenic mutations in SUCLA2. On the basis of our model, all previously described mutations were predicted to result in decreased amounts of incorrectly assembled protein or disruption of ADP phosphorylation, explaining the severe early lethal manifestations. However, the Asp333Gly change was predicted to reduce the activity of the otherwise functional enzyme. On the basis of our findings, SUCLA2 mutations should be analyzed in patients with slowly progressive encephalomyopathy, even in the absence of methylmalonic aciduria or mitochondrial DNA depletion. In addition, an encephalomyopathy in a patient with retinoblastoma suggests mutations affecting SUCLA2. PMID:24986829

An Ultraviolet Resonance Raman Spectroscopic Study of Cisplatin and Transplatin Interactions with Genomic DNA.

PubMed

Geng, Jiafeng; Aioub, Mena; El-Sayed, Mostafa A; Barry, Bridgette A

2017-09-28

Ultraviolet resonance Raman (UVRR) spectroscopy is a label-free method to define biomacromolecular interactions with anticancer compounds. Using UVRR, we describe the binding interactions of two Pt(II) compounds, cisplatin (cis-diamminedichloroplatinum(II)) and its isomer, transplatin, with nucleotides and genomic DNA. Cisplatin binds to DNA and other cellular components and triggers apoptosis, whereas transplatin is clinically ineffective. Here, a 244 nm UVRR study shows that purine UVRR bands are altered in frequency and intensity when mononucleotides are treated with cisplatin. This result is consistent with previous suggestions that purine N7 provides the cisplatin-binding site. The addition of cisplatin to DNA also causes changes in the UVRR spectrum, consistent with binding of platinum to purine N7 and disruption of hydrogen-bonding interactions between base pairs. Equally important is that transplatin treatment of DNA generates similar UVRR spectral changes, when compared to cisplatin-treated samples. Kinetic analysis, performed by monitoring decreases of the 1492 cm -1 band, reveals biphasic kinetics and is consistent with a two-step binding mechanism for both platinum compounds. For cisplatin-DNA, the rate constants (6.8 × 10 -5 and 6.5 × 10 -6 s -1 ) are assigned to the formation of monofunctional adducts and to bifunctional, intrastrand cross-linking, respectively. In transplatin-DNA, there is a 3.4-fold decrease in the rate constant of the slow phase, compared with the cisplatin samples. This change is attributed to generation of interstrand, rather than intrastrand, adducts. This longer reaction time may result in increased competition in the cellular environment and account, at least in part, for the lower pharmacological efficacy of transplatin.

Effect of short peptides containing lysine and epsilon-aminocaproic acid on fibrinolytic activity of plasmin and topoisomerase II action on supercoiled DNA.

PubMed

Midura-Nowaczek, Krystyna; Purwin, Maciej; Markowska, Agnieszka; Drozdowska, Danuta; Bruzgo, Magdalena

2013-01-01

Effects of eight short peptides containing lysine and epsilon-aminocaproic acid (EACA) on prolongation of the clot lysis time, as well as hemolytic and antibacterial activities were investigated. Interaction with plasmids pBR322 and pUC19 with the use of ethidium bromide assay and determination of influence on the activity of topoisomerase I and II were also tested. Examined compounds inhibited fibrinolytic activity of plasmin and five of them were more active than EACA. Amides of dipeptides were most active antifibrinolytics (IC50 < 0.2 mM). According to the obtained data, the significant inhibition of fibrinolytic activity of plasmin was not associated with hemolytic effects. Examined compounds did not show antibacterial activity (MIC > 512 mg/L). DNA binding effects determined with the use of ethidium bromide were weak for all peptides and similar to those observed with EACA. Six compounds inhibited topoisomerase II action on supercoiled DNA.

Novel Bacterial Topoisomerase Inhibitors with Potent Broad-Spectrum Activity against Drug-Resistant Bacteria.

PubMed

Charrier, Cédric; Salisbury, Anne-Marie; Savage, Victoria J; Duffy, Thomas; Moyo, Emmanuel; Chaffer-Malam, Nathan; Ooi, Nicola; Newman, Rebecca; Cheung, Jonathan; Metzger, Richard; McGarry, David; Pichowicz, Mark; Sigerson, Ralph; Cooper, Ian R; Nelson, Gary; Butler, Hayley S; Craighead, Mark; Ratcliffe, Andrew J; Best, Stuart A; Stokes, Neil R

2017-05-01

The novel bacterial topoisomerase inhibitor class is an investigational type of antibacterial inhibitor of DNA gyrase and topoisomerase IV that does not have cross-resistance with the quinolones. Here, we report the evaluation of the in vitro properties of a new series of this type of small molecule. Exemplar compounds selectively and potently inhibited the catalytic activities of Escherichia coli DNA gyrase and topoisomerase IV but did not block the DNA breakage-reunion step. Compounds showed broad-spectrum inhibitory activity against a wide range of Gram-positive and Gram-negative pathogens, including biodefence microorganisms and Mycobacterium tuberculosis No cross-resistance with fluoroquinolone-resistant Staphylococcus aureus and E. coli isolates was observed. Measured MIC 90 values were 4 and 8 μg/ml against a panel of contemporary multidrug-resistant isolates of Acinetobacter baumannii and E. coli , respectively. In addition, representative compounds exhibited greater antibacterial potency than the quinolones against obligate anaerobic species. Spontaneous mutation rates were low, with frequencies of resistance typically <10 -8 against E. coli and A. baumannii at concentrations equivalent to 4-fold the MIC. Compound-resistant E. coli mutants that were isolated following serial passage were characterized by whole-genome sequencing and carried a single Arg38Leu amino acid substitution in the GyrA subunit of DNA gyrase. Preliminary in vitro safety data indicate that the series shows a promising therapeutic index and potential for low human ether-a-go-go-related gene (hERG) inhibition (50% inhibitory concentration [IC 50 ], >100 μM). In summary, the compounds' distinct mechanism of action relative to the fluoroquinolones, whole-cell potency, low potential for resistance development, and favorable in vitro safety profile warrant their continued investigation as potential broad-spectrum antibacterial agents. Copyright © 2017 American Society for Microbiology.

The insecticide buprofezin induces morphological transformation and kinetochore-positive micronuclei in cultured Syrian hamster embryo cells in the absence of detectable DNA damage.

PubMed

Herrera, L A; Ostrosky-Wegman, P; Schiffmann, D; Chen, Q Y; Ziegler-Skylakakis, K; Andrae, U

1993-11-01

The insecticide buprofezin was examined for its genotoxicity in cultured Syrian hamster embryo cells in order to better understand the mechanisms underlying the genotoxicity of the compound in mammalian cells. Exposure to buprofezin concentrations of 12.5-100 microM did not significantly affect the colony-forming ability of the cells, but did result in increased frequencies of morphologically transformed colonies. Treatment with buprofezin did not cause a detectable induction of DNA repair synthesis, an indicator of DNA damage, but significantly increased the frequency of micronuclei. Immunostaining of the cells with antikinetochore antibody (CREST antibody) showed that essentially all of the buprofezin-induced micronuclei were kinetochore-positive. The results suggest that morphological transformation of Syrian hamster embryo cells by buprofezin results from an interaction of the compound or a metabolite of it with the mitotic apparatus rather than from DNA damage.

Genotoxicity Assessment of Volatile Organic Compounds in Landfill Gas Emission Using Comet Assay in Higher Terrestrial Plant.

PubMed

Na Roi-Et, Veerapas; Chiemchaisri, Wilai; Chiemchaisri, Chart

2017-02-01

Genotoxicity model is developed to assess the individual subacute toxicity of benzene, toluene, ethylbenzene, and xylene (BTEX) at very low levels as in a landfill gas. Golden Pothos (Epipremnum aureum), a higher plant, was tested under variation of benzene 54-5656 ng/L, toluene 10-4362 ng/L, ethylbenzene 28-4997 ng/L, xylene 53-4845 ng/L, for 96 h. DNA fragmentation in plant leaves were investigated via comet assay. The results show that DNA migration ratio increased with the BTEX concentrations, but at different rates. The 50% effective concentration (EC 50 ) of DNA fragmentation from the dose-response relationships indicated toluene has the highest EC 50 value and followed by benzene, xylene and ethylbenzene. Alternatively, ethylbenzene has the highest toxicity unit and followed by xylene, benzene and toluene as described by toxicity unit (TU). In conclusion, comet assay of Pothos can be used in differentiating DNA fragmentation against very low levels of BTEX in the atmosphere. Pothos is recommended for genotoxicity assessment of a low BTEX contaminated atmosphere.

Colon Cancer Chemoprevention by Sage Tea Drinking: Decreased DNA Damage and Cell Proliferation.

PubMed

Pedro, Dalila F N; Ramos, Alice A; Lima, Cristovao F; Baltazar, Fatima; Pereira-Wilson, Cristina

2016-02-01

Salvia officinalis and some of its isolated compounds have been found to be preventive of DNA damage and increased proliferation in vitro in colon cells. In the present study, we used the azoxymethane model to test effects of S. officinalis on colon cancer prevention in vivo. The results showed that sage treatment reduced the number of ACF formed only if administered before azoxymethane injection, demonstrating that sage tea drinking has a chemopreventive effect on colorectal cancer. A decrease in the proliferation marker Ki67 and in H2 O2 -induced and azoxymethane-induced DNA damage to colonocytes and lymphocytes were found with sage treatment. This confirms in vivo the chemopreventive effects of S. officinalis. Taken together, our results show that sage treatment prevented initiation phases of colon carcinogenesis, an effect due, at least in part, to DNA protection, and reduced proliferation rates of colon epithelial cell that prevent mutations and their fixation through cell replication. These chemopreventive effects of S. officinalis on colon cancer add to the many health benefits attributed to sage and encourage its consumption. Copyright © 2015 John Wiley & Sons, Ltd.

Crystal structure of checkpoint kinase 2 in complex with NSC 109555, a potent and selective inhibitor

PubMed Central

Lountos, George T; Tropea, Joseph E; Zhang, Di; Jobson, Andrew G; Pommier, Yves; Shoemaker, Robert H; Waugh, David S

2009-01-01

Checkpoint kinase 2 (Chk2), a ser/thr kinase involved in the ATM-Chk2 checkpoint pathway, is activated by genomic instability and DNA damage and results in either arrest of the cell cycle to allow DNA repair to occur or apoptosis if the DNA damage is severe. Drugs that specifically target Chk2 could be beneficial when administered in combination with current DNA-damaging agents used in cancer therapy. Recently, a novel inhibitor of Chk2, NSC 109555, was identified that exhibited high potency (IC50 = 240 nM) and selectivity. This compound represents a new chemotype and lead for the development of novel Chk2 inhibitors that could be used as therapeutic agents for the treatment of cancer. To facilitate the discovery of new analogs of NSC 109555 with even greater potency and selectivity, we have solved the crystal structure of this inhibitor in complex with the catalytic domain of Chk2. The structure confirms that the compound is an ATP-competitive inhibitor, as the electron density clearly reveals that it occupies the ATP-binding pocket. However, the mode of inhibition differs from that of the previously studied structure of Chk2 in complex with debromohymenialdisine, a compound that inhibits both Chk1 and Chk2. A unique hydrophobic pocket in Chk2, located very close to the bound inhibitor, presents an opportunity for the rational design of compounds with higher binding affinity and greater selectivity. PMID:19177354

Cardanol: toxicogenetic assessment and its effects when combined with cyclophosphamide

PubMed Central

Schneider, Beatriz Ursinos Catelan; Meza, Alisson; Beatriz, Adilson; Pesarini, João Renato; de Carvalho, Pamela Castilho; Mauro, Mariana de Oliveira; Karaziack, Caroline Bilhar; Cunha-Laura, Andréa Luiza; Monreal, Antônio Carlos Duenhas; Matuo, Renata; de Lima, Dênis Pires; Oliveira, Rodrigo Juliano

2016-01-01

Abstract Cardanol is an effective antioxidant and is a compound with antimutagenic and antitumoral activity. Here, we evaluated the genotoxic and mutagenic potential of saturated side chain cardanol and its effects in combination with cyclophosphamide in preventing DNA damage, apoptosis, and immunomodulation. Swiss mice were treated with cardanol (2.5, 5 and 10 mg/kg) alone or in combination with cyclophosphamide (100 mg/kg). The results showed that cardanol is an effective chemopreventive compound, with damage reduction percentages that ranged from 18.9 to 31.76% in the comet assay and from 45 to 97% in the micronucleus assay. Moreover, cardanol has the ability to reduce the frequency of apoptosis induced by cyclophosphamide. The compound did not show immunomodulatory activity. A final interpretation of the data showed that, despite its chemoprotective capacity, cardanol has a tendency to induce DNA damage. Hence, caution is needed if this compound is used as a chemopreventive agent. Also, this compound is likely not suitable as an adjuvant in chemotherapy treatments that use cyclophosphamide. PMID:27303909

Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines.

PubMed

Mohammed, M Z; Vyjayanti, V N; Laughton, C A; Dekker, L V; Fischer, P M; Wilson, D M; Abbotts, R; Shah, S; Patel, P M; Hickson, I D; Madhusudan, S

2011-02-15

Modulation of DNA base excision repair (BER) has the potential to enhance response to chemotherapy and improve outcomes in tumours such as melanoma and glioma. APE1, a critical protein in BER that processes potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer. In the current study, we aimed to develop small molecule inhibitors of APE1 for cancer therapy. An industry-standard high throughput virtual screening strategy was adopted. The Sybyl8.0 (Tripos, St Louis, MO, USA) molecular modelling software suite was used to build inhibitor templates. Similarity searching strategies were then applied using ROCS 2.3 (Open Eye Scientific, Santa Fe, NM, USA) to extract pharmacophorically related subsets of compounds from a chemically diverse database of 2.6 million compounds. The compounds in these subsets were subjected to docking against the active site of the APE1 model, using the genetic algorithm-based programme GOLD2.7 (CCDC, Cambridge, UK). Predicted ligand poses were ranked on the basis of several scoring functions. The top virtual hits with promising pharmaceutical properties underwent detailed in vitro analyses using fluorescence-based APE1 cleavage assays and counter screened using endonuclease IV cleavage assays, fluorescence quenching assays and radiolabelled oligonucleotide assays. Biochemical APE1 inhibitors were then subjected to detailed cytotoxicity analyses. Several specific APE1 inhibitors were isolated by this approach. The IC(50) for APE1 inhibition ranged between 30 nM and 50 μM. We demonstrated that APE1 inhibitors lead to accumulation of AP sites in genomic DNA and potentiated the cytotoxicity of alkylating agents in melanoma and glioma cell lines. Our study provides evidence that APE1 is an emerging drug target and could have therapeutic application in patients with melanoma and glioma.

Multispectroscopic investigation of the interaction of BSA and DNA with the anticancer drug, N-(6-ferrocenyl-2-naphthoyl)-gamma-amino butyric acid methyl ester

NASA Astrophysics Data System (ADS)

Rajina, S. R.; Sudhi, Geethu; Austin, P.; Praveen, S. G.; Xavier, T. S.; Kenny, Peter T. M.; Binoy, J.

2018-05-01

The interaction of a drug with DNA and BSA play a great role in studying anti cancer activity and drug transport properties, which can be effectively, investigated using vibrational spectroscopy, UV visible spectroscopy and Fluorescence spectroscopy. The present work reports the structural features of N-(6-ferrocenyl-2-naphthoyl)-gamma-amino butyric acid Methyl ester (FNGABME) based on FTIR and FTRaman spectroscopy. The absorption and fluorescence spectroscopic methods were used to study the efficiency of the interaction of the compound FNGABME with BSA and DNA and also molecular docking were performed computationally to validate the results which shows that the title compound may exhibit inhibitory activity against the cancer cells.

Synthesis and characterization of new unsymmetrical Schiff base Zn (II) and Co (II) complexes and study of their interactions with bovin serum albumin and DNA by spectroscopic techniques.

PubMed

Sedighipoor, Maryam; Kianfar, Ali Hossein; Sabzalian, Mohammad R; Abyar, Fatemeh

2018-06-05

Two novel tetra-coordinated Cobalt(II) and Zinc (II) chelate series with the general formula of [Co (L)·2H 2 O] (1) and [Zn (L)] (2) [L=N-2-hydroxyacetophenon-N'-2-hydroxynaphthaldehyde-1,2 phenylenediimine)] with biologically active Schiff base ligands were synthesized and recognized by elemental analysis and multi-nuclear spectroscopy (IR and 1 H and 13 C NMR); then, their biological activities including DNA and protein interactions were studied. The interaction of the synthesized compounds with bovine serum albumin (BSA) was investigated via fluorescence spectroscopy, showing the affinity of the complexes for these proteins with relatively high binding constant values and the changed secondary BSA structure in the presence of the complexes. The interaction of these compounds with CT-DNA was considered by UV-Vis technique, emission titration, viscosity measurements, helix melting methods, and circular dichroism (CD) spectroscopy, confirming that the complexes were bound to CT-DNA by the intercalation binding mode. Furthermore, the complexes had the capability to displace the DNA-bound MB, as shown by the competitive studies of these complexes with methylene blue (MB), thereby suggesting the intercalation mode for the competition. Finally, the theoretical studies carried out by the docking method were performed to calculate the binding constants and recognize the binding site of the BSA and DNA by the complexes. In addition, in vitro and in silico studies showed that the compounds were degradable by bacterial and fungal biodegradation activities. Copyright © 2018 Elsevier B.V. All rights reserved.

Synthesis and characterization of new unsymmetrical Schiff base Zn (II) and Co (II) complexes and study of their interactions with bovin serum albumin and DNA by spectroscopic techniques

NASA Astrophysics Data System (ADS)

Sedighipoor, Maryam; Kianfar, Ali Hossein; Sabzalian, Mohammad R.; Abyar, Fatemeh

2018-06-01

Two novel tetra-coordinated Cobalt(II) and Zinc (II) chelate series with the general formula of [Co (L)·2H2O] (1) and [Zn (L)] (2) [L = N-2-hydroxyacetophenon-N‧-2-hydroxynaphthaldehyde-1,2 phenylenediimine)] with biologically active Schiff base ligands were synthesized and recognized by elemental analysis and multi-nuclear spectroscopy (IR and 1H and 13C NMR); then, their biological activities including DNA and protein interactions were studied. The interaction of the synthesized compounds with bovine serum albumin (BSA) was investigated via fluorescence spectroscopy, showing the affinity of the complexes for these proteins with relatively high binding constant values and the changed secondary BSA structure in the presence of the complexes. The interaction of these compounds with CT-DNA was considered by UV-Vis technique, emission titration, viscosity measurements, helix melting methods, and circular dichroism (CD) spectroscopy, confirming that the complexes were bound to CT-DNA by the intercalation binding mode. Furthermore, the complexes had the capability to displace the DNA-bound MB, as shown by the competitive studies of these complexes with methylene blue (MB), thereby suggesting the intercalation mode for the competition. Finally, the theoretical studies carried out by the docking method were performed to calculate the binding constants and recognize the binding site of the BSA and DNA by the complexes. In addition, in vitro and in silico studies showed that the compounds were degradable by bacterial and fungal biodegradation activities.

Development of Novel Bifunctional Compounds that Induce Apoptosis in Prostate Cancer Cells

DTIC Science & Technology

2006-02-01

were different from those of other alkylating agents used in chemotherapy. The apoptotic responses of prostate cancer cells suggested that the 11β... alkylating DNA Damaging Agent Tethered to an Androgen Receptor Ligand. Chemistry & Biology 12; 779-787. Personnel Supported Dr. Robert Croy...our work was the design of DNA-damag- ing agents that disrupt both DNA repair and signaling pathways in prostate tumor cells. A DNA alkylator (N,N

[Effects of Aptamer-siRNA Nucleic Acid Compound on Growth and Apoptosis in Myeloid Leukemia Cell Line K562].

PubMed

Ping, Juan; Shen, Zhi-Hui; Wang, Bao-Quan; Zhao, Na; Li, Rui; Li, Mian; Pang, Xiao-Bin; Chen, Chuan-Bo

2015-04-01

To explore the effects of aptamer-siRNA nucleic acid compound on growth and apoptosis in myeloid leukemia cell line K562. the changes of cellular morphology and structure were observed by using fluorescence microscope, laser confocal microscope, JEM-4000EX transmission electron microscopy; MTT assay were performed to evaluate the sensibility of K562 cells to aptamer-siRNA compound, the apoptosis was detected by DNA gel electro-phoresis. The remarkably changes of morphology and structure of K562 cells treated with 200 µmol/L aptamer-siRNA were observed under fluorescence microscopy and electromicroscopy. As compared with control, the aptamer-siRNA compound showed more inhibitory effect on K562 cells and there was significant difference (P<0.05). The MTT assay showed that the IC50 value of aptamer-siRNA compound for K562 cells was 150 µmol/L. According to agarose gel electrophoresis observation, when the aptamer-siRNA compound showed effect on K562 cells, the typical DNA lader could be observed. The aptamer-siRNA compound can significantly induce K562 cell apoptosis, and provide reference for gene therapy of patients with chronic myelocytic lenkemia.

The in-vitro and in-vivo inhibitory activity of biflorin in melanoma.

PubMed

Vasconcellos, Marne C; Bezerra, Daniel P; Fonseca, Aluísio M; Araújo, Ana Jérsia; Pessoa, Cláudia; Lemos, Telma L G; Costa-Lotufo, Letícia V; de Moraes, Manoel Odorico; Montenegro, Raquel C

2011-04-01

Biflorin, an ortho-naphthoquinone, is an active compound found in the roots of Capraria biflora L. It has been reported that biflorin presents anticancer activity, inhibiting both tumor cell line growth in culture and tumor development in mice. The aim of this study was to examine the effectiveness of biflorin treatment using both in-vitro and in-vivo melanoma models. Biflorin displayed considerable cytotoxicity against all tested cell lines, with half maximal inhibitory concentration values ranging from 0.58 μg/ml in NCI H23 (human lung adenocarcinoma) to 14.61 μg/ml in MDA-MB-231 (human breast cancer) cell lines. In a second set of experiments using B16 melanoma cells as a model, biflorin reduced cell viability but did not cause significant increase in the number of nonviable cells. In addition, the DNA synthesis was significantly inhibited. Flow cytometry analysis showed that biflorin may lead to an apoptotic death in melanoma cells, inducing DNA fragmentation and mitochondria depolarization, without affecting membrane integrity. In B16 melanoma-bearing mice, administration of biflorin (25mg/day) for 10 days inhibited tumor growth, and also increased the mean survival rate from 33.3±0.9 days (control) to 44.5±3.4 days (treated). Our findings suggest that biflorin may be considered as a promising lead compound for designing new drugs to be used in the treatment of melanoma.

Structure elucidation and in silico docking studies of a novel furopyrimidine antibiotics synthesized by endolithic bacterium Actinomadura sp. AL2.

PubMed

Bhattacharjee, Kaushik; Kumar, Shakti; Palepu, Narasinga Rao; Patra, Pradeep Kumar; Rao, Kollipara Mohan; Joshi, Santa Ram

2017-09-20

On screening of endolithic actinobacteria from a granite rock sample of Meghalaya for antibacterial compound, a novel antibacterial compound CCp1 was isolated from the fermentation broth of Actinomadura sp. AL2. On purification of the compound based on chromatographic techniques followed by characterization with FT-IR, UV-visible, 1 H NMR, 13 C NMR and mass spectrometry, the molecular formula of the compound was generated as C 20 H 17 N 3 O 2 , a furopyrimidine derivative. In vitro antibacterial activity of the compound was evaluated against both Gram positive and negative bacteria by agar well diffusion assay. The compound had lowest MIC (2.00 µg/ml) for Bacillus subtilis and highest MIC (> 64 µg/ml) for Staphylococcus epidermidis and Pseudomonas aeruginosa. The study revealed that the compound has potential antibacterial activity. The mode of action of the antibacterial compound was evaluated through in silico studies for its ability to bind DNA gyrase, 30S RNA molecules, OmpF porins and N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU). The antibacterial compound demonstrated more favorable docking with DNA gyrase, 30S RNA molecules and OmpF porins than GlmU which support the antibacterial compound CCp1 can be as a promising broad spectrum antibiotic agent with "multitarget" characteristics.

Context Matters: Contribution of Specific DNA Adducts to the Genotoxic Properties of the Tobacco-Specific Nitrosamine NNK.

PubMed

Peterson, Lisa A

2017-01-17

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen in laboratory animals. It is classified as a Group 1 human carcinogen by the International Agency for Cancer Research. NNK is bioactivated upon cytochrome P450 catalyzed hydroxylation of the carbon atoms adjacent to the nitrosamino group to both methylating and pyridyloxobutylating agents. Both pathways generate a spectrum of DNA damage that contributes to the overall mutagenic and toxic properties of this compound. NNK is also reduced to form 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which is also carcinogenic. Like NNK, NNAL requires metabolic activation to DNA alkylating agents. Methyl hydroxylation of NNAL generates pyridylhydroxybutyl DNA adducts, and methylene hydroxylation leads to DNA methyl adducts. The consequence of this complex metabolism is that NNK generates a vast spectrum of DNA damage, any form of which can contribute to the overall carcinogenic properties of this potent pulmonary carcinogen. This Perspective reviews the chemistry and genotoxic properties of the collection of DNA adducts formed from NNK. In addition, it provides evidence that multiple adducts contribute to the overall carcinogenic properties of this chemical. The adduct that contributes to the genotoxic effects of NNK depends on the context, such as the relative amounts of each DNA alkylating pathway occurring in the model system, the levels and genetic variants of key repair enzymes, and the gene targeted for mutation.

Methods for the detection of antioxidants which prevent age related diseases: a critical review with particular emphasis on human intervention studies.

PubMed

Hoelzl, C; Bichler, J; Ferk, F; Simic, T; Nersesyan, A; Elbling, L; Ehrlich, V; Chakraborty, A; Knasmüller, S

2005-03-01

It is well documented that reactive oxygen species (ROS) are involved in the aetiology of age related diseases. Over the last decades, strong efforts have been made to identify antioxidants in human foods and numerous promising compounds have been detected which are used for the production of supplements and functional foods. The present paper describes the advantages and limitations of methods which are currently used for the identification of antioxidants. Numerous in vitro methods are available which are easy to perform and largely used in screening trials. However, the results of such tests are only partly relevant for humans as certain active compounds (e.g. those with large molecular configuration) are only poorly absorbed in the gastrointestinal tract and/or may undergo metabolic degradation. Therefore experimental models are required which provide information if protective effects take place in humans under realistic conditions. Over the last years, several methods have been developed which are increasingly used in human intervention trials. The most widely used techniques are chemical determinations of oxidised guanosine in peripheral blood cells or urine and single cell gel electrophoresis (comet) assays with lymphocytes which are based on the measurement of DNA migration in an electric field. By using of DNA-restriction enzymes (formamidopyrimidine DNA glycosylase and endonuclease III) it is possible to monitor the endogenous formation of oxidised purines and pyrimidines; recently also protocols have been developed which enable to monitor alterations in the repair of oxidised DNA. Alternatively, also the frequency of micronucleated cells can be monitored with the cytokinesis block method in peripheral human blood cells before and after intervention with putative antioxidants. To obtain information on alterations of the sensitivity towards oxidative damage, the cells can be treated ex vivo with ROS (H(2)O(2) exposure, radiation). The evaluation of currently available human studies shows that in approximately half of them protective effects of dietary factors towards oxidative DNA-damage were observed. Earlier studies focused predominantly on the effects of vitamins (A, C, E) and carotenoids, more recently also the effects of fruit juices (from grapes, kiwi) and beverages (soy milk, tea, coffee), vegetables (tomato products, berries, Brussels sprouts) and other components of the human diet (coenzyme Q(10), polyunsaturated fatty acids) were investigated. On the basis of the results of these studies it was possible to identify dietary compounds which are highly active (e.g. gallic acid). At present, strong efforts are made to elucidate whether the different parameters of oxidative DNA-damage correlates with life span, cancer and other age related diseases. The new techniques are highly useful tools which provide valuable information if dietary components cause antioxidant effects in humans and can be used to identify individual protective compounds and also to develop nutritional strategies to reduce the adverse health effects of ROS.

Effect of green tea catechins and hydrolyzable tannins on benzo[a]pyrene-induced DNA adducts and structure-activity relationship.

PubMed

Cao, Pengxiao; Cai, Jian; Gupta, Ramesh C

2010-04-19

Green tea catechins and hydrolyzable tannins are gaining increasing attention as chemopreventive agents. However, their mechanism of action is poorly understood. We investigated the effects of four green tea catechins and two hydrolyzable tannins on microsome-induced benzo[a]pyrene (BP)-DNA adducts and the possible structure-activity relationship. BP (1 microM) was incubated with rat liver microsomes and DNA in the presence of the test compound (1-200 microM) or vehicle. The purified DNA was analyzed by (32)P-postlabeling. The inhibitory activity of the catechins was in the following descending order: epigallocatechin gallate (IC(50) = 16 microM) > epicatechin gallate (24 microM) > epigallocatechin (146 microM) > epicatechin (462 microM), suggesting a correlation between the number of adjacent aromatic hydroxyl groups in the molecular structure and their potencies. Tannic acid (IC(50) = 4 microM) and pentagalloglucose (IC(50) = 26 microM) elicited as much DNA adduct inhibitory activity as the catechins or higher presumably due to the presence of more functional hydroxyl groups. To determine if the activity of these compounds was due to direct interaction of phenolic groups with electrophilic metabolite(s) of BP, DNA was incubated with anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (anti-BPDE) (0.5 microM) in the presence of test compounds (200 microM) or vehicle. Significant inhibition of DNA adduct formation was found (tannic acid > pentagalloglucose > epigallocatechin gallate > epicatechin gallate). This notion was confirmed by analysis of the reaction products of anti-BPDE with the catechins and pentagalloglucose by electrospray ionization mass spectrometry and liquid chromatography-mass spectrometry. In conclusion, our data demonstrate that green tea catechins and the hydrolyzable tannins are highly effective in inhibiting BP-DNA adduct formation at least, in part, due to direct interaction of adjacent hydroxyl groups in their structures and that the activity is higher with an increasing number of functional hydroxyl groups.

Effect of Green Tea Catechins and Hydrolyzable Tannins on Benzo[a]pyrene-Induced DNA Adducts and Structure–Activity Relationship

PubMed Central

Cao, Pengxiao; Cai, Jian; Gupta, Ramesh C.

2016-01-01

Green tea catechins and hydrolyzable tannins are gaining increasing attention as chemopreventive agents. However, their mechanism of action is poorly understood. We investigated the effects of four green tea catechins and two hydrolyzable tannins on microsome-induced benzo[a]pyrene (BP)–DNA adducts and the possible structure–activity relationship. BP (1 μM) was incubated with rat liver microsomes and DNA in the presence of the test compound (1–200 μM) or vehicle. The purified DNA was analyzed by 32P-postlabeling. The inhibitory activity of the catechins was in the following descending order: epigallocatechin gallate (IC50 = 16 μM) > epicatechin gallate (24 μM) > epigallocatechin (146 μM) > epicatechin (462 μM), suggesting a correlation between the number of adjacent aromatic hydroxyl groups in the molecular structure and their potencies. Tannic acid (IC50 = 4 μM) and pentagalloglucose (IC50 = 26 μM) elicited as much DNA adduct inhibitory activity as the catechins or higher presumably due to the presence of more functional hydroxyl groups. To determine if the activity of these compounds was due to direct interaction of phenolic groups with electrophilic metabolite(s) of BP, DNA was incubated with anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (anti-BPDE) (0.5 μM) in the presence of test compounds (200 μM) or vehicle. Significant inhibition of DNA adduct formation was found (tannic acid > pentagalloglucose > epigallocatechin gallate > epicatechin gallate). This notion was confirmed by analysis of the reaction products of anti-BPDE with the catechins and pentagalloglucose by electrospray ionization mass spectrometry and liquid chromatography–mass spectrometry. In conclusion, our data demonstrate that green tea catechins and the hydrolyzable tannins are highly effective in inhibiting BP–DNA adduct formation at least, in part, due to direct interaction of adjacent hydroxyl groups in their structures and that the activity is higher with an increasing number of functional hydroxyl groups. PMID:20218540

Efficient interrupting skills of amino acid metallointercalators with DNA at physiological pH: Evaluation of biological assays

NASA Astrophysics Data System (ADS)

Raman, Natarajan; Selvaganapathy, Muthusamy; Radhakrishnan, Srinivasan

2014-06-01

The 4-aminoantipyrine derivatives (sbnd NO2, sbnd OCH3) and their mixed-ligand complexes with amino acids have been synthesized and investigated for their binding with CT DNA using UV-visible spectroscopy, cyclic voltammetry, and viscosity measurements under physiological conditions of pH (stomach 4.7; blood 7.4). The results from all techniques i.e. binding constant (Kb), and free energy change (ΔG) were in good agreement and inferred spontaneous compound-DNA complexes formation via intercalation. Among all the compounds 1 and 4 showed comparatively greater binding at pH 7.4 as evident from its greater Kb values. All the complexes exhibit oxidative cleavage of supercoiled (SC) pBR322 plasmid DNA in the presence of H2O2 as an activator. It is remarkable that at 25 μM concentration 1 and 4 completely degrade SC DNA into undetectable minor fragments and thus they act as efficient chemical nucleases. Among the new complexes, complexes 1 and 4 have highest potential against all the microorganisms tested. The results of the above biological experiments also reveal that the choice of different metal ions has little influence on the DNA binding, DNA cleavage and antimicrobial assay.

Development of Novel Bifunctional Compounds That Induce Apoptosis in Prostate Cancer Cells

DTIC Science & Technology

2009-03-01

amol 14C/Ag DNA 11h adducts/106 DNA bases amol 14C/Ag DNA 11h adducts/106 DNA bases 1 51 F 5 1.7 F 0.2 4.1 F 1.4 0.14 F 0.05 2 52 F 9 1.7 F 0.3 6.3 F...adduct per million DNA bases was found in LNCaP cells after a 4-hour exposure to 2.5 Amol/L 11h-dichloro in culture. A single dose of 50 mg/kg 11h

Neutral and ionic platinum compounds containing a cyclometallated chiral primary amine: synthesis, antitumor activity, DNA interaction and topoisomerase I-cathepsin B inhibition.

PubMed

Albert, Joan; Bosque, Ramon; Crespo, Margarita; Granell, Jaume; López, Concepción; Martín, Raquel; González, Asensio; Jayaraman, Anusha; Quirante, Josefina; Calvis, Carme; Badía, Josefa; Baldomà, Laura; Font-Bardia, Mercè; Cascante, Marta; Messeguer, Ramon

2015-08-14

The synthesis and preliminary biological evaluation of neutral and cationic platinum derivatives of chiral 1-(1-naphthyl)ethylamine are reported, namely cycloplatinated neutral complexes [PtCl{(R or S)-NH(2)CH(CH(3))C(10)H(6)}(L)] [L = SOMe(2) ( 1-R or 1-S ), L = PPh(3) (2-R or 2-S), L = P(4-FC(6)H(4))(3) (3-R), L = P(CH(2))(3)N(3)(CH(2))(3) (4-R)], cycloplatinated cationic complexes [Pt{(R)-NH(2)CH(CH(3))C(10)H(6)}{L}]Cl [L = Ph(2)PCH(2)CH(2)PPh(2) (5-R), L = (C(6)F(5))(2)PCH(2)CH(2)P(C(6)F(5))(2) (6-R)] and the Pt(ii) coordination compound trans-[PtCl(2){(R)-NH(2)CH(CH(3))C(10)H(6)}(2)] (7-R). The X-ray molecular structure of 7-R is reported. The cytotoxic activity against a panel of human adenocarcinoma cell lines (A-549 lung, MDA-MB-231 and MCF-7 breast, and HCT-116 colon), cell cycle arrest and apoptosis, DNA interaction, topoisomerase I and cathepsin B inhibition, and Pt cell uptake of the studied compounds are presented. Remarkable cytotoxicity was observed for most of the synthesized Pt(ii) compounds regardless of (i) the absolute configuration R or S, and (ii) the coordinated/cyclometallated (neutral or cationic) nature of the complexes. The most potent compound 2-R (IC(50) = 270 nM) showed a 148-fold increase in potency with regard to cisplatin in HCT-116 colon cancer cells. Preliminary biological results point out to different biomolecular targets for the investigated compounds. Neutral cyclometallated complexes 1-R and 2-R, modify the DNA migration as cisplatin, cationic platinacycle 5-R was able to inhibit topoisomerase I-promoted DNA supercoiling, and Pt(ii) coordination compound 7-R turned out to be the most potent inhibitor of cathepsin B. Induction of G-1 phase ( 2-R and 5-R ), and S and G-2 phases (6-R) arrests are related to the antiproliferative activity of some representative compounds upon A-549 cells. Induction of apoptosis is also observed for 2-R and 6-R.

Photo-oxidation of 6-thioguanine by UVA: the formation of addition products with low molecular weight thiol compounds.

PubMed

Ren, Xiaolin; Xu, Yao-Zhong; Karran, Peter

2010-01-01

The thiopurine, 6-thioguanine (6-TG) is present in the DNA of patients treated with the immunosuppressant and anticancer drugs azathioprine or mercaptopurine. The skin of these patients is selectively sensitive to UVA radiation-which comprises >90% of the UV light in incident sunlight-and they suffer high rates of skin cancer. UVA irradiation of DNA 6-TG produces DNA lesions that may contribute to the development of cancer. Antioxidants can protect 6-TG against UVA but 6-TG oxidation products may undergo further reactions. We characterize some of these reactions and show that addition products are formed between UVA-irradiated 6-TG and N-acetylcysteine and other low molecular weight thiol compounds including β-mercaptoethanol, cysteine and the cysteine-containing tripeptide glutathione (GSH). GSH is also adducted to 6-TG-containing oligodeoxynucleotides in an oxygen- and UVA-dependent nucleophilic displacement reaction that involves an intermediate oxidized 6-TG, guanine sulfonate (G(SO3) ). These photochemical reactions of 6-TG, particularly the formation of a covalent oligodeoxynucleotide-GSH complex, suggest that crosslinking of proteins or low molecular weight thiol compounds to DNA may be a previously unrecognized hazard in sunlight-exposed cells of thiopurine-treated patients. © 2010 The Authors. Journal Compilation. The American Society of Photobiology.

Synthesis, characterization and biological activities of some Ru(II) complexes with substituted chalcones and their applications as chemotherapeutics against breast cancer

NASA Astrophysics Data System (ADS)

Singh, Ashok K.; Saxena, Gunjan; Dixit, Shivani; Hamidullah; Singh, Sachin K.; Singh, Sudheer K.; Arshad, M.; Konwar, Rituraj

2016-05-01

Four new Ru(II) DMSO complexes with substituted chalcone ligands viz. (E)-1-(2-hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (HL1), (E)-1-(2-hydroxyphenyl)-3-(4-nitrophenyl)prop-2-en-1-one (HL2), (E)-3-(4-(dimethylamino)phenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (HL3) and (E)-1-(2-hydroxyphenyl)-3-(4-Chlorophenyl)prop-2-en-1-one (HL4) have been synthesized, and characterized by micro-analyses, IR, 1H NMR, UV-Vis and ESI-MS and screened for anti-cancer activity against breast cancer cell lines (MCF-7 and MDA MB-231). Compounds HL4 and [Ru(HL1) (O-DMSO)3(S-DMSO)]Cl (M1R) showed significant anti-breast cancer activity as evident from cytotoxicity, morphological and nuclear changes, DNA fragmentation and cell cycle arrest in breast cancer cells. UV-Vis and CD-spectra analysis showed HL4 and M1R interfered with DNA absorption spectra possibly due to DNA binding whereas these compounds were devoid of DNA topoisomerase inhibiting activity. Thus, these Ru(II) compounds have been established as new leads for future optimization by improving anti-cancer potency and safety.

Cytotoxicity, DNA binding and localisation of novel bis-naphthalimidopropyl polyamine derivatives.

PubMed

Pavlov, V; Kong Thoo Lin, P; Rodilla, V

2001-07-31

Bis-naphthalimidopropyl spermidine (BNIPSpd), spermine (BNIPSpm) and oxa-spermine (BNIPOSpm) showed high in vitro cytotoxicity against human breast cancer MCF-7 cells with IC(50) values of 1.38, 2.91 and 8.45 microM, respectively. These compounds were found to effectively displace the intercalating agent ethidium bromide bound to the calf thymus DNA using fluorimetric methods (C(50) 0.08-0.12 microM) and their apparent equilibrium binding constants (K(app)) were calculated to be in the range of 10.5-18 x 10(7) M(-1). Furthermore, strong stabilisation of calf thymus DNA duplex in the presence of bis-naphthalimidopropyl polyamine derivatives (BNIPSpd, BNIPSpm and BNIPOSpm) was observed by UV spectrophotometric analysis (T(m)=93.3-97 degrees C compared with 75 degrees C for calf thymus DNA without drug). Because of their inherent fluorescence, these compounds were localised preferentially inside the nucleus as evidenced by their direct observation under the fluorescence microscope. The results obtained suggest that the cytotoxic activity of the bis-naphthalimidopropyl polyamines may be in part, caused by their effects on DNA.

Identification of genotoxic compounds in crude oil using fractionation according to distillation, polarity and Kow.

PubMed

Park, Shin Yeong; Lee, Hyo Jin; Khim, Jong Seong; Kim, Gi Beum

2017-01-30

We examined the degree of DNA damage caused by fractions of crude oil in accordance with the boiling points, polarity and log K ow . Relatively high DNA damage was observed in the aromatic fraction (290-330°C) and resin and polar fraction (350-400°C). The resin and polar fraction showed relatively high genotoxicity compared with the aliphatic and aromatic fraction at the 1-4 log K ow range. At the 6-7 log K ow range, the aromatic fraction showed relatively high DNA damage compared with the aliphatic and resin and polar fraction. In particular, every detailed fraction in accordance with the log K ow values (aliphatic and aromatic (310-320°C) and resins and polar fractions (370-380°C)) showed one or less than one DNA damage. However, the fractions before separation in accordance with log K ow values (aliphatic and aromatic (310-320°C) and resin and polar (370-380°C) fractions) showed high DNA damage. Thus, we confirm the synergistic action between the detailed compounds. Copyright © 2016 Elsevier Ltd. All rights reserved.

Time-Resolved Fluorescence Resonance Energy Transfer Assay for Discovery of Small-Molecule Inhibitors of Methyl-CpG Binding Domain Protein 2.

PubMed

Wyhs, Nicolas; Walker, David; Giovinazzo, Hugh; Yegnasubramanian, Srinivasan; Nelson, William G

2014-08-01

Methylated DNA binding proteins such as Methyl-CpG Binding Domain Protein 2 (MBD2) can transduce DNA methylation alterations into a repressive signal by recruiting transcriptional co-repressor complexes. Interfering with MBD2 could lead to reactivation of tumor suppressor genes and therefore represents an attractive strategy for epigenetic therapy. We developed and compared fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET)-based high-throughput screening (HTS) assays to identify small-molecule inhibitors of the interaction between the methyl binding domain of MBD2 (MBD2-MBD) and methylated DNA. Although both assays performed well in 96-well format, the TR-FRET assay (Z' factor = 0.58) emerged as a superior screening strategy compared with FP (Z' factor = 0.08) when evaluated in an HTS 384-well plate format. Using TR-FRET, we screened the Sigma LOPAC library for MBD2-MBD inhibitors and identified four compounds that also validated in a dose-response series. This included two known DNA intercalators (mitoxantrone and idarubicin) among two other inhibitory compounds (NF449 and aurintricarboxylic acid). All four compounds also inhibited the binding of SP-1, a transcription factor with a GC-rich binding sequence, to a methylated oligonucleotide, demonstrating that the activity was nonspecific. Our results provide proof of principle for using TR-FRET-based HTS to identify small-molecule inhibitors of MBD2 and other DNA-protein interactions. © 2014 Society for Laboratory Automation and Screening.

Plant extracts and natural compounds used against UVB-induced photoaging.

PubMed

Cavinato, Maria; Waltenberger, Birgit; Baraldo, Giorgia; Grade, Carla V C; Stuppner, Hermann; Jansen-Dürr, Pidder

2017-08-01

Skin is continuously exposed to a variety of environmental stresses, including ultraviolet (UV) radiation. UVB is an inherent component of sunlight that crosses the epidermis and reaches the upper dermis, leading to increased oxidative stress, activation of inflammatory response and accumulation of DNA damage among other effects. The increase in UVB radiation on earth due to the destruction of stratospheric ozone poses a major environmental threat to the skin, increasing the risk of damage with long-term consequences, such as photoaging and photocarcinogenesis. Extracts from plants and natural compounds have been historically used in traditional medicine in the form of teas and ointments but the effect of most of these compounds has yet to be verified. Regarding the increasing concern of the population with issues related to quality of life and appearance, the cosmetic market for anti-aging and photoprotective products based on natural compounds is continuously growing, and there is increasing requirement of expansion on research in this field. In this review we summarized the most current and relevant information concerning plant extracts and natural compounds that are able to protect or mitigate the deleterious effects caused by photoaging in different experimental models.

Mechanisms of inhibition of zinc-finger transcription factors by selenium compounds ebselen and selenite.

PubMed

Larabee, Jason L; Hocker, James R; Hanas, Jay S

2009-03-01

The anti-inflammatory selenium compounds, ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one) and selenite, were found to alter the DNA binding mechanisms and structures of cysteine-rich zinc-finger transcription factors. As assayed by DNase I protection, DNA binding by TFIIIA (transcription factor IIIA, prototypical Cys(2)His(2) zinc finger protein), was inhibited by micromolar amounts of ebselen. In a gel shift assay, ebselen inhibited the Cys(2)His(2) zinc finger-containing DNA binding domain (DBD) of the NF-kappaB mediated transcription factor Sp1. Ebselen also inhibited DNA binding by the p50 subunit of the pro-inflammatory Cys-containing NF-kappaB transcription factor. Electrospray ionization mass spectrometry (ESI-MS) was utilized to elucidate mechanisms of chemical interaction between ebselen and a zinc-bound Cys(2)His(2) zinc finger polypeptide modeled after the third finger of Sp1 (Sp1-3). Exposing Sp1-3 to micromolar amounts of ebselen resulted in Zn(2+) release from this peptide and the formation of a disulfide bond by oxidation of zinc finger SH groups, the likely mechanism for DNA binding inhibition. Selenite was shown by ESI-MS to also eject zinc from Sp1-3 as well as induce disulfide bond formation through SH oxidation. The selenite-dependent inhibition/oxidation mechanism differed from that of ebselen by inducing the formation of a stable selenotrisulfide bond. Selenite-induced selenotrisulfide formation was dependent upon the structure of the Cys(2)His(2) zinc finger as alteration in the finger structure enhanced this reaction as well as selenite-dependent zinc release. Ebselen and selenite-dependent inhibition/oxidation of Cys-rich zinc finger proteins, with concomitant release of zinc and finger structural changes, points to mechanisms at the atomic and protein level for selenium-induced alterations in Cys-rich proteins, and possible amelioration of certain inflammatory, neurodegenerative, and oncogenic responses.

Novel 3-Aminothiazolquinolones: Design, Synthesis, Bioactive Evaluation, SARs, and Preliminary Antibacterial Mechanism.

PubMed

Cui, Sheng-Feng; Addla, Dinesh; Zhou, Cheng-He

2016-05-26

A series of novel 3-aminothiazolquinolones as analogues of quinolone antibacterial agents were designed and synthesized in an effort to circumvent quinolone resistance. Among these 3-aminothiazolquinolones, 3-(2-aminothiazol-4-yl)-7-chloro-6-(pyrrolidin-1-yl) quinolone 12b exhibited potent antibacterial activity, low cytotoxicity to hepatocyte cells, strong inhibitory potency to DNA gyrase, and a broad antimicrobial spectrum including against multidrug-resistant strains. This active molecule 12b also induced bacterial resistance more slowly than norfloxacin. Analysis of structure-activity relationships (SARs) disclosed that the 2-aminothiazole fragment at the 3-position of quinolone plays an important role in exerting antibacterial activity. Molecular modeling and experimental investigation of aminothiazolquinolone 12b with DNA from a sensitive methicillin-resistant Staphylococcus aureus (MRSA) strain revealed that the possible antibacterial mechanism might be related to the formation of a compound 12b-Cu(2+)-DNA ternary complex in which the Cu(2+) ion acts as a bridge between the backbone of 3-aminothiazolquinolone and the phosphate group of the nucleic acid.

Hydrolysis of N3-methyl-2'-deoxycytidine: model compound for reactivity of protonated cytosine residues in DNA.

PubMed

Sowers, L C; Sedwick, W D; Shaw, B R

1989-11-01

Protonation of cytosine residues at physiological pH may occur in DNA as a consequence of both alkylation and aberrant base-pair formation. When cytosine derivatives are protonated, they undergo hydrolysis reactions at elevated rates and can either deaminate to form the corresponding uracil derivatives or depyrimidinate generating abasic sites. The kinetic parameters for reaction of protonated cytosine are derived by studying the hydrolysis of N3-methyl-2'-deoxycytidine (m3dC), a cytosine analogue which is predominantly protonated at physiological pH. Both deamination and depyrimidimation reaction rates are shown to be linearly dependent upon the fraction of protonated molecules. We present here thermodynamic parameters which allow determination of hydrolysis rates of m3dC as functions of pH and temperature. Protonation of cytosine residues in DNA, as induced by aberrant base-pair formation or base modification, may accelerate the rate of both deamination and depyrimidation up to several thousand-fold under physiological conditions.

Deoxynybomycins inhibit mutant DNA gyrase and rescue mice infected with fluoroquinolone-resistant bacteria

PubMed Central

Parkinson, Elizabeth I.; Bair, Joseph S.; Nakamura, Bradley A.; Lee, Hyang Y.; Kuttab, Hani I.; Southgate, Emma H.; Lezmi, Stéphane; Lau, Gee W.; Hergenrother, Paul J.

2015-01-01

Fluoroquinolones are one of the most commonly prescribed classes of antibiotics, but fluoroquinolone resistance (FQR) is widespread and increasing. Deoxynybomycin (DNM) is a natural-product antibiotic with an unusual mechanism of action, inhibiting the mutant DNA gyrase that confers FQR. Unfortunately, isolation of DNM is difficult and DNM is insoluble in aqueous solutions, making it a poor candidate for development. Here we describe a facile chemical route to produce DNM and its derivatives. These compounds possess excellent activity against FQR methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci clinical isolates and inhibit mutant DNA gyrase in-vitro. Bacteria that develop resistance to DNM are re-sensitized to fluoroquinolones, suggesting that resistance that emerges to DNM would be treatable. Using a DNM derivative, the first in-vivo efficacy of the nybomycin class is demonstrated in a mouse infection model. Overall, the data presented suggest the promise of DNM derivatives for the treatment of FQR infections. PMID:25907309

Novel pregnenolone derivatives modulate apoptosis via Bcl-2 family genes in hepatocellular carcinoma in vitro.

PubMed

Elhinnawi, Manar A; Mohareb, Rafat M; Rady, Hanaa M; Khalil, Wagdy K B; Abd Elhalim, Mervat M; Elmegeed, Gamal A

2018-06-10

A series of pregnenolone derivatives were synthesized and assessed for anti-cancer activity against hepatocellular carcinoma cell line (HepG2). The synthesized hetero-steroids (compounds 3, 4, 5, 6, 7, 8a and 8b) were evaluated for their cytotoxic activities using MTT (3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assay. Apoptotic activity was assessed using dual acridine orange/ethidium bromide staining method and DNA fragmentation assay. Pro-apoptotic genes (Bax and Bak) and anti-apoptotic genes (Bcl-2 and Bcl-xL) were analyzed using quantitative real time PCR. The results revealed that compounds 4 and 6 displayed cytotoxic activity (IC 50s , 36.97 ± 2.18 and 18.46 ± 0.64 µM, respectively), while compounds 5 and 7 exhibited weak cytotoxic activity (IC 50s , 93.87 ± 8.30 µM and 93.48 ± 4.14 µM, respectively). All synthesized heterocyclic pregnenolone derivatives induced apoptosis through DNA fragmentation. Compounds 4 and 6 increased early and late apoptotic cell percentages while compounds 3, 5, 7 and 8b increased either early or late apoptotic cell percentage. Moreover, compounds 3, 6 and 8b up-regulated the expression level of Bak gene. On the other hand, compounds 4, 5, 7 and 8a down-regulated the Bcl-2 expression level, besides, compounds 5, 7 and 8a down-regulated the Bcl-xL expression level. Compounds 5, 7, 8a and 8b increased the Bak/Bcl-xL ratio, besides, compound 8a raised the Bax/Bcl-xL ratio whereas compound 5 elevated Bax/Bcl-2 and Bak/Bcl-2 ratios. The present work introduced novel pro-apoptotic pregnenolone derivatives that acted against HepG2 cells through DNA fragmentation, apoptotic morphological changes and were able to increase the pro-apoptotic/anti-apoptotic ratios of Bcl-2 family genes. This study particularly revealed that the cytotoxic compound 4 is the most promising pro-apoptotic compound among other synthesized derivatives where it induced apoptosis (late and early) through the down-regulation of Bcl-2 gene expression level. Copyright © 2018 Elsevier Ltd. All rights reserved.

Biological evaluation of omega-(dialkylamino)alkyl derivatives of 6H-indolo[2,3-b]quinoline--novel cytotoxic DNA topoisomerase II inhibitors.

PubMed

Godlewska, Joanna; Luniewski, Wojciech; Zagrodzki, Bogdan; Kaczmarek, Lukasz; Bielawska-Pohl, Aleksandra; Dus, Danuta; Wietrzyk, Joanna; Opolski, Adam; Siwko, Magdalena; Jaromin, Anna; Jakubiak, Anna; Kozubek, Arkadiusz; Peczyñska-Czoch, Wanda

2005-01-01

A series of novel 6H-indolo[2,3-b]quinoline derivatives, substituted at C-2, C-9 or N-6 position with dialkyl(alkylamino)alkyl chains differing in the number of methylene groups, was prepared. These compounds were evaluated in vitro for their antimicrobial and cytotoxic activity against several cell lines of different origin and tested for their ability to influence the cell cycle and inhibit topoisomerase II activity. Liphophilic and calf thymus DNA-binding properties of these compounds were also investigated. All the compounds tested inhibited the growth of Gram-positive bacteria and fungi at MIC values ranging between 0.25 and 1 mM. They also showed cytotoxic activity against KB (human cervix carcinoma) cells (ID50 varied from 2.1 to 9.0 microM) and were able to overcome multidrug resistance in colorectal adenocarcinoma LoVo/DX, uterine sarcoma MES-SA/DX5 and promyelocytic leukemia HL-60/MX2 cells (the values of the resistance index RI fell between 0.54 and 2.4). The compounds induced G2M-phase cell cycle arrest in Jurkat T-cell leukemia cells, revealed DNA-binding properties and inhibited topoisomerase II activity.

Cellular evaluation of diselenonicotinamide (DSNA) as a radioprotector against cell death and DNA damage.

PubMed

Raghuraman, M; Verma, Prachi; Kunwar, Amit; Phadnis, Prasad P; Jain, V K; Priyadarsini, K Indira

2017-06-01

Diselenonicotinamide (DSNA), a synthetic organoselenium compound, was evaluated for its radioprotective effect in cellular models. A clonogenic assay in Chinese Hamster Ovary (CHO) cells and an apoptosis assay in murine splenic lymphocytes indicated that pre-treatment with DSNA at a concentration of 25 μM significantly protected them from radiation-induced cell death. Upon irradiation (1-12 Gy), dose-response studies were carried out under similar treatment conditions, and its dose modification factor (DMF) was estimated to be 1.26. Furthermore, DSNA showed its radioprotective effect, even when administered after exposure to radiation. Mechanistic investigation revealed that DSNA increased the intracellular levels of GPx and GSH in irradiated cells. In line with this observation, the addition of a pharmacological inhibitor of GPx cycle, abrogated the activity of DSNA. The radioprotective effect of DSNA was also complemented by its ability to prevent radiation-induced DNA damage as monitored by micronucleus and γ-H2AX assays. Furthermore, treatment with DSNA did not show much change in the expressions of Nrf2 dependent genes (γ-GCL and HO-1), but the presence of a pharmacological inhibitor of Nrf2 abrogated the radioprotective activity of DSNA against cell death and DNA damage. Additionally, ATRA treatment also inhibited the DSNA-mediated up-regulation of a repair gene RAD51, suggesting possible involvement of basal Nrf2 in the anti-genotoxic effect of DSNA. In conclusion, the present study demonstrates radioprotection by a synthetic organoselenium compound containing nutritionally important moieties like selenium and nicotinamide.

Quantum-mechanical parameters for the risk assessment of multi-walled carbon-nanotubes: A study using adsorption of probe compounds and its application to biomolecules.

PubMed

Chayawan; Vikas

2016-11-01

This work forwards new insights into the risk-assessment of multi-walled carbon-nanotubes (MWCNTs) while analysing the role of quantum-mechanical interactions between the electrons in the adsorption of probe compounds and biomolecules by MWCNTs. For this, the quantitative models are developed using quantum-chemical descriptors and their electron-correlation contribution. The major quantum-chemical factors contributing to the adsorption are found to be mean polarizability, electron-correlation energy, and electron-correlation contribution to the absolute electronegativity and LUMO energy. The proposed models, based on only three quantum-chemical factors, are found to be even more robust and predictive than the previously known five or four factors based linear free-energy and solvation-energy relationships. The proposed models are employed to predict the adsorption of biomolecules including steroid hormones and DNA bases. The steroid hormones are predicted to be strongly adsorbed by the MWCNTs, with the order: hydrocortisone > aldosterone > progesterone > ethinyl-oestradiol > testosterone > oestradiol, whereas the DNA bases are found to be relatively less adsorbed but follow the order as: guanine > adenine > thymine > cytosine > uracil. Besides these, the developed electron-correlation based models predict several insecticides, pesticides, herbicides, fungicides, plasticizers and antimicrobial agents in cosmetics, to be strongly adsorbed by the carbon-nanotubes. The present study proposes that the instantaneous inter-electronic interactions may be quite significant in various physico-chemical processes involving MWCNTs, and can be used as a reliable predictor for their risk assessment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Medicinal Plants Recommended by the World Health Organization: DNA Barcode Identification Associated with Chemical Analyses Guarantees Their Quality

PubMed Central

Palhares, Rafael Melo; Gonçalves Drummond, Marcela; dos Santos Alves Figueiredo Brasil, Bruno; Pereira Cosenza, Gustavo; das Graças Lins Brandão, Maria; Oliveira, Guilherme

2015-01-01

Medicinal plants are used throughout the world, and the regulations defining their proper use, such as identification of the correct species and verification of the presence, purity and concentration of the required chemical compounds, are widely recognized. Herbal medicines are made from vegetal drugs, the processed products of medicinal species. These processed materials present a number of challenges in terms of botanical identification, and according to the World Health Organization (WHO), the use of incorrect species is a threat to consumer safety. The samples used in this study consisted of the dried leaves, flowers and roots of 257 samples from 8 distinct species approved by the WHO for the production of medicinal herbs and sold in Brazilian markets. Identification of the samples in this study using DNA barcoding (matK, rbcL and ITS2 regions) revealed that the level of substitutions may be as high as 71%. Using qualitative and quantitative chemical analyses, this study identified situations in which the correct species was being sold, but the chemical compounds were not present. Even more troubling, some samples identified as substitutions using DNA barcoding contained the chemical compounds from the correct species at the minimum required concentration. This last situation may lead to the use of unknown species or species whose safety for human consumption remains unknown. This study concludes that DNA barcoding should be used in a complementary manner for species identification with chemical analyses to detect and quantify the required chemical compounds, thus improving the quality of this class of medicines. PMID:25978064

In Vitro Evaluation of Antileishmanial Activity of Computationally Screened Compounds against Ascorbate Peroxidase To Combat Amphotericin B Drug Resistance

PubMed Central

Mansuri, Rani; Kumar, Ashish; Rana, Sindhuprava; Panthi, Bhavana; Ansari, M. Yousuf; Das, Sushmita; Dikhit, Manas Ranjan

2017-01-01

ABSTRACT In visceral leishmaniasis (VL), the host macrophages generate oxidative stress to destroy the pathogen, while Leishmania combats the harmful effect of radicals by redox homeostasis through its unique trypanothione cascade. Leishmania donovani ascorbate peroxidase (LdAPx) is a redox enzyme that regulates the trypanothione cascade and detoxifies the effect of H2O2. The absence of an LdAPx homologue in humans makes it an excellent drug target. In this study, the homology model of LdAPx was built, including heme, and diverse compounds were prefiltered (PAINS, ADMET, and Lipinski's rule of five) and thereafter screened against the LdAPx model. Compounds having good affinity in terms of the Glide XP (extra precision) score were clustered to select diverse compounds for experimental validation. A total of 26 cluster representatives were procured and tested on promastigote culture, yielding 12 compounds with good antileishmanial activity. Out of them, six compounds were safer on the BALB/c peritoneal macrophages and were also effective against disease-causing intracellular amastigotes. Three out of six compounds inhibited recombinant LdAPx in a noncompetitive manner and also demonstrated partial reversion of the resistance property in an amphotericin B (AmB)-resistant strain, which may be due to an increased level of reactive oxygen species (ROS) and decrease of glutathione (GSH) content. However, inhibition of LdAPx in resistant parasites enhanced annexin V staining and activation of metacaspase-like protease activity, which may help in DNA fragmentation and apoptosis-like cell death. Thus, the present study will help in the search for specific hits and templates of potential therapeutic interest and therefore may facilitate the development of new drugs for combination therapy against VL. PMID:28461317

Theory of electron transfer and molecular state in DNA

NASA Astrophysics Data System (ADS)

Endres, Robert Gunter

2002-09-01

In this thesis, a mechanism for long-range electron transfer in DNA and a systematic search for high conductance DNA are developed. DNA is well known for containing the genetic code of all living species. On the other hand, there are some experimental indications that DNA can mediate effectively long-range electron transfer leading to the concept of chemistry at a distance. This can be important for DNA damage and healing. In the first part of the thesis, a possible mechanism for long-range electron transfer is introduced. The weak distance dependent electron transfer was experimentally observed using transition metal intercalators for donor and acceptor. In our model calculations, the transfer is mediated by the molecular analogue of a Kondo bound state well known from solid state physics of mixed-valence rare-earth compounds. We believe this is quite realistic, since localized d orbitals of the transition metal ions could function as an Anderson impurity embedded in a reservoir of rather delocalized molecular orbitals of the intercalator ligands and DNA pi orbitals. The effective Anderson model is solved with a physically intuitive variational ansatz as well as with the essentially exact DMRG method. The electronic transition matrix element, which is important because it contains the donor-acceptor distance dependence, is obtained with the Mulliken-Hush algorithm as well as from Born-Oppenheimer potential energy surfaces. Our possible explanation of long-range electron transfer is put in context to other more conventional mechanisms which also could lead to similar behavior. Another important issue of DNA is its possible use for nano-technology. Although DNA's mechanical properties are excellent, the question whether it can be conducting and be used for nano-wires is highly controversial. Experimentally, DNA shows conducting, semi-conducting and insulating properties. Motivated by these wide ranging experimental results on the conductivity of DNA, we have embarked on a theoretical effort to ascertain what conditions might induce such remarkable behavior. We use a combination of an ab initio density functional theory method and a parameterized Huckel-Slater-Koster model. Our focus here is to examine whether any likely DNA structures or environments can yield reduced activation gaps to conduction or enhanced electronic overlaps. In particular, we study a hypothetical stretched ribbon structure, A-, and B-form DNA, and the effects of counterions and humidity. Unlike solids, DNA and other molecules are considered soft condensed matter. Hence, we study the influence of vibrations upon the electronic structure of DNA. We calculate parameters for charge transfer rates between adjacent bases. We find good agreement between our estimated rates and recent experimental data assuming that torsional vibrations limit the charge transfer most significantly.

The effects of strawberry tree water leaf extract, arbutin and hydroquinone on haematological parameters and levels of primary DNA damage in white blood cells of rats.

PubMed

Jurica, Karlo; Brčić Karačonji, Irena; Kopjar, Nevenka; Shek-Vugrovečki, Ana; Cikač, Tihana; Benković, Vesna

2018-04-06

Strawberry tree (Arbutus unedo L., Ericaceae) leaves represent a potent source of biologically active compounds and have been used for a long to relieve symptoms of various health impairments and diseases. Two major compounds related to their beneficial activities in animals and humans are arbutin and hydroquinone. To establish potential benefit/risk ratio associated with daily oral administration of strawberry tree water leaf extract, arbutin and hydroquinone in doses expected to be non-toxic. We performed a 14-day and a 28-day study on male and female Lewis rats and evaluated main haematological parameters and the effects of treatments on the levels of primary DNA damage in white blood cells (WBC) using the alkaline comet assay. Our findings suggest no significant changes in the haematological parameters following prolonged exposure to strawberry tree water leaf extract, arbutin, and hydroquinone. However, hydroquinone causes increased, and extract as well as arbutin decreased WBC count in male rats compared to control after 14 days of treatment. DNA damage measured in WBC of rats treated with all compounds was below 10% of the DNA in the comet tail, which indicates low genotoxicity. The genotoxic potential of strawberry water leaf extract was within acceptable limits and reflected effects of a complex chemical composition upon DNA. We also observed slight gender- and exposure time- related differences in primary DNA damage in the leucocytes of control and treated rats. Future studies should investigate which doses of strawberry tree water leaf extract would be most promising for the potential use as a substitute for bearberry leaves for treatment of urinary infection. Copyright © 2017 Elsevier B.V. All rights reserved.

Ionization of biomolecular targets by ion impact: input data for radiobiological applications

NASA Astrophysics Data System (ADS)

de Vera, Pablo; Abril, Isabel; Garcia-Molina, Rafael; Solov'yov, Andrey V.

2013-06-01

In this work we review and further develop a semiempirical model recently proposed for the ion impact ionization of complex biological media. The model is based on the dielectric formalism, and makes use of a semiempirical parametrization of the optical energy-loss function of bioorganic compounds, allowing the calculation of single and total ionization cross sections and related quantities for condensed biological targets, such as liquid water, DNA and its components, proteins, lipids, carbohydrates or cell constituents. The model shows a very good agreement with experimental data for water, adenine and uracil, and allows the comparison of the ionization efficiency of different biological targets, and also the average kinetic energy of the ejected secondary electrons.

Novel design strategy for checkpoint kinase 2 inhibitors using pharmacophore modeling, combinatorial fusion, and virtual screening.

PubMed

Lin, Chun-Yuan; Wang, Yen-Ling

2014-01-01

Checkpoint kinase 2 (Chk2) has a great effect on DNA-damage and plays an important role in response to DNA double-strand breaks and related lesions. In this study, we will concentrate on Chk2 and the purpose is to find the potential inhibitors by the pharmacophore hypotheses (PhModels), combinatorial fusion, and virtual screening techniques. Applying combinatorial fusion into PhModels and virtual screening techniques is a novel design strategy for drug design. We used combinatorial fusion to analyze the prediction results and then obtained the best correlation coefficient of the testing set (r test) with the value 0.816 by combining the Best(train)Best(test) and Fast(train)Fast(test) prediction results. The potential inhibitors were selected from NCI database by screening according to Best(train)Best(test) + Fast(train)Fast(test) prediction results and molecular docking with CDOCKER docking program. Finally, the selected compounds have high interaction energy between a ligand and a receptor. Through these approaches, 23 potential inhibitors for Chk2 are retrieved for further study.

Optimization of potent hepatitis C virus NS3 helicase inhibitors isolated from the yellow dyes thioflavine S and primuline.

PubMed

Li, Kelin; Frankowski, Kevin J; Belon, Craig A; Neuenswander, Ben; Ndjomou, Jean; Hanson, Alicia M; Shanahan, Matthew A; Schoenen, Frank J; Blagg, Brian S J; Aubé, Jeffrey; Frick, David N

2012-04-12

A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2 ± 1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because P4 also interacted with DNA, more specific analogues were synthesized from the abundant dimeric component of primuline. Some of the 32 analogues prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6 ± 1 μM. © 2012 American Chemical Society

Novel pyrimidinic selenourea induces DNA damage, cell cycle arrest, and apoptosis in human breast carcinoma.

PubMed

Barbosa, Flavio A R; Siminski, Tâmila; Canto, Rômulo F S; Almeida, Gabriela M; Mota, Nádia S R S; Ourique, Fabiana; Pedrosa, Rozangela Curi; Braga, Antonio Luiz

2018-06-11

Novel pyrimidinic selenoureas were synthesized and evaluated against tumour and normal cell lines. Among these, the compound named 3j initially showed relevant cytotoxicity and selectivity for tumour cells. Three analogues of 3j were designed and synthesized keeping in view the structural requirements of this compound. Almost all the tested compounds displayed considerable cytotoxicity. However, 8a, one of the 3j analogues, was shown to be highly selective and cytotoxic, especially for breast carcinoma cells (MCF-7) (IC 50  = 3.9 μM). Furthermore, 8a caused DNA damage, inhibited cell proliferation, was able to arrest cell cycle in S phase, and induced cell death by apoptosis in human breast carcinoma cells. Moreover, predictions of pharmacokinetic properties showed that 8a may present good absorption and permeation characteristics for oral administration. Overall, the current study established 8a as a potential drug prototype to be employed as a DNA interactive cytotoxic agent for the treatment of breast cancer. Copyright © 2018. Published by Elsevier Masson SAS.

Novel cholinesterase modulators and their ability to interact with DNA

NASA Astrophysics Data System (ADS)

Janockova, Jana; Gulasova, Zuzana; Musilek, Kamil; Kuca, Kamil; Kozurkova, Maria

2013-11-01

In the present work, an interaction of four cholinesterase modulators (1-4) with calf thymus DNA was studied via spectroscopic techniques (UV-Vis, fluorescent spectroscopy and circular dichroism). From UV-Vis spectroscopic analysis, the binding constants for DNA-pyridinium oximes complexes were calculated (K = 3.5 × 104 to 1.4 × 105 M-1). All these measurements indicated that the compounds behave as effective DNA-interacting agents. Electrophoretic techniques proved that ligand 2 inhibited topoisomerase I at a concentration 5 μM.

Comparative Genotoxicity and Cytotoxicity of Four Hexavalent Chromium Compounds in Human Bronchial Cells

PubMed Central

Wise, Sandra S.; Holmes, Amie L.; Qin, Qin; Xie, Hong; Katsifis, Spiros P.; Thompson, W. Douglas; Wise, John Pierce

2010-01-01

Hexavalent chromium (Cr(VI)) compounds are well-established human lung carcinogens. Solubility plays an important role in their carcinogenicity with the particulate Cr(VI) compounds being the most carcinogenic. Epidemiology and animal studies suggest that zinc chromate is the most potent particulate Cr(VI) compound, however, there are few comparative data to support these observations. The purpose of this study was to compare the genotoxicity of zinc chromate with two other particulate Cr(VI) compounds, barium chromate and lead chromate, and one soluble Cr(VI) compound, sodium chromate. The clastogenic effects of barium chromate and zinc chromate were similar but lead chromate induced significantly less damage. The levels of DNA damage measured by gamma-H2A.X foci formation were similar for the three particulate chromium compounds. Corrected for chromium uptake differences, we found that zinc chromate and barium chromate were the most cytotoxic and lead chromate and sodium chromate were less cytotoxic. Zinc chromate was more clastogenic than all other chromium compounds and lead chromate was the least clastogenic. There was no significant difference between any of the compounds for the induction of DNA double strand breaks. All together, these data suggest that the difference in the carcinogenic potency of zinc chromate over the other chromium compounds is not due solely to a difference in chromium ion uptake and the zinc cation may in fact have an important role in its carcinogenicity. PMID:20000473

Probing the electrostatics and pharmacologic modulation of sequence-specific binding by the DNA-binding domain of the ETS-family transcription factor PU.1: a binding affinity and kinetics investigation

PubMed Central

Munde, Manoj; Poon, Gregory M. K.; Wilson, W. David

2013-01-01

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally-conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤105 M−1 s−1), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt-dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>107 M−1 s−1). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes. PMID:23416556

Thinner inhalation effects on oxidative stress and DNA repair in a rat model of abuse.

PubMed

Martínez-Alfaro, Minerva; Cárabez-Trejo, Alfonso; Gallegos-Corona, Marco-Antonio; Pedraza-Aboytes, Gustavo; Hernández-Chan, Nancy Georgina; Leo-Amador, Guillermo Enrique

2010-04-01

Humans can come into contact with thinner by occupational exposure or by intentional inhalation abuse. Numerous studies of workers for genotoxic effects of thinner exposure have yielded conflicting results, perhaps because co-exposure to variable other compounds cannot be avoided in workplace exposure studies. In contrast, there is no data concerning the genotoxic effects of intentional inhalation abuse. The aim of this project was to examine the genotoxic effects of thinner inhalation in an animal model of thinner abuse (rats exposed to 3000 ppm toluene, a high solvent concentration over a very short, 15 min time period, twice a day for 6 weeks). The data presented here provides evidence that thinner inhalation in our experimental conditions is able to induce weight loss, lung abnormalities and oxidative stress. This oxidative stress induces oxidative DNA damage that is not a characteristic feature of genotoxic damage. No significant difference in DNA damage and DNA repair (biomarkers of genotoxicity) in lymphocytes from thinner-treated and control rats was found. Lead treatment was used as a positive control in these assays. Finally, bone marrow was evaluated as a biomarker of cellular alteration associated with thinner inhalation. The observed absence of hemopoietic and genetic toxicity could be explained in part by the absence of benzene, the only carcinogenic component of thinner; however, benzene is no longer a common component of thinner. In conclusion, thinner did not cause genotoxic effects in an experimental model of intentional abuse despite the fact that thinner inhalation induces oxidative stress. (c) 2009 John Wiley & Sons, Ltd.

Compound Heterozygosity for Y Box Proteins Causes Sterility Due to Loss of Translational Repression

PubMed Central

Sharma, Manju; Dearth, Andrea; Smith, Benjamin; Braun, Robert E.

2015-01-01

The Y-box proteins YBX2 and YBX3 bind RNA and DNA and are required for metazoan development and fertility. However, possible functional redundancy between YBX2 and YBX3 has prevented elucidation of their molecular function as RNA masking proteins and identification of their target RNAs. To investigate possible functional redundancy between YBX2 and YBX3, we attempted to construct Ybx2 -/- ;Ybx3 -/- double mutants using a previously reported Ybx2 -/- model and a newly generated global Ybx3 -/- model. Loss of YBX3 resulted in reduced male fertility and defects in spermatid differentiation. However, homozygous double mutants could not be generated as haploinsufficiency of both Ybx2 and Ybx3 caused sterility characterized by extensive defects in spermatid differentiation. RNA sequence analysis of mRNP and polysome occupancy in single and compound Ybx2/3 heterozygotes revealed loss of translational repression almost exclusively in the compound Ybx2/3 heterozygotes. RNAseq analysis also demonstrated that Y-box protein dose-dependent loss of translational regulation was inversely correlated with the presence of a Y box recognition target sequence, suggesting that Y box proteins bind RNA hierarchically to modulate translation in a range of targets. PMID:26646932

Regulated transport into the nucleus of herpesviridae DNA replication core proteins.

PubMed

Gualtiero, Alvisi; Jans, David A; Camozzi, Daria; Avanzi, Simone; Loregian, Arianna; Ripalti, Alessandro; Palù, Giorgio

2013-09-16

The Herpesvirdae family comprises several major human pathogens belonging to three distinct subfamilies. Their double stranded DNA genome is replicated in the nuclei of infected cells by a number of host and viral products. Among the latter the viral replication complex, whose activity is strictly required for viral replication, is composed of six different polypeptides, including a two-subunit DNA polymerase holoenzyme, a trimeric primase/helicase complex and a single stranded DNA binding protein. The study of herpesviral DNA replication machinery is extremely important, both because it provides an excellent model to understand processes related to eukaryotic DNA replication and it has important implications for the development of highly needed antiviral agents. Even though all known herpesviruses utilize very similar mechanisms for amplification of their genomes, the nuclear import of the replication complex components appears to be a heterogeneous and highly regulated process to ensure the correct spatiotemporal localization of each protein. The nuclear transport process of these enzymes is controlled by three mechanisms, typifying the main processes through which protein nuclear import is generally regulated in eukaryotic cells. These include cargo post-translational modification-based recognition by the intracellular transporters, piggy-back events allowing coordinated nuclear import of multimeric holoenzymes, and chaperone-assisted nuclear import of specific subunits. In this review we summarize these mechanisms and discuss potential implications for the development of antiviral compounds aimed at inhibiting the Herpesvirus life cycle by targeting nuclear import of the Herpesvirus DNA replicating enzymes.

DNA damage in mouse and rat liver by caprolactam and benzoin, evaluated with three different methods.

PubMed

Parodi, S; Abelmoschi, M L; Balbi, C; De Angeli, M T; Pala, M; Russo, P; Taningher, M; Santi, L

1989-11-01

Benzoin and caprolactam were examined for their capability of inducing alkaline DNA fragmentation in mouse and rat liver DNA after treatment in vivo. Three different methods were used. With the alkaline elution technique we measured an effect presumably related to the conformation of the DNA coil. With a viscometric and a fluorometric unwinding method we measured an effect presumably related to the number of unwinding points in DNA. For both compounds only the alkaline elution technique was clearly positive. The results suggest that both caprolactam and benzoin can induce an important change in the conformation of the DNA coil without inducing true breaks in DNA.

Local epigenetic reprograming induced by G-quadruplex ligands

PubMed Central

Recolin, Bénédicte; Campbell, Beth C.; Maiter, Ahmed; Sale, Julian E.; Balasubramanian, Shankar

2017-01-01

DNA and histone modifications regulate transcriptional activity and thus represent valuable targets to reprogram the activity of genes. Current epigenetic therapies target the machinery that regulates these modifications, leading to global transcriptional reprogramming with the potential for extensive undesired effects. Epigenetic information can also be modified as a consequence of disrupting processive DNA replication. Here we demonstrate that impeding replication by small molecule-mediated stabilisation of G-quadruplex nucleic acid secondary structures triggers local epigenetic plasticity. We report the use of the BU-1 locus of chicken DT40 cells to screen for small molecules able to induce G-quadruplex-dependent transcriptional reprogramming. Further characterisation of the top hit compound revealed its ability to induce a dose-dependent inactivation of BU-1 expression in two steps, first loss of H3K4me3 and subsequently DNA cytosine methylation, changes that were heritable across cell divisions even after the compound was removed. Targeting DNA secondary structures thus represents a potentially new approach for locus-specific epigenetic reprogramming. PMID:29064488

Bioactive components, antioxidant and DNA damage inhibitory activities of honeys from arid regions.

PubMed

Habib, Hosam M; Al Meqbali, Fatima T; Kamal, Hina; Souka, Usama D; Ibrahim, Wissam H

2014-06-15

Honey serves as a good source of natural antioxidants, which are effective in reducing the risk of occurrence of several diseases. This study was undertaken to address the limited knowledge regarding the polyphenolic content, antioxidant and DNA damage inhibitory activities of honeys produced in arid regions and compare them with well-recognized honeys from non-arid regions. Different types of honey were assessed for their contents of total phenolics, total flavonoids, and certain types of phenolic compounds. The antioxidant capacity of honey was evaluated by ferric-reducing/antioxidant power assay (FRAP), free radical-scavenging activity (DPPH), nitric oxide (NO) radical-scavenging assay, total antioxidant activity, and DNA damage. Results clearly showed significant differences among honeys with all the evaluated parameters. Results also showed that one or more types of honey from arid regions contained higher levels of phenolic compounds, free radical-scavenging activities, or DNA damage inhibitory activities compared with the evaluated honeys from non-arid regions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Local epigenetic reprogramming induced by G-quadruplex ligands

NASA Astrophysics Data System (ADS)

Guilbaud, Guillaume; Murat, Pierre; Recolin, Bénédicte; Campbell, Beth C.; Maiter, Ahmed; Sale, Julian E.; Balasubramanian, Shankar

2017-11-01

DNA and histone modifications regulate transcriptional activity and thus represent valuable targets to reprogram the activity of genes. Current epigenetic therapies target the machinery that regulates these modifications, leading to global transcriptional reprogramming with the potential for extensive undesired effects. Epigenetic information can also be modified as a consequence of disrupting processive DNA replication. Here, we demonstrate that impeding replication by small-molecule-mediated stabilization of G-quadruplex nucleic acid secondary structures triggers local epigenetic plasticity. We report the use of the BU-1 locus of chicken DT40 cells to screen for small molecules able to induce G-quadruplex-dependent transcriptional reprogramming. Further characterization of the top hit compound revealed its ability to induce a dose-dependent inactivation of BU-1 expression in two steps: the loss of H3K4me3 and then subsequent DNA cytosine methylation, changes that were heritable across cell divisions even after the compound was removed. Targeting DNA secondary structures thus represents a potentially new approach for locus-specific epigenetic reprogramming.

Novel coumarins and related copper complexes with biological activity: DNA binding, molecular docking and in vitro antiproliferative activity.

PubMed

Pivetta, Tiziana; Valletta, Elisa; Ferino, Giulio; Isaia, Francesco; Pani, Alessandra; Vascellari, Sarah; Castellano, Carlo; Demartin, Francesco; Cabiddu, Maria Grazia; Cadoni, Enzo

2017-12-01

Coumarins show biological activity and are widely exploited for their therapeutic effects. Although a great number of coumarins substituted by heterocyclic moieties have been prepared, few studies have been carried out on coumarins containing pyridine heterocycle, which is known to modulate their physiological activities. We prepared and characterized three novel 3-(pyridin-2-yl)coumarins and their corresponding copper(II) complexes. We extended our investigations also to three known similar coumarins, since no data about their biochemical activity was previously been reported. The antiproliferative activity of the studied compounds was tested against human derived tumor cell lines and one human normal cell line. The DNA binding constants were determined and docking studies with DNA carried out. Selected Quantitative Structure-Activity Relationship (QSAR) descriptors were calculated in order to relate a set of structural and topological descriptors of the studied compounds to their DNA interaction and cytotoxic activity. Copyright © 2017 Elsevier Inc. All rights reserved.

Synthesis and characterization of nitrile functionalized silver(I)-N-heterocyclic carbene complexes: DNA binding, cleavage studies, antibacterial properties and mosquitocidal activity against the dengue vector, Aedes albopictus.

PubMed

Asekunowo, Patrick O; Haque, Rosenani A; Razali, Mohd R; Avicor, Silas W; Wajidi, Mustafa F F

2018-04-25

A series of four benzimidazolium based nitrile-functionalized mononuclear-Ag(I)-N-heterocyclic carbene and binuclear-Ag(I)-N-heterocyclic carbene (Ag(I)-NHC) hexafluorophosphate complexes (5b-8b) were synthesized by reacting the corresponding hexafluorophosphate salts (1b-4b) with Ag 2 O in acetonitrile, respectively. These compounds were characterized by 1 H NMR, 13 C NMR, IR, UV-visible spectroscopic techniques, elemental analyses and molar conductivity. Additionally, 8b was structurally characterized by single crystal X-ray diffraction technique. Preliminary in vitro antibacterial evaluation was conducted for all the compounds against two standard bacteria; gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacterial strains. Most of the Ag(I)-NHC complexes (5b-8b) showed moderate to good antibacterial activity with MIC values in the range of 12.5-100 μg/mL. Especially, compound 8b exhibited promising anti-Staphylococcus aureus activity with a low MIC value (12.5 μg/mL). However, all the hexafluorophosphate salts (1b-4b) were inactive against the bacteria strains. The preliminary interactive investigation revealed that the most active compound, 8b, could effectively intercalate into DNA to form 8b-DNA complex which shows a better binding ability for DNA (K b  = 3.627 × 10 6 ) than the complexes 5b-7b (2.177 × 10 6 , 8.672 × 10 5 and 6.665 × 10 5 , respectively). Nuclease activity of the complexes on plasmid DNA and Aedes albopictus genomic DNA was time-dependent, although minimal. The complexes were larvicidal to the mosquito, with 5b, 6b and 8b being highly active. Developmental progression from the larval to the adult stage was affected by the complexes, progressively being toxic to the insect's development with increasing concentration. These indicate the potential use of these complexes as control agents against bacteria and the dengue mosquito Ae. albopictus. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Synthesis of trimethoprim metal complexes: Spectral, electrochemical, thermal, DNA-binding and surface morphology studies.

PubMed

Demirezen, Nihat; Tarınç, Derya; Polat, Duygu; Ceşme, Mustafa; Gölcü, Ayşegül; Tümer, Mehmet

2012-08-01

Complexes of trimethoprim (TMP), with Cu(II), Zn(II), Pt(II), Ru(III) and Fe(III) have been synthesized. Then, these complexes have been characterized by spectroscopic techniques involving UV-vis, IR, mass and (1)H NMR. CHN elemental analysis, electrochemical and thermal behavior of complexes have also been investigated. The electrochemical properties of all complexes have been investigated by cyclic voltammetry (CV) using glassy carbon electrode. The biological activity of the complexes has been evaluated by examining their ability to bind to calf-thymus DNA (CT DNA) with UV spectroscopy and cyclic voltammetry. UV studies of the interaction of the complexes with DNA have shown that these compounds can bind to CT DNA. The binding constants of the complexes with CT DNA have also been calculated. The cyclic voltammograms of the complexes in the presence of CT DNA have shown that the complexes can bind to CT DNA by both the intercalative and the electrostatic binding mode. The antimicrobial activity of these complexes has been evaluated against three Gram-positive and four Gram-negative bacteria. Antifungal activity against two different fungi has been evaluated and compared with the reference drug TMP. Almost all types of complexes show excellent activity against all type of bacteria and fungi. The morphology of the CT DNA, TMP, metal ions and metal complexes has been investigated by scanning electron microscopy (SEM). To get the SEM images, the interaction of compounds with CT DNA has been studied by means of differential pulse voltammetry (DPV) at CT DNA modified pencil graphite electrode (PGE). The decrease in intensity of the guanine oxidation signals has been used as an indicator for the interaction mechanism. Copyright © 2012 Elsevier B.V. All rights reserved.

Targeting copper(II)-induced oxidative stress and the acetylcholinesterase system in Alzheimer's disease using multifunctional tacrine-coumarin hybrid molecules.

PubMed

Hamulakova, Slavka; Poprac, Patrik; Jomova, Klaudia; Brezova, Vlasta; Lauro, Peter; Drostinova, Lenka; Jun, Daniel; Sepsova, Vendula; Hrabinova, Martina; Soukup, Ondrej; Kristian, Pavol; Gazova, Zuzana; Bednarikova, Zuzana; Kuca, Kamil; Valko, Marian

2016-08-01

Alzheimer's disease is a multifactorial disease that is characterized mainly by Amyloid-β (A-β) deposits, cholinergic deficit and extensive metal (copper, iron)-induced oxidative stress. In this work we present details of the synthesis, antioxidant and copper-chelating properties, DNA protection study, cholinergic activity and amyloid-antiaggregation properties of new multifunctional tacrine-7-hydroxycoumarin hybrids. The mode of interaction between copper(II) and hybrids and interestingly, the reduction of Cu(II) to Cu(I) species (for complexes Cu-5e-g) were confirmed by EPR measurements. EPR spin trapping on the model Fenton reaction, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, demonstrated a significantly suppressed formation of hydroxyl radicals for the Cu-5e complex in comparison with free copper(II). This suggests that compound 5e upon coordination to free copper ion prevents the Cu(II)-catalyzed decomposition of hydrogen peroxide, which in turn may alleviate oxidative stress-induced damage. Protective activity of hybrids 5c and 5e against DNA damage in a Fenton system (copper catalyzed) was found to be in excellent agreement with the EPR spin trapping study. Compound 5g was the most effective in the inhibition of acetylcholinesterase (hAChE, IC50=38nM) and compound 5b was the most potent inhibitor of butyrylcholinesterase (hBuChE, IC50=63nM). Compound 5c was the strongest inhibitor of A-β1-40 aggregation, although a significant inhibition (>50%) was detected for compounds 5b, 5d, 5e and 5g. Collectively, these results suggest that the design and investigation of multifunctional agents containing along with the acetylcholinesterase inhibitory segment also an antioxidant moiety capable of alleviating metal (copper)-induced oxidative stress, may be of importance in the treatment of Alzheimer's disease. Copyright © 2016 Elsevier Inc. All rights reserved.

Discriminating model for skin cancer diagnosis in vivo through Raman spectroscopy

NASA Astrophysics Data System (ADS)

Silveira, Fabrício Luiz; Pacheco, Marcos Tadeu T.; Bodanese, Benito; Zângaro, Renato Amaro; Silveira, Landulfo

2013-03-01

This work aimed the development of a discriminating model, using Raman spectroscopy, based on the estimated concentration of biochemical components presented in skin, for in vivo diagnosis. Raman spectra were collected in patients who underwent excision surgery of suspicious lesions at the lesion site and at a normal circumjacent site. It has been estimated the relative amount of selected biochemical compounds presented in skin. The Raman spectra of normal and malignant (basal cell carcinoma - BCC and squamous cell carcinoma - SCC) skin are quite similar, with some spectral differences in the regions of lipids, nucleic acids, and hemoglobin. Some biochemicals showed statistically significant differences among N, BCC and SCC, such as elastin, ceramide, melanin, nucleid acid, actin and phenylalanine. Elastin and ceramide presented significant differences between N and BCC, melanin, DNA and actin presented significant differences between N and BCC and between N and SCC, being melanin and DNA decreased in neoplasias, in contrast with actin, that increased in neoplasias. Concentration of phenylalanine was significantly increased for SCC compared to N and BCC. The relative concentration of melanin, DNA and phenylalanine showed sensitivity, specificity and accuracy of about 81%, 65% and 60%, respectively, using Mahalanobis distance as a discriminator. This model is being incorporated to a Raman system with automated data collection and processing that could be used for a future in vivo, real time discrimination algorithm.

Spectroscopic studies on the interactions of 5-ethyl-6-phenyl-3,8-bis((3-aminoalkyl)propanamido)phenanthridin-5-ium derivatives with G-quadruplex DNA

NASA Astrophysics Data System (ADS)

Yalçın, Ergin; Duyar, Halil; Ihmels, Heiko; Seferoğlu, Zeynel

2018-05-01

An improved microwave-induced synthesis of five ethidium derivatives (Ethidium derivatives, 2a-d) is presented. As the derivatives 2a-d have been proposed previously to be telomerase inhibitors, the binding interactions of these ethidium derivatives with G-quadruplex DNA were evaluated by means of photometric and fluorimetric titration, thermal DNA denaturation, CD and 1H NMR spectroscopy. In particular, the compound bearing 3,8-bis(pyrrolidin-1-yl)propanamido substituent 2a exhibits high selectivity for G-quadruplex DNA relative to duplex DNA.

DNA attachment to support structures

DOEpatents

Balhorn, Rodney L.; Barry, Christopher H.

2002-01-01

Microscopic beads or other structures are attached to nucleic acids (DNA) using a terminal transferase. The transferase adds labeled dideoxy nucleotide bases to the ends of linear strands of DNA. The labels, such as the antigens digoxigenin and biotin, bind to the antibody compounds or other appropriate complementary ligands, which are bound to the microscopic beads or other support structures. The method does not require the synthesis of a synthetic oligonucleotide probe. The method can be used to tag or label DNA even when the DNA has an unknown sequence, has blunt ends, or is a very large fragment (e.g., >500 kilobase pairs).

Use of LC-MS/MS and Stable Isotopes to Differentiate Hydroxymethyl and Methyl DNA Adducts from Formaldehyde and Nitrosodimethylamine

PubMed Central

Lu, Kun; Craft, Sessaly; Nakamura, Jun; Moeller, Benjamin C.; Swenberg, James A.

2012-01-01

Formaldehyde is a known human and animal carcinogen that forms DNA adducts, and causes mutations. While there is widespread exposure to formaldehyde in the environment, formaldehyde is also an essential biochemical in all living cells. The presence of both endogenous and exogenous sources of formaldehyde makes it difficult to develop exposure-specific DNA biomarkers. Furthermore, chemicals such as nitrosodimethylamine form one mole of formaldehyde for every mole of methylating agent, raising questions about potential co-carcinogenesis. Formaldehyde-induced hydroxymethyl DNA adducts are not stable and need to be reduced to stable methyl adducts for detection, which adds another layer of complexity to identifying the origins of these adducts. In this study, highly sensitive mass spectrometry methods and isotope labeled compounds were used to differentiate between endogenous and exogenous hydroxymethyl and methyl DNA adducts. We demonstrate that N2-hydroxymethyl-dG is the primary DNA adduct formed in cells following formaldehyde exposure. In addition, we show that alkylating agents induce methyl adducts at N2-dG and N6-dA positions, which are identical to the reduced forms of hydroxymethyl adducts arising from formaldehyde. The use of highly sensitive LC-MS/MS and isotope labeled compounds for exposure solves these challenges and provides mechanistic insights on the formation and role of these DNA adducts. PMID:22148432

S - and N-alkylating agents diminish the fluorescence of fluorescent dye-stained DNA.

PubMed

Giesche, Robert; John, Harald; Kehe, Kai; Schmidt, Annette; Popp, Tanja; Balzuweit, Frank; Thiermann, Horst; Gudermann, Thomas; Steinritz, Dirk

2017-01-25

Sulfur mustard (SM), a chemical warfare agent, causes DNA alkylation, which is believed to be the main cause of its toxicity. SM DNA adducts are commonly used to verify exposure to this vesicant. However, the required analytical state-of-the-art mass-spectrometry methods are complex, use delicate instruments, are not mobile, and require laboratory infrastructure that is most likely not available in conflict zones. Attempts have thus been made to develop rapid detection methods that can be used in the field. The analysis of SM DNA adducts (HETE-G) by immunodetection is a convenient and suitable method. For a diagnostic assessment, HETE-G levels must be determined in relation to the total DNA in the sample. Total DNA can be easily visualized by the use of fluorescent DNA dyes. This study examines whether SM and related compounds affect total DNA staining, an issue that has not been investigated before. After pure DNA was extracted from human keratinocytes (HaCaT cells), DNA was exposed to different S- and N-alkylating agents. Our experiments revealed a significant, dose-dependent decrease in the fluorescence signal of fluorescent dye-stained DNA after exposure to alkylating agents. After mass spectrometry and additional fluorescence measurements ruled out covalent modifications of ethidium bromide (EthBr) by SM, we assumed that DNA crosslinks caused DNA condensation and thereby impaired access of the fluorescent dyes to the DNA. DNA digestion by restriction enzymes restored fluorescence, a fact that strengthened our hypothesis. However, monofunctional agents, which are unable to crosslink DNA, also decreased the fluorescence signal. In subsequent experiments, we demonstrated that protons produced during DNA alkylation caused a pH decrease that was found responsible for the reduction in fluorescence. The use of an appropriate buffer system eliminated the adverse effect of alkylating agents on DNA staining with fluorescent dyes. An appropriate buffer system is thus crucial for DNA quantification with fluorescent dyes in the presence of alkylating compounds. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Determinants for Tight and Selective Binding of a Medicinal Dicarbene Gold(I) Complex to a Telomeric DNA G-Quadruplex: a Joint ESI MS and XRD Investigation.

PubMed

Bazzicalupi, Carla; Ferraroni, Marta; Papi, Francesco; Massai, Lara; Bertrand, Benoît; Messori, Luigi; Gratteri, Paola; Casini, Angela

2016-03-18

The dicarbene gold(I) complex [Au(9-methylcaffein-8-ylidene)2 ]BF4 is an exceptional organometallic compound of profound interest as a prospective anticancer agent. This gold(I) complex was previously reported to be highly cytotoxic toward various cancer cell lines in vitro and behaves as a selective G-quadruplex stabilizer. Interactions of the gold complex with various telomeric DNA models have been analyzed by a combined ESI MS and X-ray diffraction (XRD) approach. ESI MS measurements confirmed formation of stable adducts between the intact gold(I) complex and Tel 23 DNA sequence. The crystal structure of the adduct formed between [Au(9-methylcaffein-8-ylidene)2 ](+) and Tel 23 DNA G-quadruplex was solved. Tel 23 maintains a characteristic propeller conformation while binding three gold(I) dicarbene moieties at two distinct sites. Stacking interactions appear to drive noncovalent binding of the gold(I) complex. The structural basis for tight gold(I) complex/G-quadruplex recognition and its selectivity are described. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genotoxic and antigenotoxic effects of catechin and tannins from the bark of Hamamelis virginiana L. in metabolically competent, human hepatoma cells (Hep G2) using single cell gel electrophoresis.

PubMed

Dauer, Andreas; Hensel, Andreas; Lhoste, Evelyne; Knasmüller, Siegfried; Mersch-Sundermann, Volker

2003-05-01

The genotoxic and antigenotoxic activities of catechin, hamamelitannin and two proanthocyanidin fractions prepared from the bark of Hamamelis virginiana L. were investigated in a human derived, metabolically competent hepatoma cell line (Hep G2) using single cell gel electrophoresis (SCGE) for the detection of DNA-damage. DNA-migration was calculated as Olive tail moment (OTM). Catechin and a low-molecular weight proanthocyandin fraction (W(M)) caused only slight increases of OTM up to concentrations of 166 microg/ml whereas hamamelitannin and the proanthocyandin fraction with higher molecular weight (W(A)) led to a two-fold enhancement of OTM at the same concentrations. These effects were dose-independent. Treatment of the cells with the test compounds in a dose-range of 2-166 microg/ml prior to the exposure to benzo(a)pyrene (B(a)P, 10 microM, 2.5 microg/ml) led to a significant reduction of induced DNA damage which was dose-dependent for all test compounds, except for hamamelitannin. The inhibitory effects of proanthocyanidins were stronger than those of catechin and hamamelitannin; the lowest effective concentrations were about 2 microg/ml. In order to clarify the mechanisms of protection, possible effects of the test compounds on enzymes involved in toxification and detoxification of B(a)P were investigated. While B(a)P toxification by cytochrome P450 was not inhibited by the test compounds, detoxification by glutathion-S-transferase (GST) was induced by catechin and W(M). Combination experiments with the ultimate metabolite of B(a)P, (+/-)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE; 5 microM, 1.5 microg/ml), revealed strong inhibitory effects, indicating that the observed protective effects were caused by scavenging of the ultimate mutagen by the test compounds. Exposure of Hep G2 cells to the test compounds after B(a)P treatment did not influence B(a)P induced DNA damage, demonstrating that repair mechanisms were not affected.

[Stimulation of DNA molecules association with amphiphilic derivatives of 1,3-diazaadamantane containing hydrophobic side chanins].

PubMed

Mamaeva, O K; Gabrielian, A G; Arutiunian, G L; Bocharova, T N; Smirnova, E A; Volodin, A A; Shchelkina, A K; Kaliuzhnyĭ, D N

2014-01-01

Earlier, a new class of compounds--amphiphilic derivatives of 1,3-diazaadamantanes, capable of facilitating the strand exchange in the system of short oligonucleotides was revealed. Longer hydrophobic side chains of 1,3-diazaadamantanes promoted stronger acceleration of the reaction. In this study, interaction with DNA of two 1,3-diazaadamantane derivatives containing different side chains was investigated by use of optical methods. Concentration of the investigated 1,3-diazaadamantans micelles formation were determined by the means of monitoring fluorescence intensity enhancement of 1-anilinonaphtalene-8-sulphonate probe; as well as the ranges of concentrations where the compounds/water mixtures existed as true solutions. 1,3-diazaadamantanes affinity to DNA was determined with Fluorescent Intercalator Displacement (FID) approach. Significant increase in hydrodynamic volume of short DNA hairpins in the complexes with 1,3-diazaadamantanes was revealed by estimation of the fluorescence polarization of ethidium bromide probe bound to the hairpins. Intermolecular association of DNA hairpins upon binding with 1,3-diazaadamantans was confirmed by Förster resonance energy transfer in system of an equimolar mixture of fluorescently labeled with Cy-3 and Cy-5 hairpins. In this study, the number of positive charges at 1,3-diazaadamantane derivatives containing side chains of different lengths was demonstrated to affect their affinity to DNA, whereas longer length of the hydrophobic side chains ensured more efficient interaction between the DNA duplexes that may facilitate, in particular, DNA strand exchange.

Anthocyanin-rich blackberry extract suppresses the DNA-damaging properties of topoisomerase I and II poisons in colon carcinoma cells.

PubMed

Esselen, Melanie; Boettler, Ute; Teller, Nicole; Bachler, Simone; Hutter, Melanie; Rufer, Corinna E; Skrbek, Susanne; Marko, Doris

2011-07-13

In the present study, we addressed the question whether cyanidin-3-glucoside (C3G) or complex C3G-rich blackberry extracts affect human topoisomerases with special emphasis on the contribution of the potential degradation products phloroglucinol aldehyde (PGA) and protocatechuic acid (PCA). In HT29 colon carcinoma cells a C3G-rich blackberry extract suppressed camptothecin- (CPT-) or doxorubicin- (DOX-) induced stabilization of the covalent DNA-topoisomerase intermediate, thus antagonizing the effects of these classical topoisomerase poisons on DNA integrity. As a single compound, C3G (100 μM) decreased the DNA-damaging effects of CPT as well, but did not significantly affect those induced by DOX. At the highest applied concentration (100 μM), cyanidin protected DNA from CPT- and DOX-induced damage. Earlier reports on DNA-damaging properties of cyanidin were found to result most likely from the formation of hydrogen peroxide as an artifact in the cell culture medium when the incubation was performed in the absence of catalase. The suppression of hydrogen peroxide accumulation, achieved by the addition of catalase, demonstrated that cyanidin does not exhibit DNA-damaging properties in HT29 cells (up to 100 μM). The observed effects on topoisomerase interference and DNA protection against CPT or DOX were clearly limited to the parent compound and were not observed for the potential cyanidin degradation products PGA and PCA.

ACCUMULATION OF M1DG DNA ADDUCTS AFTER CHRONIC EXPOSURE TO PCBS, BUT NOT FROM ACUTE EXPOSURE TO DIOXIN-LIKE COMPOUNDS

EPA Science Inventory

ABSTRACT: Oxidative DNA damage is one of the key events leading to mutation and cancer. The present study examined the accumulation of M1dG DNA adducts, 3-(2’-deoxy-β-D-erythro-pentofuranosyl)-pyrimido[1,2-a]-purin-10(3H)-one, after single or yearly exposur...

Antibacterial activity of lichen secondary metabolite usnic acid is primarily caused by inhibition of RNA and DNA synthesis.

PubMed

Maciąg-Dorszyńska, Monika; Węgrzyn, Grzegorz; Guzow-Krzemińska, Beata

2014-04-01

Usnic acid, a compound produced by various lichen species, has been demonstrated previously to inhibit growth of different bacteria and fungi; however, mechanism of its antimicrobial activity remained unknown. In this report, we demonstrate that usnic acid causes rapid and strong inhibition of RNA and DNA synthesis in Gram-positive bacteria, represented by Bacillus subtilis and Staphylococcus aureus, while it does not inhibit production of macromolecules (DNA, RNA, and proteins) in Escherichia coli, which is resistant to even high doses of this compound. However, we also observed slight inhibition of RNA synthesis in a Gram-negative bacterium, Vibrio harveyi. Inhibition of protein synthesis in B. subtilis and S. aureus was delayed, which suggest indirect action (possibly through impairment of transcription) of usnic acid on translation. Interestingly, DNA synthesis was halted rapidly in B. subtilis and S. aureus, suggesting interference of usnic acid with elongation of DNA replication. We propose that inhibition of RNA synthesis may be a general mechanism of antibacterial action of usnic acid, with additional direct mechanisms, such as impairment of DNA replication in B. subtilis and S. aureus. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

In vitro characterization of DNA gyrase inhibition by microcin B17 analogs with altered bisheterocyclic sites

PubMed Central

Zamble, Deborah B.; Miller, Deborah A.; Heddle, Jonathan G.; Maxwell, Anthony; Walsh, Christopher T.; Hollfelder, Florian

2001-01-01

Microcin B17 (MccB17) is a 3.1-kDa Escherichia coli antibiotic that contains thiazole and oxazole heterocycles in a peptide backbone. MccB17 inhibits its cellular target, DNA gyrase, by trapping the enzyme in a complex that is covalently bound to double-strand cleaved DNA, in a manner similar to the well-known quinolone drugs. The identification of gyrase as the target of MccB17 provides an opportunity to analyze the relationship between the structure of this unusual antibiotic and its activity. In this report, steady-state parameters are used to describe the induction of the cleavable complex by MccB17 analogs containing modified bisheterocyclic sites. The relative potency of these analogs corresponds to the capacity of the compounds to prevent growth of sensitive cells. In contrast to previously reported experiments, inhibition of DNA gyrase supercoiling activity by wild-type MccB17 also was observed. These results suggest that DNA gyrase is the main intracellular target of MccB17. This study probes the structure-function relationship of a new class of gyrase inhibitors and demonstrates that these techniques could be used to analyze compounds in the search for clinically useful antibiotics that block DNA gyrase. PMID:11427730

The naphthoquinone diospyrin is an inhibitor of DNA gyrase with a novel mechanism of action.

PubMed

Karkare, Shantanu; Chung, Terence T H; Collin, Frederic; Mitchenall, Lesley A; McKay, Adam R; Greive, Sandra J; Meyer, Jacobus J M; Lall, Namrita; Maxwell, Anthony

2013-02-15

Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents.

The Naphthoquinone Diospyrin Is an Inhibitor of DNA Gyrase with a Novel Mechanism of Action*

PubMed Central

Karkare, Shantanu; Chung, Terence T. H.; Collin, Frederic; Mitchenall, Lesley A.; McKay, Adam R.; Greive, Sandra J.; Meyer, Jacobus J. M.; Lall, Namrita; Maxwell, Anthony

2013-01-01

Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents. PMID:23275348

Synthesis of bifunctional molecules containing [12]aneN3 and coumarin moieties as effective DNA condensation agents and new non-viral gene vectors.

PubMed

Yue, Pan; Zhang, Ying; Guo, Zhi-Fo; Cao, Ao-Cheng; Lu, Zhong-Lin; Zhai, Yong-Gong

2015-04-21

A series of bifunctional molecules with different combinations of macrocyclic polyamine [12]aneN3 and coumarin moieties, 4a/b and 5a/b, were synthesized by a two-step copper(I)-mediated alkyne–azide click reactions between 1,3,5-tris(azidomethyl)benzene and Boc-protected N-propynyl-[12]aneN3/7-propynyloxycoumarins. Agarose gel electrophoresis experiments indicated that bifunctional molecules 4b and 5b effectively induced complete plasmid DNA condensation at concentrations up to 40 μM. It was found that the structural variation had a major impact on the condensation behavior of these compounds. The electrostatic interaction involving the [12]aneN3 moiety can be compensated by the binding contribution of the coumarin units during the DNA condensation process. These two types of interaction showed different effects on the reversibility of DNA condensation. Results from studies using dynamic laser scattering, atomic force microscopy, and EB replacement assay further supported the above conclusion. Cytotoxicity assays on bifunctional compounds 4a/b and 5a/b indicated their low cytotoxicity. Results from cellular uptake and cell transfection experiments proved that bifunctional compounds 4b and 5b successfully served as non-viral gene vectors. Furthermore, methyl substituents attached to the coumarin unit (4b and 5b) greatly enhanced their DNA condensation capability and gene transfection. These bifunctional molecules, with the advantages of lower cytotoxicity, good water solubility, and potential structural modification, will have great potential for the development of new non-viral gene delivery agents.

1,2,3-Triazole Tagged 3H-Pyrano[2,3-d]pyrimidine-6-carboxylate Derivatives: Synthesis, in Vitro Cytotoxicity, Molecular Docking and DNA Interaction Studies.

PubMed

Boda, Sathish Kumar; Pishka, Vasantha; Lakshmi, P V Anantha; Chinde, Srinivas; Grover, Paramjit

2018-06-01

A series of novel ethyl 2,7-dimethyl-4-oxo-3-[(1-phenyl-1H-1,2,3-triazol-4-yl)methyl]-4,5-dihydro-3H-pyrano[2,3-d]pyrimidine-6-carboxylate derivatives 7a - 7m were efficiently synthesized employing click chemistry approach and evaluated for in vitro cytotoxic activity against four tumor cell lines: A549 (human lung adenocarcinoma cell line), HepG2 (human hematoma), MCF-7 (human breast adenocarcinoma), and SKOV3 (human ovarian carcinoma cell line). Among the compounds tested, the compounds 7a, 7b, 7f, 7l, and 7m have shown potential and selective activity against human lung adenocarcinoma cell line (A549) with IC 50 ranging from 0.69 to 6.74 μm. Molecular docking studies revealed that the compounds 7a, 7b, 7f, 7l, and 7m are potent inhibitors of human DNA topoisomerase-II and also showed compliance with stranded parameters of drug likeness. The calculated binding constants, k b , from UV/VIS absorptional binding studies of 7a and 7l with CT-DNA were 10.77 × 10 4 , 6.48 × 10 4 , respectively. Viscosity measurements revealed that the binding could be surface binding mainly due to groove binding. DNA cleavage study showed that 7a and 7l have the potential to cleave pBR322 plasmid DNA without any external agents. © 2018 Wiley-VHCA AG, Zurich, Switzerland.

Effects of cytotoxic cis- and trans-diammine monochlorido platinum(II) complexes on selenium-dependent redox enzymes and DNA.

PubMed

Lemmerhirt, Heidi; Behnisch, Steven; Bodtke, Anja; Lillig, Christopher H; Pazderova, Lucia; Kasparkova, Jana; Brabec, Viktor; Bednarski, Patrick J

2018-01-01

Here we present the preparation of 14 pairs of cis- and trans-diammine monochlorido platinum(II) complexes, coordinated to heterocycles (i.e., imidazole, 2-methylimidazole and pyrazole) and linked to various acylhydrazones, which were designed as potential inhibitors of the selenium-dependent enzymes glutathione peroxidase 1 (GPx-1) and thioredoxin reductase 1 (TrxR-1). However, no inhibition of bovine GPx-1 and only weak inhibition of murine TrxR-1 was observed in in vitro assays. Nonetheless, the cis configured diammine monochlorido Pt(II) complexes exhibited cytotoxic and apoptotic properties on various human cancer cell lines, whereas the trans configured complexes generally showed weaker potency with a few exceptions. On the other hand, the trans complexes were generally more likely to lack cross-resistance to cisplatin than the cis analogues. Platinum was found bound to the nuclear DNA of cancer cells treated with representative Pt complexes, suggesting that DNA might be a possible target. Thus, detailed in vitro binding experiments with DNA were conducted. Interactions of the compounds with calf thymus DNA were investigated, including Pt binding kinetics, circular dichroism (CD) spectral changes, changes in DNA melting temperatures, unwinding of supercoiled plasmids and ethidium bromide displacement in DNA. The CD results indicate that the most active cis configured pyrazole-derived complex causes unique structural changes in the DNA compared to the other complexes as well as to those caused by cisplatin, suggesting a denaturation of the DNA structure. This may be important for the antiproliferative activity of this compound in the cancer cells. Copyright © 2017. Published by Elsevier Inc.

Protective effect of Uncaria tomentosa extract against oxidative stress and genotoxicity induced by glyphosate-Roundup® using zebrafish (Danio rerio) as a model.

PubMed

Santo, Glaucia Dal; Grotto, Alan; Boligon, Aline A; Da Costa, Bárbara; Rambo, Cassiano L; Fantini, Emily A; Sauer, Elisa; Lazzarotto, Luan M V; Bertoncello, Kanandra T; Júnior, Osmar Tomazelli; Garcia, Solange C; Siebel, Anna M; Rosemberg, Denis B; Magro, Jacir Dal; Conterato, Greicy M M; Zanatta, Leila

2018-04-01

Oxidative stress and DNA damage are involved in the glyphosate-based herbicide toxicity. Uncaria tomentosa (UT; Rubiaceae) is a plant species from South America containing bioactive compounds with known beneficial properties. The objective of this work was to evaluate the antioxidant and antigenotoxic potential of UT extract in a model of acute exposure to glyphosate-Roundup® (GR) in zebrafish (Danio rerio). We showed that UT (1.0 mg/mL) prevented the decrease of brain total thiols, the increase of lipid peroxidation in both brain and liver, and the decrease of liver GPx activity caused after 96 h of GR (5.0 mg/L) exposure. In addition, UT partially protected against the increase of micronucleus frequency induced by GR exposure in fish brain. Overall, our results indicate that UT protects against damage induced by a glyphosate-based herbicide by providing antioxidant and antigenotoxic effects, which may be related to the phenolic compounds identified in the extract.

[Novel hybrid inhibitors of the phage T7 RNA polymerase: synthesis, docking and screening in vitro].

PubMed

Kostina, V H; Pal'chykovs'ka, L H; Platonov, M O; Vasyl'chenko, O V; Lysenko, N A; Alekseeva, I V

2012-01-01

A number of new hybrid heteroaromatic compounds, consisting of tricyclic fragments (acridone, thioxanthone and phenazine) and bicyclic fragments (benzimidazole, benzothiazole and benzoxazole) were synthesized using the method, developed by the authors. As a result of screening against the transcription model system of the phage T7 DNA-dependent RNA polymerase three effective inhibitors of the RNA syntheses with the IC50 value of 8.9, 5.7 and 19.8 microM were detected. To cast light on the mode of interaction between the synthesized compounds and the target, the molecular docking was applied to the model pocket of the phage T7 RNA polymerase transcription complex. It was established that these ligands form networks of H-bonds with residues of the pocket conservative amino acids and pi-interaction with the Mg2+ ion. A planar geometry of the hybrid molecules, realized due to the intramolecular H-bonds, proved to be an important structural feature, which correlates with an efficacious inhibitory activity.

Characterization of Ofloxacin Interaction with Mutated (A91V) Quinolone Resistance Determining Region of DNA Gyrase in Mycobacterium Leprae through Computational Simulation.

PubMed

Nisha, J; Shanthi, V

2018-06-01

Mycobacterium leprae, the causal agent of leprosy is non-cultivable in vitro. Thus, the assessment of antibiotic activity against Mycobacterium leprae depends primarily upon the time-consuming mouse footpad system. The GyrA protein of Mycobacterium leprae is the target of the antimycobacterial drug, Ofloxacin. In recent times, the GyrA mutation (A91V) has been found to be resistant to Ofloxacin. This phenomenon has necessitated the development of new, long-acting antimycobacterial compounds. The underlying mechanism of drug resistance is not completely known. Currently, experimentally crystallized GyrA-DNA-OFLX models are not available for highlighting the binding and mechanism of Ofloxacin resistance. Hence, we employed computational approaches to characterize the Ofloxacin interaction with both the native and mutant forms of GyrA complexed with DNA. Binding energy measurements obtained from molecular docking studies highlights hydrogen bond-mediated efficient binding of Ofloxacin to Asp47 in the native GyrA-DNA complex in comparison with that of the mutant GyrA-DNA complex. Further, molecular dynamics studies highlighted the stable binding of Ofloxacin with native GyrA-DNA complex than with the mutant GyrA-DNA complex. This mechanism provided a plausible reason for the reported, reduced effect of Ofloxacin to control leprosy in individuals with the A91V mutation. Our report is the first of its kind wherein the basis for the Ofloxacin drug resistance mechanism has been explored with the help of ternary Mycobacterium leprae complex, GyrA-DNA-OFLX. These structural insights will provide useful information for designing new drugs to target the Ofloxacin-resistant DNA gyrase.

Studies on free radical scavenging, cancer cell antiproliferation, and calf thymus DNA interaction of Schiff bases.

PubMed

da Silva, Cleiton M; Silva, Marina M; Reis, Fabiano S; Ruiz, Ana Lúcia T G; de Carvalho, João E; Santos, Josué C C; Figueiredo, Isis M; Alves, Rosemeire B; Modolo, Luzia V; de Fátima, Ângelo

2017-07-01

Thirty-nine Schiff bases were synthesized by performing microwave-assisted condensation of the corresponding aldehydes and aromatic amines. Their reactive nitrogen species (RNS) scavenging activity and inhibitory effects against cancer cell growth were then subsequently investigated. Additionally, the interaction between the calf thymus DNA (ctDNA) and selected Schiff bases was evaluated using fluorescence spectroscopy, and their binding parameters were determined. The yields of the various compounds ranged from moderate to excellent (43-99%) after only a 2-min reaction. The hydroxylated Schiff bases 2, 8, 15, 16, 18, 20, 29, 32, 34, and 37 were found to be potent scavengers of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals with half-maximal scavenging concentration (SC 50 ) values lower than that of the positive control, resveratrol. The presence of hydroxyl substituents on the aromatic rings also proved essential to the cytotoxicity of the compounds. The binding constants (K b ) obtained using fluorescence spectroscopy ranged from 0.37 to 3.07×10 5 Lmol -1 , and were strongly influenced by the structure and hydroxylation degree. Schiff bases 3 and 8 showed promising cytotoxic activity, with half-maximal growth inhibitory (GI 50 ) values in the same order of magnitude as those exhibited by the reference drug, doxorubicin against various cell lines. Interestingly, these compounds also showed the highest K b , suggesting that the cytotoxic activity could be related to their interaction with the DNA of the tumor cells. The results of this study highlighted some Schiff bases as potential lead compounds for the design of new free radical scavengers and anticancer agents. Copyright © 2017 Elsevier B.V. All rights reserved.

Phototoxicity of phenylenediamine hair dye chemicals in Salmonella typhimurium TA102 and human skin keratinocytes.

PubMed

Mosley-Foreman, Charity; Choi, Jaehwa; Wang, Shuguang; Yu, Hongtao

2008-12-01

Phenylenediamines (PD) are dye precursors used to manufacture hair dyes. The three PDs, 1,2-,1,3-, and 1,4-PD and three chlorinated PDs, 4-chloro-1,2-PD, 4-chloro-1,3-PD, and 4,5-dichloro-1,2-PD were studied for their mutagenic effect in Salmonella typhimurium TA 102, cytotoxicity in human skin keratinocyte cells, and for DNA cleavage. The results show that all six compounds are not toxic/mutagenic in TA 102 bacteria or skin cells, and do not cause DNA cleavage in PhiX 174 phage DNA. If the same tests are carried out by exposing them to light irradiation concurrently, all three chlorinated PDs cause mutation in TA 102 bacteria and single strand cleavage in PhiX174 phage DNA. This indicates that chlorination of the PDs makes these compounds more photochemically active and produces reactive species that cause DNA damage and mutation. For the photocytotoxicity test in skin cells, it appears there is no such structure-activity relationship. Two chlorinated PDs and two non-chlorinated PDs are cytotoxic at a fairly high concentration (1000microM) upon exposure to light irradiation.

High-throughput genotoxicity assay identifies antioxidants as inducers of DNA damage response and cell death

EPA Science Inventory

Human ATAD5 is an excellent biomarker for identifying genotoxic compounds because ATADS protein levels increase post-transcriptionally following exposure to a variety of DNA damaging agents. Here we report a novel quantitative high-throughput ATAD5-Iuciferase assay that can moni...

Characterization of Thermally Activated Metalloenediyne Cytotoxicity in Human Melanoma Cells.

PubMed

Keller, Eric J; Porter, Meghan; Garrett, Joy E; Varie, Meredith; Wang, Haiyan; Pollok, Karen E; Turchi, John J; Zaleski, Jeffrey M; Dynlacht, Joseph R

2018-05-15

Enediynes are a highly cytotoxic class of compounds. However, metallation of these compounds may modulate their activation, and thus their cytotoxicity. We previously demonstrated that cytotoxicity of two different metalloenediynes, including (Z)-N,N'-bis[1-pyridyl-2-yl-meth-(E)-ylidene]octa-4-ene-2,6-diyne-1,8-diamine] (PyED), is potentiated when the compounds are administered to HeLa cells during hyperthermia treatment at concentrations that are minimally or not cytotoxic at 37°C. In this study, we further characterized the concentration, time and temperature dependence of cytotoxicity of PyED on human U-1 melanoma cells. We also investigated the potential mechanisms by which PyED cytotoxicity is enhanced during hyperthermia treatment. Cell killing with PyED was dependent on concentration, temperature during treatment and time of exposure. Potentiation of cytotoxicity was observed when cells were treated with PyED at temperatures ≥39.5°C, and enhancement of cell killing increased with temperature and with increasing time at a given temperature. All cells treated with PyED were shown to have DNA damage, but substantially more damage was observed in cells treated with PyED during heating. DNA repair was also inhibited in cells treated with the drug during hyperthermia. Thus, potentiation of PyED cytotoxicity by hyperthermia may be due to enhancement of drug-induced DNA lesions, and/or the inhibition of repair of sublethal DNA damage. While the selective thermal activation of PyED supports the potential clinical utility of metalloenediynes as cancer thermochemotherapeutic agents, therapeutic gain could be optimized by identifying compounds that produce minimal toxicity at 37°C but which become activated and show enhancement of cytotoxicity within a tumor subjected to localized hyperthermic or thermal ablative treatment, or which might act as bifunctional agents. We thus also describe the development and initial characterization of a novel cofactor complex of PyED, platinated PyED (Pt-PyED). Pt-PyED binds to DNA-like cisplatin, and much like PyED, cytotoxicity is greatly enhanced after treatment with the drug at elevated temperatures. However, in contrast to PyED, Pt-PyED is only minimally cytotoxic at 37°C, at concentrations at which cytotoxicity is enhanced by hyperthermia. Further development of cisplatin-based enediynes may result in compounds which, when activated, will possess multiple DNA binding modalities similar to cisplatin, but produce less side effects in tissues at normothermic temperatures.

Unraveling a mechanism of honey antibacterial action: polyphenol/H₂O₂-induced oxidative effect on bacterial cell growth and on DNA degradation.

PubMed

Brudzynski, Katrina; Abubaker, Kamal; Miotto, Danielle

2012-07-15

Several compounds with antibacterial activities were identified in honey however, a mechanism by which they lead to bacterial growth inhibition and bacterial death remains still unknown. We recently found that honeys possess DNA degrading activity mediated by honey hydrogen peroxide and an unknown honey component(s). Here we provide evidence that active honeys (MIC90 of 6.25-12.5% v/v) possessed significantly higher levels of phenolics (p<0.02) of higher radical scavenging activities (p<0.005) than honeys of average activity. Removal of H2O2 by catalase eliminated bacteriostatic activities caused by both phenolics and H2O2 suggesting that the growth inhibition resulted from the coupling chemistry between these compounds. Both phenolics and H2O2 were involved in DNA degradation by honeys. Treatment of plasmid DNA with H2O2 alone did not affect the DNA integrity but H2O2 removal from honey by catalase prevented DNA degradation. Polyphenols extracted from honeys degraded plasmid DNA in the presence of H2O2 and Cu(II) in the Fenton-type reaction. The extent of DNA degradation was inversely related to the polyphenol concentration in this system as well as in honeys. At low content, honey polyphenols exerted pro-oxidant activity damaging to DNA. In conclusion, honey phenolics with pro-oxidant activities were necessary intermediates that conferred oxidative action of H2O2. Phenolic/H2O2-induced oxidative stress constituted the mechanism of honey bacteriostatic and DNA damaging activities. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

DNA-protein crosslinks in peripheral lymphocytes of individuals exposed to hexavalent chromium compounds.

PubMed

Zhitkovich, A; Lukanova, A; Popov, T; Taioli, E; Cohen, H; Costa, M; Toniolo, P

1996-01-01

Abstract DNA-protein crosslinks were measured in peripheral blood lymphocytes of chrome-platers and controls from Bulgaria in order to evaluate a genotoxic effect of human exposure to carcinogenic Cr(VI) compounds. Chrome-platers and most of the unexposed controls were from the industrial city of Jambol; some additional controls were recruited from the seaside town of Burgas. The chrome-platers had significantly elevated levels of chromium in pre- and post-shift urine, erythrocytes and lymphocytes compared with the control subjects. The largest differences between the two groups were found in erythrocyte chromium concentrations which are considered to be indicative of Cr(VI) exposure. Despite the significant differences in internal chromium doses, levels of DNA-protein crosslinks were not significantly different between the combined controls and exposed workers. Individual DNA-protein crosslinks, however, correlated strongly with chromium in erythrocytes at low and moderate doses but at high exposures, such as among the majority of chrome-platers, these DNA adducts were saturated at maximum levels. The saturation of DNA-protein crosslinks seems to occur at 7-8 μg I-(1) chromium in erythrocytes whereas a mean erythrocyte chromium among the chrome platers was as high as 22.8 μg l(-1). Occupationally unexposed subjects exhibited a significant variability with respect to the erythrocyte chromium concentration, however erythrocyte chromium levels correlated closely with DNA-protein crosslinks in lymphocytes. The controls from Jambol had higher chromium concentrations in erythrocytes and elevated levels of DNA-protein crosslinks compared with Burgas controls. Occupational exposure to formaldehyde among furniture factory workers did not change levels of DNA-protein crosslinks in peripheral lymphocytes. DNA-protein crosslink measurements showed a low intraindividual variability and their levels among both controls and exposed indivduals were not affected by smoking, age or weight.

Novel human topoisomerase I inhibitors, topopyrones A, B, C and D. I. Producing strain, fermentation, isolation, physico-chemical properties and biological activity.

PubMed

Kanai, Y; Ishiyama, D; Senda, H; Iwatani, W; Takahashi, H; Konno, H; Tokumasu, S; Kanazawa, S

2000-09-01

In the course of a screening program for specific inhibitors of human topoisomerase I using a recombinant yeast, we have discovered four new active compounds. All four compounds were isolated from the culture broth of a fungus, Phoma sp. BAUA2861, and two of them were isolated from the culture broth of a fungus, Penicillium sp. BAUA4206. We designated these compounds as topopyrones A, B, C and D. Topopyrones A, B, C and D selectively inhibited recombinant yeast growth dependent on expression of human topoisomerase I with IC50 values of 1.22, 0.15, 4.88 and 19.63 ng/ml, respectively. The activity and selectivity of topopyrone B were comparable to those of camptothecin. The relaxation of supercoiled pBR322 DNA by human DNA topoisomerase I was inhibited by these compounds, however they did not inhibit human DNA topoisomerase II. Topopyrones A, B, C and D were cytotoxic to all tumor cell lines when tested in vitro. Topopyrone B has potent inhibitory activity against herpesvirus, especially varicella zoster virus (VZV). It inhibited VZV growth with EC50 value of 0.038 microg/ml, which is 24-fold stronger than that of acyclovir (0.9 microg/ml). Topopyrones A, B, and C were inhibitory to Gram-positive bacteria.

Identification of early target genes of aflatoxin B1 in human hepatocytes, inter-individual variability and comparison with other genotoxic compounds

DOE Office of Scientific and Technical Information (OSTI.GOV)

Josse, Rozenn; Dumont, Julie; Fautrel, Alain

Gene expression profiling has recently emerged as a promising approach to identify early target genes and discriminate genotoxic carcinogens from non-genotoxic carcinogens and non-carcinogens. However, early gene changes induced by genotoxic compounds in human liver remain largely unknown. Primary human hepatocytes and differentiated HepaRG cells were exposed to aflatoxin B1 (AFB1) that induces DNA damage following enzyme-mediated bioactivation. Gene expression profile changes induced by a 24 h exposure of these hepatocyte models to 0.05 and 0.25 μM AFB1 were analyzed by using oligonucleotide pangenomic microarrays. The main altered signaling pathway was the p53 pathway and related functions such as cellmore » cycle, apoptosis and DNA repair. Direct involvement of the p53 protein in response to AFB1 was verified by using siRNA directed against p53. Among the 83 well-annotated genes commonly modulated in two pools of three human hepatocyte populations and HepaRG cells, several genes were identified as altered by AFB1 for the first time. In addition, a subset of 10 AFB1-altered genes, selected upon basis of their function or tumor suppressor role, was tested in four human hepatocyte populations and in response to other chemicals. Although they exhibited large variable inter-donor fold-changes, several of these genes, particularly FHIT, BCAS3 and SMYD3, were found to be altered by various direct and other indirect genotoxic compounds and unaffected by non-genotoxic compounds. Overall, this comprehensive analysis of early gene expression changes induced by AFB1 in human hepatocytes identified a gene subset that included several genes representing potential biomarkers of genotoxic compounds. -- Highlights: ► Gene expression profile changes induced by aflatoxin B1 in human hepatocytes. ► AFB1 modulates various genes including tumor suppressor genes and proto-oncogenes. ► Important inter-individual variations in the response to AFB1. ► Some genes also altered by other genotoxic compounds requiring or not bioactivation.« less

Using phosphine ligands with a biological role to modulate reactivity in novel platinum complexes

NASA Astrophysics Data System (ADS)

Echeverri, Marcelo; Alvarez-Valdés, Amparo; Navas, Francisco; Perles, Josefina; Sánchez-Pérez, Isabel; Quiroga, A. G.

2018-02-01

Three platinum complexes with cis and trans configuration cis-[Pt(TCEP)2Cl2], cis-[Pt(tmTCEP)2Cl2] and trans-[Pt(TCEP)2Cl2], where TCEP is tris(2-carboxyethyl)phosphine, have been synthesized and fully characterized by usual techniques including single-crystal X-ray diffraction for trans-[Pt(TCEP)2Cl2] and cis-[Pt(tmTCEP)2Cl2]. Here, we also report on an esterification process of TCEP, which takes place in the presence of alcohols, leading to a platinum complex coordinated to an ester tmTCEP (2-methoxycarbonylethyl phosphine) ligand. The stability in solution of the three compounds and their interaction with biological models such as DNA (pBR322 and calf thymus DNA) and proteins (lysozyme and RNase) have also been studied.

Genomic Models of Short-Term Exposure Accurately Predict Long-Term Chemical Carcinogenicity and Identify Putative Mechanisms of Action

PubMed Central

Gusenleitner, Daniel; Auerbach, Scott S.; Melia, Tisha; Gómez, Harold F.; Sherr, David H.; Monti, Stefano

2014-01-01

Background Despite an overall decrease in incidence of and mortality from cancer, about 40% of Americans will be diagnosed with the disease in their lifetime, and around 20% will die of it. Current approaches to test carcinogenic chemicals adopt the 2-year rodent bioassay, which is costly and time-consuming. As a result, fewer than 2% of the chemicals on the market have actually been tested. However, evidence accumulated to date suggests that gene expression profiles from model organisms exposed to chemical compounds reflect underlying mechanisms of action, and that these toxicogenomic models could be used in the prediction of chemical carcinogenicity. Results In this study, we used a rat-based microarray dataset from the NTP DrugMatrix Database to test the ability of toxicogenomics to model carcinogenicity. We analyzed 1,221 gene-expression profiles obtained from rats treated with 127 well-characterized compounds, including genotoxic and non-genotoxic carcinogens. We built a classifier that predicts a chemical's carcinogenic potential with an AUC of 0.78, and validated it on an independent dataset from the Japanese Toxicogenomics Project consisting of 2,065 profiles from 72 compounds. Finally, we identified differentially expressed genes associated with chemical carcinogenesis, and developed novel data-driven approaches for the molecular characterization of the response to chemical stressors. Conclusion Here, we validate a toxicogenomic approach to predict carcinogenicity and provide strong evidence that, with a larger set of compounds, we should be able to improve the sensitivity and specificity of the predictions. We found that the prediction of carcinogenicity is tissue-dependent and that the results also confirm and expand upon previous studies implicating DNA damage, the peroxisome proliferator-activated receptor, the aryl hydrocarbon receptor, and regenerative pathology in the response to carcinogen exposure. PMID:25058030

Direct inhibition of excision/synthesis DNA repair activities by cadmium: analysis on dedicated biochips.

PubMed

Candéias, S; Pons, B; Viau, M; Caillat, S; Sauvaigo, S

2010-12-10

The well established toxicity of cadmium and cadmium compounds results from their additive effects on several key cellular processes, including DNA repair. Mammalian cells have evolved several biochemical pathways to repair DNA lesions and maintain genomic integrity. By interfering with the homeostasis of redox metals and antioxidant systems, cadmium promotes the development of an intracellular environment that results in oxidative DNA damage which can be mutagenic if unrepaired. Small base lesions are recognised by specialized glycosylases and excised from the DNA molecule. The resulting abasic sites are incised, and the correct sequences restored by DNA polymerases using the opposite strands as template. Bulky lesions are recognised by a different set of proteins and excised from DNA as part of an oligonucleotide. As in base repair, the resulting gaps are filled by DNA polymerases using the opposite strands as template. Thus, these two repair pathways consist in excision of the lesion followed by DNA synthesis. In this study, we analysed in vitro the direct effects of cadmium exposure on the functionality of base and nucleotide DNA repair pathways. To this end, we used recently described dedicated microarrays that allow the parallel monitoring in cell extracts of the repair activities directed against several model base and/or nucleotide lesions. Both base and nucleotide excision/repair pathways are inhibited by CdCl₂, with different sensitivities. The inhibitory effects of cadmium affect mainly the recognition and excision stages of these processes. Furthermore, our data indicate that the repair activities directed against different damaged bases also exhibit distinct sensitivities, and the direct comparison of cadmium effects on the excision of uracile in different sequences even allows us to propose a hierarchy of cadmium sensibility within the glycosylases removing U from DNA. These results indicate that, in our experimental conditions, cadmium is a very potent DNA repair poison. Copyright © 2010 Elsevier B.V. All rights reserved.

Nonadiabatic tapered optical fiber sensor for measuring interaction nicotine with DNA

NASA Astrophysics Data System (ADS)

Zibaii, M. I.; Latifi, H.; Pourbeyram, H.; Gholami, M.; Taghipour, Z.; Saeedian, Z.; Hosseini, S. M.

2011-05-01

A nonadiabatic tapered optical fiber sensor was utilized for studying of bimolecular interactions including DNA-DNA and DNA-Drug interaction. This work presents a simple evanescent wave sensing system based on an interferometric approach, suitable to meet the requirements of lable-free sensor systems for detecting biomolecular interactions. We have demonstrated the measuring refractive index and the real time detection of interactions between biomolecules. Furthermore basic experiments were carried out, for detecting the hybridization of 25-mer DNA with an immobilized counterpart on the surface. The overall shift after the successful DNA hybridization was 9.5 nm. In this work, a new approach for studying DNA-drug interactions was successfully tested. Nicotine as a carcinogenic compound in cigarette smoke plays an important role in interaction with DNA. Different concentrations of nicotine were applied to observe the Longmuir interaction with DNA.

Chemosensitization of Breast Cancer Cells to Chemotherapeutic Agents by 3,3’-Diindolylmethane (DIM)

DTIC Science & Technology

2007-08-01

activity (Fig. 4A). More importantly, our animal studies showed that dietary B-DIM in combination with Taxotere also inhibited NF-κB DNA binding...κB DNA-binding activity (Fig. 4C). Importantly, our animal studies showed that dietary B-DIM in combination with Taxotere inhibited NF-κB DNA...Rahman et al 15 DISCUSSION Current reports have shown that plant-derived dietary compounds provide protection against the

Strategy for Imidazotetrazine Prodrugs with Anticancer Activity Independent of MGMT and MMR

PubMed Central

2012-01-01

The imidazotetrazine ring is an acid-stable precursor and prodrug of highly reactive alkyl diazonium ions. We have shown that this reactivity can be managed productively in an aqueous system for the generation of aziridinium ions with 96% efficiency. The new compounds are potent DNA alkylators and have antitumor activity independent of the O6-methylguanine-DNA methyltransferase and DNA mismatch repair constraints that limit the use of Temozolomide. PMID:24900418

Structural relationship of curcumin derivatives binding to the BRCT domain of human DNA polymerase lambda.

PubMed

Takeuchi, Toshifumi; Ishidoh, Tomomi; Iijima, Hiroshi; Kuriyama, Isoko; Shimazaki, Noriko; Koiwai, Osamu; Kuramochi, Kouji; Kobayashi, Susumu; Sugawara, Fumio; Sakaguchi, Kengo; Yoshida, Hiromi; Mizushina, Yoshiyuki

2006-03-01

We previously reported that phenolic compounds, petasiphenol and curcumin (diferuloylmethane), were a selective inhibitor of DNA polymerase lambda (pol lambda) in vitro. The purpose of this study was to investigate the molecular structural relationship of curcumin and 13 chemically synthesized derivatives of curcumin. The inhibitory effect on pol lambda (full-length, i.e. intact pol lambda including the BRCA1 C- terminal [BRCT] domain) by some derivatives was stronger than that by curcumin, and monoacetylcurcumin (compound 13) was the strongest pol lambda inhibitor of all the compounds tested, achieving 50% inhibition at a concentration of 3.9 microm. The compound did not influence the activities of replicative pols such as alpha, delta, and epsilon. It had no effect on pol beta activity either, although the three-dimensional structure of pol beta is thought to be highly similar to that of pol lambda. Compound 13 did not inhibit the activity of the C-terminal catalytic domain of pol lambda including the pol beta-like core, in which the BRCT motif was deleted from its N-terminal region. MALDI-TOF MS analysis demonstrated that compound 13 bound selectively to the N-terminal domain of pol lambda, but did not bind to the C-terminal region. Based on these results, the pol lambda-inhibitory mechanism of compound 13 is discussed.

Evaluation of new iodinated acridine derivatives for targeted radionuclide therapy of melanoma using 125I, an Auger electron emitter.

PubMed

Gardette, Maryline; Papon, Janine; Bonnet, Mathilde; Desbois, Nicolas; Labarre, Pierre; Wu, Ting-Dee; Miot-Noirault, Elisabeth; Madelmont, Jean-Claude; Guerquin-Kern, Jean-Luc; Chezal, Jean-Michel; Moins, Nicole

2011-12-01

The increasing incidence of melanoma and the lack of effective therapy on the disseminated form have led to an urgent need for new specific therapies. Several iodobenzamides or analogs are known to possess specific affinity for melanoma tissue. New heteroaromatic derivatives have been designed with a cytotoxic moiety and termed DNA intercalating agents. These compounds could be applied in targeted radionuclide therapy using (125)I, which emits Auger electrons and gives high-energy, localized irradiation. Two iodinated acridine derivatives have been reported to present an in vivo kinetic profile conducive to application in targeted radionuclide therapy. The aim of the present study was to perform a preclinical evaluation of these compounds. The DNA intercalating property was confirmed for both compounds. After radiolabeling with (125)I, the two compounds induced in vitro a significant radiotoxicity to B16F0 melanoma cells. Nevertheless, the acridine compound appeared more radiotoxic than the acridone compound. While cellular uptake was similar for both compounds, SIMS analysis and in vitro protocol showed a stronger affinity for melanin with acridone derivative, which was able to induce a predominant scavenging process in the melanosome and restrict access to the nucleus. In conclusion, the acridine derivative with a higher nuclear localization appeared a better candidate for application in targeted radionuclide therapy using (125)I.

Protective effects of pulmonary epithelial lining fluid on oxidative stress and DNA single-strand breaks caused by ultrafine carbon black, ferrous sulphate and organic extract of diesel exhaust particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuang, Hsiao-Chi; Division of Pulmonary Medicine, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan; Cheng, Yi-Ling

Pulmonary epithelial lining fluid (ELF) is the first substance to make contact with inhaled particulate matter (PM) and interacts chemically with PM components. The objective of this study was to determine the role of ELF in oxidative stress, DNA damage and the production of proinflammatory cytokines following physicochemical exposure to PM. Ultrafine carbon black (ufCB, 15 nm; a model carbonaceous core), ferrous sulphate (FeSO{sub 4}; a model transition metal) and a diesel exhaust particle (DEP) extract (a model organic compound) were used to examine the acellular oxidative potential of synthetic ELF and non-ELF systems. We compared the effects of exposuremore » to ufCB, FeSO{sub 4} and DEP extract on human alveolar epithelial Type II (A549) cells to determine the levels of oxidative stress, DNA single-strand breaks and interleukin-8 (IL-8) production in ELF and non-ELF systems. The effects of ufCB and FeSO{sub 4} on the acellular oxidative potential, cellular oxidative stress and DNA single-strand breakage were mitigated significantly by the addition of ELF, whereas there was no decrease following treatment with the DEP extract. There was no significant effect on IL-8 production following exposure to samples that were suspended in ELF/non-ELF systems. The results of the present study indicate that ELF plays an important role in the initial defence against PM in the pulmonary environment. Experimental components, such as ufCB and FeSO{sub 4}, induced the production of oxidative stress and led to DNA single-strand breaks, which were moderately prevented by the addition of ELF. These findings suggest that ELF plays a protective role against PM-driven oxidative stress and DNA damage. -- Highlights: ► To determine the role of ELF in ROS, DNA damage and IL-8 after exposure to PM. ► ufCB, FeSO{sub 4} and DEP extract were used to examine the protective effects of ELF. ► PM-driven oxidative stress and DNA single-strand breakage were mitigated by ELF. ► The findings suggest that ELF has a protective role against PM. ► The synthetic ELF system could reduce the use of animals in PM-driven ROS testing.« less

EXPRESSION PROFILING OF ESTROGENIC COMPOUNDS USING A SHEEPSHEAD MINNOW CDNA MACROARRAY

EPA Science Inventory

Larkin, Patrick, Leroy C. Folmar, Michael J. Hemmer, Arianna J. Poston and Nancy D. Denslow. 2003. Expression Profiling of Estrogenic Compounds Using a Sheepshead Minnow cDNA Macroarray. Environ. Health Perspect. 111(6):839-846. (ERL,GB 1171).

A variety of anthropogenic c...

DNA tests for strawberry: mesifurane "sherry" aroma - FaOMT-SI/NO

USDA-ARS?s Scientific Manuscript database

The amazing flavor and texture in strawberries is caused by a complex balance of numerous sugars and aromatic compounds. One of the most important aromatic compounds contributing to the flavor we have come to love in strawberries is mesifurane. Mesifurane produces a sweet sherry-like aroma and incre...

Platinated DNA oligonucleotides: new probes forming ultrastable conjugates with graphene oxide

NASA Astrophysics Data System (ADS)

Wang, Feng; Liu, Juewen

2014-05-01

Metal containing polymers have expanded the property of polymers by involving covalently associated metal complexes. DNA is a special block copolymer. While metal ions are known to influence DNA, little is explored on its polymer property when strong metal complexes are associated. In this work, we study cisplatin modified DNA as a new polymer and probe. Out of the complexes formed between cisplatin-A15, HAuCl4-A15, Hg2+-T15 and Ag+-C15, only the cisplatin adduct is stable under the denaturing gel electrophoresis condition. Each Pt-nucleobase bond gives a positive charge and thus makes DNA a zwitterionic polymer. This allows ultrafast adsorption of DNA by graphene oxide (GO) and the adsorbed complex is highly stable. Non-specific DNA, protein, surfactants and thiolated compounds cannot displace platinated DNA from GO, while non-modified DNA is easily displaced in most cases. The stable GO/DNA conjugate is further tested for surface hybridization. This is the first demonstration of using metallated DNA as a polymeric material for interfacing with nanoscale materials.Metal containing polymers have expanded the property of polymers by involving covalently associated metal complexes. DNA is a special block copolymer. While metal ions are known to influence DNA, little is explored on its polymer property when strong metal complexes are associated. In this work, we study cisplatin modified DNA as a new polymer and probe. Out of the complexes formed between cisplatin-A15, HAuCl4-A15, Hg2+-T15 and Ag+-C15, only the cisplatin adduct is stable under the denaturing gel electrophoresis condition. Each Pt-nucleobase bond gives a positive charge and thus makes DNA a zwitterionic polymer. This allows ultrafast adsorption of DNA by graphene oxide (GO) and the adsorbed complex is highly stable. Non-specific DNA, protein, surfactants and thiolated compounds cannot displace platinated DNA from GO, while non-modified DNA is easily displaced in most cases. The stable GO/DNA conjugate is further tested for surface hybridization. This is the first demonstration of using metallated DNA as a polymeric material for interfacing with nanoscale materials. Electronic supplementary information (ESI) available: Methods, additional gels, kinetics, mass spectrum. See DOI: 10.1039/c4nr00867g

Discovery of a Potent BTK Inhibitor with a Novel Binding Mode by Using Parallel Selections with a DNA-Encoded Chemical Library.

PubMed

Cuozzo, John W; Centrella, Paolo A; Gikunju, Diana; Habeshian, Sevan; Hupp, Christopher D; Keefe, Anthony D; Sigel, Eric A; Soutter, Holly H; Thomson, Heather A; Zhang, Ying; Clark, Matthew A

2017-05-04

We have identified and characterized novel potent inhibitors of Bruton's tyrosine kinase (BTK) from a single DNA-encoded library of over 110 million compounds by using multiple parallel selection conditions, including variation in target concentration and addition of known binders to provide competition information. Distinct binding profiles were observed by comparing enrichments of library building block combinations under these conditions; one enriched only at high concentrations of BTK and was competitive with ATP, and another enriched at both high and low concentrations of BTK and was not competitive with ATP. A compound representing the latter profile showed low nanomolar potency in biochemical and cellular BTK assays. Results from kinetic mechanism of action studies were consistent with the selection profiles. Analysis of the co-crystal structure of the most potent compound demonstrated a novel binding mode that revealed a new pocket in BTK. Our results demonstrate that profile-based selection strategies using DNA-encoded libraries form the basis of a new methodology to rapidly identify small molecule inhibitors with novel binding modes to clinically relevant targets. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Polyphenol Compound as a Transcription Factor Inhibitor.

PubMed

Park, Seyeon

2015-10-30

A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor-DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein-protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1), c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and β-catenin/T cell factor (Tcf)).

Development of Derivatives of 3, 3′-Diindolylmethane as Potent Leishmania donovani Bi-Subunit Topoisomerase IB Poisons

PubMed Central

Sengupta, Souvik; Mandal, Madhumita; Jaisankar, Parasuraman; D'Annessa, Ilda; Desideri, Alessandro; Majumder, Hemanta K.

2011-01-01

Background The development of 3, 3′-diindolyl methane (DIM) resistant parasite Leishmania donovani (LdDR50) by adaptation with increasing concentrations of the drug generates random mutations in the large and small subunits of heterodimeric DNA topoisomerase I of Leishmania (LdTOP1LS). Mutation of large subunit of LdTOP1LS at F270L is responsible for resistance to DIM up to 50 µM concentration. Methodology/Principal Findings In search of compounds that inhibit the growth of the DIM resistant parasite and inhibit the catalytic activity of mutated topoisomerase I (F270L), we have prepared three derivatives of DIM namely DPDIM (2,2′-diphenyl 3,3′-diindolyl methane), DMDIM (2,2′-dimethyl 3,3′-diindolyl methane) and DMODIM (5,5′-dimethoxy 3,3′-diindolyl methane) from parent compound DIM. All the compounds inhibit the growth of DIM resistant parasites, induce DNA fragmentation and stabilize topo1-DNA cleavable complex with the wild type and mutant enzyme. Conclusion The results suggest that the three derivatives of DIM can act as promising lead molecules for the generation of new anti-leishmanial agents. PMID:22174820

Arsenic Exposure Is Associated with Decreased DNA Repair in Vitro and in Individuals Exposed to Drinking Water Arsenic

PubMed Central

Andrew, Angeline S.; Burgess, Jefferey L.; Meza, Maria M.; Demidenko, Eugene; Waugh, Mary G.; Hamilton, Joshua W.; Karagas, Margaret R.

2006-01-01

The mechanism(s) by which arsenic exposure contributes to human cancer risk is unknown; however, several indirect cocarcinogenesis mechanisms have been proposed. Many studies support the role of As in altering one or more DNA repair processes. In the present study we used individual-level exposure data and biologic samples to investigate the effects of As exposure on nucleotide excision repair in two study populations, focusing on the excision repair cross-complement 1 (ERCC1) component. We measured drinking water, urinary, or toenail As levels and obtained cryopreserved lymphocytes of a subset of individuals enrolled in epidemiologic studies in New Hampshire (USA) and Sonora (Mexico). Additionally, in corroborative laboratory studies, we examined the effects of As on DNA repair in a cultured human cell model. Arsenic exposure was associated with decreased expression of ERCC1 in isolated lymphocytes at the mRNA and protein levels. In addition, lymphocytes from As-exposed individuals showed higher levels of DNA damage, as measured by a comet assay, both at baseline and after a 2-acetoxyacetylaminofluorene (2-AAAF) challenge. In support of the in vivo data, As exposure decreased ERCC1 mRNA expression and enhanced levels of DNA damage after a 2-AAAF challenge in cell culture. These data provide further evidence to support the ability of As to inhibit the DNA repair machinery, which is likely to enhance the genotoxicity and mutagenicity of other directly genotoxic compounds, as part of a cocarcinogenic mechanism of action. PMID:16882524

Group-based QSAR and molecular dynamics mechanistic analysis revealing the mode of action of novel piperidinone derived protein-protein inhibitors of p53-MDM2.

PubMed

Goyal, Sukriti; Grover, Sonam; Dhanjal, Jaspreet Kaur; Tyagi, Chetna; Goyal, Manisha; Grover, Abhinav

2014-06-01

Tumour suppressor p53 is known to play a central role in prevention of tumour development, DNA repair, senescence and apoptosis which is in normal cells maintained by negative feedback regulator MDM2 (Murine Double Minute 2). In case of dysfunctioning of this regulatory loop, tumour development starts thus resulting in cancerous condition. Inhibition of p53-MDM2 binding would result in activation of the tumour suppressor. In this study, a novel robust fragment-based QSAR model has been developed for piperidinone derived compounds experimentally known to inhibit p53-MDM2 interaction. The QSAR model developed showed satisfactory statistical parameters for the experimentally reported dataset (r(2)=0.9415, q(2)=0.8958, pred_r(2)=0.8894 and F-test=112.7314), thus judging the robustness of the model. Low standard error values (r(2)_se=0.3003, q(2)_se=0.4009 and pred_r(2)_se=0.3315) confirmed the accuracy of the developed model. The regression equation obtained constituted three descriptors (R2-DeltaEpsilonA, R1-RotatableBondCount and R2-SssOCount), two of which had positive contribution while third showed negative correlation. Based on the developed QSAR model, a combinatorial library was generated and activities of the compounds were predicted. These compounds were docked with MDM2 and two top scoring compounds with binding affinities of -10.13 and -9.80kcal/mol were selected. The binding modes of actions of these complexes were analyzed using molecular dynamics simulations. Analysis of the developed fragment-based QSAR model revealed that addition of unsaturated electronegative groups at R2 site and groups with more rotatable bonds at R1 improved the inhibitory activity of these potent lead compounds. The detailed analysis carried out in this study provides a considerable basis for the design and development of novel piperidinone-based lead molecules against cancer and also provides mechanistic insights into their mode of actions. Copyright © 2014 Elsevier Inc. All rights reserved.

Annonalide and derivatives: Semisynthesis, cytotoxic activities and studies on interaction of annonalide with DNA.

PubMed

Marques, Ricardo A; Gomes, Akenaton O C V; de Brito, Maria V; Dos Santos, Ana L P; da Silva, Gladyane S; de Lima, Leandro B; Nunes, Fátima M; de Mattos, Marcos C; de Oliveira, Fátima C E; do Ó Pessoa, Cláudia; de Moraes, Manoel O; de Fátima, Ângelo; Franco, Lucas L; Silva, Marina de M; Dantas, Maria Dayanne de A; Santos, Josué C C; Figueiredo, Isis M; da Silva-Júnior, Edeíldo F; de Aquino, Thiago M; de Araújo-Júnior, João X; de Oliveira, Maria C F; Leslie Gunatilaka, A A

2018-02-01

The cytotoxic activity of the pimarane diterpene annonalide (1) and nine of its semisynthetic derivatives (2-10) was investigated against the human tumor cell lines HL-60 (leukemia), PC-3 (prostate adenocarcinoma), HepG2 (hepatocellular carcinoma), SF-295 (glioblastoma) and HCT-116 (colon cancer), and normal mouse fibroblast (L929) cells. The preparation of 2-10 involved derivatization of the side chain of 1 at C-13. Except for 2, all derivatives are being reported for the first time. Most of the tested compounds presented IC 50 s below 4.0 μM, being considered potential antitumor agents. The structures of all new compounds were elucidated by spectroscopic analyses including 2D NMR and HRMS. Additionally, the interaction of annonalide (1) with ctDNA was evaluated using spectroscopic techniques, and the formation of a supramolecular complex with the macromolecule was confirmed. Competition assays with fluorescent probes (Hoechst and ethidium bromide) and theoretical studies confirmed that 1 interacts preferentially via DNA intercalation with stoichiometric ratio of 1:1 (1:ctDNA). The ΔG value was calculated as -28.24 kJ mol -1 , and indicated that the interaction process occurs spontaneously. Docking studies revealed that van der Walls is the most important interaction in 1-DNA and EB-DNA complexes, and that both ligands (1 and EB) interact with the same DNA residues (DA6, DA17 and DT19). Copyright © 2018. Published by Elsevier B.V.

Interaction between bioactive compound 11a-N-tosyl-5-deoxi-pterocarpan (LQB-223) and Calf thymus DNA: Spectroscopic approach, electrophoresis and theoretical studies.

PubMed

Silva, Marina M; Nascimento, Eduarda O O; Silva, Edeíldo F; Araújo, João Xavier de; Santana, Camilla C; Grillo, Luciano Aparecido M; de Oliveira, Rafaela S; R R Costa, Paulo; Buarque, Camilla D; Santos, Josué Carinhanha C; Figueiredo, Isis M

2017-03-01

The interaction of small molecules with DNA has been quite important, since this biomolecule is currently the major target for a wide range of drugs in clinical use or advanced clinical research phase. Thus, the present work aimed to assess the interaction process between the bioactive compound 11a-N-tosyl-5-carba-pterocarpan, (LQB-223), that presents antitumor activity, with DNA, employing spectroscopic techniques, electrophoresis, viscosity and theoretical studies. Through UV-vis and molecular fluorescence spectroscopy, it was possible to infer that the preferential quenching mechanism was static, characterized by non-fluorescent supramolecular complex formation between the LQB-223 and DNA. The binding constant was 1.94∙10 3 Lmol -1 (30°C) and, according to the thermodynamic parameters, the main forces involved in the interaction process are hydrophobic. Potassium iodide assay, competition with ethidium bromide, fluorescence contact energy transfer and melting temperature profile of DNA were employed to evaluate the binding mode. Except for KI assay, all results obtained indicated minor groove as the preferential binding mode of LQB-223 to DNA. These observations were supported by ionic strength assay, viscosity and molecular dynamics and docking studies. Finally, electrophoresis analysis demonstrated that the interaction does not promote DNA fragmentation, but it leads to variation in the migration profile after increasing the ligand concentration. Copyright © 2016 Elsevier B.V. All rights reserved.

Fanconi Anemia Proteins, DNA Interstrand Crosslink Repair Pathways, and Cancer Therapy

PubMed Central

Andreassen, Paul R.; Ren, Keqin

2016-01-01

DNA interstrand crosslinkers, a chemically diverse group of compounds which also induce alkylation of bases and DNA intrastrand crosslinks, are extensively utilized for cancer therapy. Understanding the cellular response to DNA damage induced by these agents is critical for more effective utilization of these compounds and for the identification of novel therapeutic targets. Importantly, the repair of DNA interstrand crosslinks (ICLs) involves many distinct DNA repair pathways, including nucleotide excision repair, translesion synthesis (TLS), and homologous recombination (HR). Additionally, proteins implicated in the pathophysiology of the multigenic disease Fanconi anemia (FA) have a role in the repair of ICLs that is not well understood. Cells from FA patients are hypersensitive to agents that induce ICLs, therefore FA proteins are potentially novel therapeutic targets. Here we will review current research directed at identifying FA genes and understanding the function of FA proteins in DNA damage responses. We will also examine interactions of FA proteins with other repair proteins and pathways, including signaling networks, which are potentially involved in ICL repair. Potential approaches to the modulation of FA protein function to enhance therapeutic outcome will be discussed. Also, mutation of many genes that encode proteins involved in ICL repair, including FA genes, increases susceptibility to cancer. A better understanding of these pathways is therefore critical for the design of individualized therapies tailored to the genetic profile of a particular malignancy. For this purpose, we will also review evidence for the association of mutation of FA genes with cancer in non-FA patients. PMID:19200054

Synthesis and evaluation of 7-substituted-5,6-dihydrobenzo[c]acridine derivatives as new c-KIT promoter G-quadruplex binding ligands.

PubMed

Guo, Qian-Liang; Su, Hua-Fei; Wang, Ning; Liao, Sheng-Rong; Lu, Yu-Ting; Ou, Tian-Miao; Tan, Jia-Heng; Li, Ding; Huang, Zhi-Shu

2017-04-21

It has been shown that treatment of cancer cells with c-KIT G-quadruplex binding ligands can reduce their c-KIT expression levels thus inhibiting cell proliferation and inducing cell apoptosis. Herein, a series of new 7-substituted-5,6-dihydrobenzo[c]acridine derivatives were designed and synthesized. Subsequent biophysical evaluation demonstrated that the derivatives could effectively bind to and stabilize c-KIT G-quadruplex with good selectivity against duplex DNA. It was found that 12-N-methylated derivatives with a positive charge introduced at 12-position of 5,6-dihydrobenzo[c]acridine ring had similar binding affinity but lower stabilizing ability to c-KIT G-quadruplex DNA, compared with those of nonmethylated derivatives. Further molecular modeling studies showed possible binding modes of G-quadruplex with the ligands. RT-PCR assay and Western blot showed that compound 2b suppressed transcription and translation of c-KIT gene in K562 cells, which was consistent with the property of an effective G-quadruplex binding ligand targeting c-KIT oncogene promoter. Further biological evaluation showed that compound 2b could induce apoptosis through activation of the caspase-3 cascade pathway. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

AN EVALUATION OF THE MUTAGENICITY, METABOLISM AND DNA ADDUCT FORMATION OF 5-NITROBENZO[B]NAPHTHO[2,1-D]THIOPHENE

EPA Science Inventory

An Evaluation of the Mutagenicity, Metabolism and DNA Adduct Formation of 5-Nitrobenzo[b ]naphtho[2, I-d]thiophene

Thioarenes, sulfur containing polycyclic aromatic compounds, are environmental contaminants suspected of posing human health risks. In this study, 5-nitroben...

Selenium compounds activate ATM-dependent DNA damage responses via the mismatch repair protein hMLH1 in colorectal cancer cells

USDA-ARS?s Scientific Manuscript database

Epidemiological and animal studies indicate that selenium supplementation suppresses risk of colorectal and other cancers. The majority of colorectal cancers are characterized by a defective DNA mismatch repair (MMR) process. Here, we have employed the MMR-deficient HCT 116 colorectal cancer cells ...

Method of controlling gene expression

DOEpatents

Peters, Norman K.; Frost, John W.; Long, Sharon R.

1991-12-03

A method of controlling expression of a DNA segment under the control of a nod gene promoter which comprises administering to a host containing a nod gene promoter an amount sufficient to control expression of the DNA segment of a compound of the formula: ##STR1## in which each R is independently H or OH, is described.

Interaction between a cationic porphyrin and ctDNA investigated by SPR, CV and UV-vis spectroscopy.

PubMed

Xu, Zi-Qiang; Zhou, Bo; Jiang, Feng-Lei; Dai, Jie; Liu, Yi

2013-10-01

The interaction between ctDNA and a cationic porphyrin was studied in this work. The binding process was monitored by surface plasmon resonance (SPR) spectroscopy in detail. The association, dissociation rate constants and the binding constants calculated by global analysis were 2.4×10(2)±26.4M(-1)s(-1), 0.011±0.0000056s(-1) and 2.18×10(4)M(-1), respectively. And the results were confirmed by cyclic voltammetry and UV-vis absorption spectroscopy. The binding constants obtained from cyclic voltammetry and UV-vis absorption spectroscopy were 8.28×10(4)M(-1) and 6.73×10(4)M(-1) at 298K, respectively. The covalent immobilization methodology of ctDNA onto gold surface modified with three different compounds was also investigated by SPR. These compounds all contain sulfydryl but with different terminated functional groups. The results indicated that the 11-MUA (HS(CH2)10COOH)-modified gold film is more suitable for studying the DNA-drug interaction. Copyright © 2013 Elsevier B.V. All rights reserved.

Use of human peripheral blood lymphocytes to measure DNA binding capacity of chemical carcinogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gupta, R.C.; Earley, K.; Sharma, S.

1988-05-01

Although animal models have been used successfully to study metabolic activation and binding of carcinogens to DNA, only limited studies have been done in human systems. To circumvent the problems associated with the inaccessibility of human tissues and a lack of sensitive methods to detect DNA damage, the authors have investigated the capability of human peripheral blood lymphocytes in vitro to metabolize carcinogens to their DNA binding species by a {sup 32}P-labeled adduct assay. Freshly isolated lymphocytes were exposed at 37{degree}C for 18 hr to 4-aminobiphenyl, 2-aminofluorene, 2-anthramine, 2-acetylaminophenanthrene, benzidine, 1-nitropyrene, 1,2-benzanthracene, triphenylene, 7,12-dimethylbenz(a)anthracene, or benzo(a)pyrene at 30 {mu}M each,more » compounds that are shown or suspected to be carcinogenic in experimental animals. The data indicate that all test carcinogens formed readily measurable levels of DNA adducts. Analysis of exposed DNAs by {sup 32}P-labeling after digestion and adduct enrichment showed exclusively or predominantly one major adduct for all test carcinogens, except for 2-anthramine, triphenylene, and 7,12-dimethylbenz(a)anthracene, which showed two or three adducts. From 12 lymphocyte specimens studied thus far, significant interindividual variations were observed. The lymphocyte system in combination with the {sup 32}P-adduct assay may prove to be an ultrasensitive means to determine interindividual variations in the ability to biotransform carcinogens.« less

Evaluation of DNA extraction methods for PCR-based detection of Listeria monocytogenes from vegetables.

PubMed

Vojkovska, H; Kubikova, I; Kralik, P

2015-03-01

Epidemiological data indicate that raw vegetables are associated with outbreaks of Listeria monocytogenes. Therefore, there is a demand for the availability of rapid and sensitive methods, such as PCR assays, for the detection and accurate discrimination of L. monocytogenes. However, the efficiency of PCR methods can be negatively affected by inhibitory compounds commonly found in vegetable matrices that may cause false-negative results. Therefore, the sample processing and DNA isolation steps must be carefully evaluated prior to the introduction of such methods into routine practice. In this study, we compared the ability of three column-based and four magnetic bead-based commercial DNA isolation kits to extract DNA of the model micro-organism L. monocytogenes from raw vegetables. The DNA isolation efficiency of all isolation kits was determined using a triplex real-time qPCR assay designed to specifically detect L. monocytogenes. The kit with best performance, the PowerSoil(™) Microbial DNA Isolation Kit, is suitable for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. Coupled with the triplex real-time qPCR assay, this DNA isolation kit is applicable to the samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes. Several recent outbreaks of Listeria monocytogenes have been associated with the consumption of fruits and vegetables. Real-time PCR assays allow fast detection and accurate quantification of microbes. However, the success of real-time PCR is dependent on the success with which template DNA can be extracted. The results of this study suggest that the PowerSoil(™) Microbial DNA Isolation Kit can be used for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. This method is applicable to samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes. © 2014 The Society for Applied Microbiology.

Multispectroscopic DNA-Binding studies and antimicrobial evaluation of new mixed-ligand Silver(I) complex and nanocomplex: A comparative study

NASA Astrophysics Data System (ADS)

Movahedi, Elaheh; Rezvani, Ali Reza

2018-05-01

A novel mixed-ligand Ag(I) complex, , has been synthesized and characterized by the elemental analysis, IR spectroscopy and 1HNMR. In the formula, dian and phen are N-(4,5-diazafluoren-9-ylidene)aniline and 1,10-phenanthroline, respectively. This complex also has been prepared at nano size by sonochemical technique and characterized by the FTIR and scanning electron microscopy (SEM). To evaluate the biological preferences of the Ag(I) complex and nanocomplex and verify the relationships between the structure and biological function, in vitro DNA binding and antibacterial experiments have been carried out. DNA-complex interaction has been pursued by electronic absorption titration, luminescence titration, competitive binding experiment, effect of ionic strength, thermodynamic studies, viscometric evaluation and circular dichroism spectroscopy in the physiological pH. Each compound displays significant binding trend to the CT-DNA. The mode of binding to the CT-DNA probably is a moderate intercalation mode with the partial insertion of the planar ligands between the base stacks of double-stranded DNA. The relative viscosities and circular dichroism spectra of the CT-DNA with the complex solutions, confirm the intense interactions of the Ag(I) complex and nanocomplex with DNA. An in vitro antibacterial test of the complex and nanocomplex on a series of the Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis) and the Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) shows a remarkable antibacterial feature of the Ag(I) complex. The MIC values (minimum inhibitory concentration) of the compounds compare with silver nitrate and silver sulfadiazine. The bacterial inhibitions of the Ag(I) complex and nanocomplex are agreed to their DNA binding affinities.

The Lambda Select cII Mutation Detection System.

PubMed

Besaratinia, Ahmad; Tommasi, Stella

2018-04-26

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic mice and the Lambda (λ) Select cII Mutation Detection System have been employed for mutagenicity experiments by many research groups worldwide. Here, we describe a detailed protocol for the Lambda Select cII mutation assay, which can be applied to cultured cells of transgenic mice/rats or the corresponding animals treated with a chemical/physical agent of interest. The protocol consists of the following steps: (1) isolation of genomic DNA from the cells or organs/tissues of transgenic animals treated in vitro or in vivo, respectively, with a test compound; (2) recovery of the lambda shuttle vector carrying a mutational reporter gene (i.e., cII transgene) from the genomic DNA; (3) packaging of the rescued vectors into infectious bacteriophages; (4) infecting a host bacteria and culturing under selective conditions to allow propagation of the induced cII mutations; and (5) scoring the cII-mutants and DNA sequence analysis to determine the cII mutant frequency and mutation spectrum, respectively.

High-Throughput Genetic Analysis and Combinatorial Chiral Separations Based on Capillary Electrophoresis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhong, Wenwan

2003-01-01

Capillary electrophoresis (CE) offers many advantages over conventional analytical methods, such as speed, simplicity, high resolution, low cost, and small sample consumption, especially for the separation of enantiomers. However, chiral method developments still can be time consuming and tedious. They designed a comprehensive enantioseparation protocol employing neutral and sulfated cyclodextrins as chiral selectors for common basic, neutral, and acidic compounds with a 96-capillary array system. By using only four judiciously chosen separation buffers, successful enantioseparations were achieved for 49 out of 54 test compounds spanning a large variety of pKs and structures. Therefore, unknown compounds can be screened in thismore » manner to identify optimal enantioselective conditions in just one rn. In addition to superior separation efficiency for small molecules, CE is also the most powerful technique for DNA separations. Using the same multiplexed capillary system with UV absorption detection, the sequence of a short DNA template can be acquired without any dye-labels. Two internal standards were utilized to adjust the migration time variations among capillaries, so that the four electropherograms for the A, T, C, G Sanger reactions can be aligned and base calling can be completed with a high level of confidence. the CE separation of DNA can be applied to study differential gene expression as well. Combined with pattern recognition techniques, small variations among electropherograms obtained by the separation of cDNA fragments produced from the total RNA samples of different human tissues can be revealed. These variations reflect the differences in total RNA expression among tissues. Thus, this Ce-based approach can serve as an alternative to the DNA array techniques in gene expression analysis.« less

Dual Targeting of GyrB and ParE by a Novel Aminobenzimidazole Class of Antibacterial Compounds▿

PubMed Central

Grossman, Trudy H.; Bartels, Douglas J.; Mullin, Steve; Gross, Christian H.; Parsons, Jonathan D.; Liao, Yusheng; Grillot, Anne-Laure; Stamos, Dean; Olson, Eric R.; Charifson, Paul S.; Mani, Nagraj

2007-01-01

A structure-guided drug design approach was used to optimize a novel series of aminobenzimidazoles that inhibit the essential ATPase activities of bacterial DNA gyrase and topoisomerase IV and that show potent activities against a variety of bacterial pathogens. Two such compounds, VRT-125853 and VRT-752586, were characterized for their target specificities and preferences in bacteria. In metabolite incorporation assays, VRT-125853 inhibited both DNA and RNA synthesis but had little effect on protein synthesis. Both compounds inhibited the maintenance of negative supercoils in plasmid DNA in Escherichia coli at the MIC. Sequencing of DNA corresponding to the GyrB and ParE ATP-binding regions in VRT-125853- and VRT-752586-resistant mutants revealed that their primary target in Staphylococcus aureus and Haemophilus influenzae was GyrB, whereas in Streptococcus pneumoniae it was ParE. In Enterococcus faecalis, the primary target of VRT-125853 was ParE, whereas for VRT-752586 it was GyrB. DNA transformation experiments with H. influenzae and S. aureus proved that the mutations observed in gyrB resulted in decreased susceptibilities to both compounds. Novobiocin resistance-conferring mutations in S. aureus, H. influenzae, and S. pneumoniae were found in gyrB, and these mutants showed little or no cross-resistance to VRT-125853 or VRT-752586 and vice versa. Furthermore, gyrB and parE double mutations increased the MICs of VRT-125853 and VRT-752586 significantly, providing evidence of dual targeting. Spontaneous frequencies of resistance to VRT-752586 were below detectable levels (<5.2 × 10−10) for wild-type E. faecalis but were significantly elevated for strains containing single and double target-based mutations, demonstrating that dual targeting confers low levels of resistance emergence and the maintenance of susceptibility in vitro. PMID:17116675

Luminescent Carbon Dot Mimics Assembled on DNA

PubMed Central

Chan, Ke Min; Xu, Wang; Kwon, Hyukin; Kietrys, Anna M.; Kool, Eric T.

2018-01-01

Nanometer-sized fragments of carbon in the form of multilayer graphene (“carbon dots”) have been under highly active study for applications in imaging. While offering advantages of low toxicity and photostability, such nanomaterials are inhomogeneous and have limited wavelengths of emission. Here we address these issues by assembling luminescent aromatic C16–C38 hydrocarbons together on a DNA scaffold in homogeneous, soluble molecular compounds. Monomer deoxyribosides of five different aromatic hydrocarbons were synthesized and assembled into a library of 1296 different tetramer compounds on PEG-polystyrene beads. These were screened for photostability and a range of emission colors using 365 nm excitation, observing visible light (>400 nm) emission. We identified a set of six oligomers (DNA-carbon assemblies, DNA-CAs) with exceptional photostability that emit from 400 to 680 nm in water, with Stokes shifts of up to 110 nm, quantum yields ranging from 0.01 to 0.29, and fluorescence lifetimes from 3 to 42 ns. In addition, several of these DNA-CAs exhibited white emission in aqueous solution. The molecules were used in multispectral cell imaging experiments and were taken up into cells passively. The results expand the range of emission properties that can be achieved in water with all-hydrocarbon chromophores and establish the use of the DNA scaffold to arrange carbon layers in homogeneous, rapidly synthesized assemblies. PMID:28841010

Anticancer effect and structure-activity analysis of marine products isolated from metabolites of mangrove fungi in the South China Sea.

PubMed

Tao, Li-yang; Zhang, Jian-ye; Liang, Yong-ju; Chen, Li-ming; Zhen, Li-sheng; Wang, Fang; Mi, Yan-jun; She, Zhi-gang; To, Kenneth Kin Wah; Lin, Yong-cheng; Fu, Li-wu

2010-04-01

Marine-derived fungi provide plenty of structurally unique and biologically active secondary metabolites. We screened 87 marine products from mangrove fungi in the South China Sea for anticancer activity by MTT assay. 14% of the compounds (11/86) exhibited a potent activity against cancer in vitro. Importantly, some compounds such as compounds 78 and 81 appeared to be promising for treating cancer patients with multidrug resistance, which should encourage more efforts to isolate promising candidates for further development as clinically useful chemotherapeutic drugs. Furthermore, DNA intercalation was not involved in their anticancer activities, as determined by DNA binding assay. On the other hand, the structure-activity analysis indicated that the hydroxyl group was important for their cytotoxic activity and that bulky functional groups such as phenyl rings could result in a loss of biological activity, which will direct the further development of marine product-based derivatives.

Specific inhibitors of mammalian DNA polymerase species.

PubMed

Mizushina, Yoshiyuki

2009-06-01

In screening of selective inhibitors of eukaryotic DNA polymerases (pols) for 15 years, more than 100 inhibitors have been discovered from natural and chemical sources. Some compounds selectively inhibit the activities of mammalian pols, and in particular, dehydroaltenusin and curcumin derivatives, such as monoacetyl-curcumin, were found to be specific inhibitors of pol alpha and pol lambda, respectively. Dehydroaltenusin was isolated from a fungus (Alternaria tennuis), and this compound inhibited cell proliferation of human cancer cell lines by arresting the cells at the S-phase, and was effective in suppressing the growth on nude mice of solid tumors of human cervical cancer cell line HeLa. Curcumin derivatives had anti-12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammatory activity with the same tendency as pol lambda inhibitory activity. These compounds might be useful not only as "molecular probes" for pol research, but also as biomedical and chemotherapeutic drugs for anti-cancer or anti-inflammation.

Detection of influenza A virus using carbon nanotubes field effect transistor based DNA sensor

NASA Astrophysics Data System (ADS)

Tran, Thi Luyen; Nguyen, Thi Thuy; Huyen Tran, Thi Thu; Chu, Van Tuan; Thinh Tran, Quang; Tuan Mai, Anh

2017-09-01

The carbon nanotubes field effect transistor (CNTFET) based DNA sensor was developed, in this paper, for detection of influenza A virus DNA. Number of factors that influence the output signal and analytical results were investigated. The initial probe DNA, decides the available DNA strands on CNTs, was 10 μM. The hybridization time for defined single helix was 120 min. The hybridization temperature was set at 30 °C to get a net change in drain current of the DNA sensor without altering properties of any biological compounds. The response time of the DNA sensor was less than one minute with a high reproducibility. In addition, the DNA sensor has a wide linear detection range from 1 pM to 10 nM, and a very low detection limit of 1 pM. Finally, after 7-month storage in 7.4 pH buffer, the output signal of DNA sensor recovered 97%.

Assessment of configurations and chemistries of bridged nucleic acids-containing oligomers as external guide sequences: a methodology for inhibition of expression of antibiotic resistance genes

PubMed Central

Jackson, Alexis; Jani, Saumya; Davies-Sala, Carol; Soler-Bistué, Alfonso J. C.; Zorreguieta, Angeles; Tolmasky, Marcelo E.

2016-01-01

External guide sequences (EGSs) are short antisense oligoribonucleotides that elicit RNase P-mediated cleavage of a target mRNA, which results in inhibition of gene expression. EGS technology is used to inhibit expression of a wide variety of genes, a strategy that may lead to development of novel treatments of numerous diseases, including multidrug-resistant bacterial and viral infections. Successful development of EGS technology depends on finding nucleotide analogs that resist degradation by nucleases present in biological fluids and the environment but still elicit RNase P-mediated degradation when forming a duplex with a target mRNA. Previous results suggested that locked nucleic acids (LNA)/DNA chimeric oligomers have these properties. LNA are now considered the first generation of compounds collectively known as bridged nucleic acids (BNAs) – modified ribonucleotides that contain a bridge at the 2ʹ,4ʹ-position of the ribose. LNA and the second-generation BNA, known as BNANC, differ in the chemical nature of the bridge. Chimeric oligomers containing LNA or BNANC and deoxynucleotide monomers in different configurations are nuclease resistant and could be excellent EGS compounds. However, not all configurations may be equally active as EGSs. RNase P cleavage assays comparing LNA/DNA and BNANC/DNA chimeric oligonucleotides that share identical nucleotide sequence but with different configurations were carried out using as target the amikacin resistance aac(6ʹ)-Ib mRNA. LNA/DNA gapmers with 5 and 3/4 LNA residues at the 5ʹ- and 3ʹ-ends, respectively, were the most efficient EGSs while all BNANC/DNA gapmers showed very poor activity. When the most efficient LNA/DNA gapmer was covalently bound to a cell-penetrating peptide, the hybrid compound conserved the EGS activity as determined by RNase P cleavage assays and reduced the levels of resistance to amikacin when added to Acinetobacter baumannii cells in culture, an indication of cellular uptake and biological activity. PMID:27857983

Global structure–activity relationship model for nonmutagenic carcinogens using virtual ligand-protein interactions as model descriptors

PubMed Central

Cunningham, Albert R.; Trent, John O.

2012-01-01

Structure–activity relationship (SAR) models are powerful tools to investigate the mechanisms of action of chemical carcinogens and to predict the potential carcinogenicity of untested compounds. We describe the use of a traditional fragment-based SAR approach along with a new virtual ligand-protein interaction-based approach for modeling of nonmutagenic carcinogens. The ligand-based SAR models used descriptors derived from computationally calculated ligand-binding affinities for learning set agents to 5495 proteins. Two learning sets were developed. One set was from the Carcinogenic Potency Database, where chemicals tested for rat carcinogenesis along with Salmonella mutagenicity data were provided. The second was from Malacarne et al. who developed a learning set of nonalerting compounds based on rodent cancer bioassay data and Ashby’s structural alerts. When the rat cancer models were categorized based on mutagenicity, the traditional fragment model outperformed the ligand-based model. However, when the learning sets were composed solely of nonmutagenic or nonalerting carcinogens and noncarcinogens, the fragment model demonstrated a concordance of near 50%, whereas the ligand-based models demonstrated a concordance of 71% for nonmutagenic carcinogens and 74% for nonalerting carcinogens. Overall, these findings suggest that expert system analysis of virtual chemical protein interactions may be useful for developing predictive SAR models for nonmutagenic carcinogens. Moreover, a more practical approach for developing SAR models for carcinogenesis may include fragment-based models for chemicals testing positive for mutagenicity and ligand-based models for chemicals devoid of DNA reactivity. PMID:22678118

Global structure-activity relationship model for nonmutagenic carcinogens using virtual ligand-protein interactions as model descriptors.

PubMed

Cunningham, Albert R; Carrasquer, C Alex; Qamar, Shahid; Maguire, Jon M; Cunningham, Suzanne L; Trent, John O

2012-10-01

Structure-activity relationship (SAR) models are powerful tools to investigate the mechanisms of action of chemical carcinogens and to predict the potential carcinogenicity of untested compounds. We describe the use of a traditional fragment-based SAR approach along with a new virtual ligand-protein interaction-based approach for modeling of nonmutagenic carcinogens. The ligand-based SAR models used descriptors derived from computationally calculated ligand-binding affinities for learning set agents to 5495 proteins. Two learning sets were developed. One set was from the Carcinogenic Potency Database, where chemicals tested for rat carcinogenesis along with Salmonella mutagenicity data were provided. The second was from Malacarne et al. who developed a learning set of nonalerting compounds based on rodent cancer bioassay data and Ashby's structural alerts. When the rat cancer models were categorized based on mutagenicity, the traditional fragment model outperformed the ligand-based model. However, when the learning sets were composed solely of nonmutagenic or nonalerting carcinogens and noncarcinogens, the fragment model demonstrated a concordance of near 50%, whereas the ligand-based models demonstrated a concordance of 71% for nonmutagenic carcinogens and 74% for nonalerting carcinogens. Overall, these findings suggest that expert system analysis of virtual chemical protein interactions may be useful for developing predictive SAR models for nonmutagenic carcinogens. Moreover, a more practical approach for developing SAR models for carcinogenesis may include fragment-based models for chemicals testing positive for mutagenicity and ligand-based models for chemicals devoid of DNA reactivity.

Mitochondrial bioenergetics and drug-induced toxicity in a panel of mouse embryonic fibroblasts with mitochondrial DNA single nucleotide polymorphisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pereira, Claudia V.; Oliveira, Paulo J.; Will, Yvonne

2012-10-15

Mitochondrial DNA (mtDNA) variations including single nucleotide polymorphisms (SNPs) have been proposed to be involved in idiosyncratic drug reactions. However, current in vitro and in vivo models lack the genetic diversity seen in the human population. Our hypothesis is that different cell strains with distinct mtDNA SNPs may have different mitochondrial bioenergetic profiles and may therefore vary in their response to drug-induced toxicity. Therefore, we used an in vitro system composed of four strains of mouse embryonic fibroblasts (MEFs) with mtDNA polymorphisms. We sequenced mtDNA from embryonic fibroblasts isolated from four mouse strains, C57BL/6J, MOLF/EiJ, CZECHII/EiJ and PERA/EiJ, with themore » latter two being sequenced for the first time. The bioenergetic profile of the four strains of MEFs was investigated at both passages 3 and 10. Our results showed that there were clear differences among the four strains of MEFs at both passages, with CZECHII/EiJ having a lower mitochondrial robustness when compared to C57BL/6J, followed by MOLF/EiJ and PERA/EiJ. Seven drugs known to impair mitochondrial function were tested for their effect on the ATP content of the four strains of MEFs in both glucose- and galactose-containing media. Our results showed that there were strain-dependent differences in the response to some of the drugs. We propose that this model is a useful starting point to study compounds that may cause mitochondrial off-target toxicity in early stages of drug development, thus decreasing the number of experimental animals used. -- Highlights: ► mtDNA SNPs may be linked to individual predisposition to drug-induced toxicity. ► CZECHII/EiJ and PERA/EiJ mtDNA was sequenced for the first time in this study. ► Strain-dependent mitochondrial capacity differences were measured. ► Strain-dependent differences in response to mitochondrial toxicants were observed.« less

Citral and eugenol modulate DNA damage and pro-inflammatory mediator genes in murine peritoneal macrophages.

PubMed

Porto, Marilia de Paula; da Silva, Glenda Nicioli; Luperini, Bruno Cesar Ottoboni; Bachiega, Tatiana Fernanda; de Castro Marcondes, João Paulo; Sforcin, José Maurício; Salvadori, Daisy Maria Fávero

2014-11-01

Citral and eugenol have been broadly studied because of their anti-inflammatory, antioxidant and antiparasitic potentials. In this study, the effects of citral (25, 50 and 100 µg/mL) and eugenol (0.31, 0.62, 1.24 and 2.48 µg/mL) on the expression (RT-PCR) of the pro-inflammatory mediator genes NF-κB1, COX-2 and TNF-α were evaluated in mouse peritoneal macrophages with or without activation by a bacterial lipopolysaccharide (LPS). Additionally, the genotoxic potentials of two compounds and their capacities to modulate the DNA damage induced by doxorubicin (DXR) were investigated using the comet assay. The data revealed that neither citral nor eugenol changed COX-2, NF-κB1 or TNF-α expression in resting macrophages. However, in LPS-activated cells, citral induced the hypoexpression of COX-2 (100 µg/mL) and TNF-α (50 and 100 µg/mL). Hypoexpression of TNF-α was also detected after cellular exposure to eugenol at the highest concentration (2.48 µg/mL). Both compounds exhibited genotoxic potential (citral at 50 and 100 µg/mL and eugenol at all concentrations) but also showed chemopreventive effects, in various treatment protocols. Both citral and eugenol might modulate inflammatory processes and DXR-induced DNA damage, but the use of these compounds must be viewed with caution because they are also able to induce primary DNA lesions.

Beta-ketoacyl-acyl carrier protein synthase III from pea (Pisum sativum L.): properties, inhibition by a novel thiolactomycin analogue and isolation of a cDNA clone encoding the enzyme.

PubMed

Jones, A Lesley; Gane, Andy M; Herbert, Derek; Willey, David L; Rutter, Andrew J; Kille, Peter; Dancer, Jane E; Harwood, John L

2003-03-01

A beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III; short-chain condensing enzyme) has been partly purified from pea leaves. The enzyme, which had acetyl-CoA:ACP acyltransferase (ACAT) activity, was resolved from a second, specific, ACAT protein. The KAS III enzyme had a derived molecular mass of 42 kDa (from its cDNA sequence) and operated as a dimer. Its enzymological characteristics were similar to those of two other plant KAS III enzymes except for its inhibition by thiolactomycin. A derivative of thiolactomycin containing a longer (C8 saturated) hydrophobic side-chain (compound 332) was a more effective inhibitor of pea KAS III and showed competitive inhibition towards malonyl-ACP whereas thiolactomycin showed uncompetitive characteristics at high concentrations. This difference may be due to the better fit of compound 332 into a hydrophobic pocket at the active site. A full-length cDNA for the pea KAS III was isolated. This was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in order to facilitate subsequent purification. Demonstrated activity in preparations from E. coli confirmed that the cDNA encoded a KAS III enzyme. Furthermore, the expressed KAS III had ACAT activity, showing that the latter was inherent. The derived amino acid sequence of the pea cDNA showed 81-87% similarity to that for other plant dicotyledon KAS IIIs, somewhat less for Allium porrum (leek, 71%) and for Porphyra spp. (62%), Synechocystis spp. (65%) and various bacteria (42-65%). The pea KAS III exhibited four areas of homology, three of which were around the active-site Cys(123), His(323) and Asn(353). In addition, a stretch of 23 amino acids (residues 207-229 in the pea KAS III) was almost completely conserved in the plant KAS IIIs. Modelling this stretch showed they belonged to a peptide fragment that fitted over the active site and contained segments suggested to be involved in substrate binding and in conformational changes during catalysis, as well as an arginine suggested to participate in the acid-base catalytic mechanism.

Biomimetic trapping cocktail to screen reactive metabolites: use of an amino acid and DNA motif mixture as light/heavy isotope pairs differing in mass shift.

PubMed

Hosaka, Shuto; Honda, Takuto; Lee, Seon Hwa; Oe, Tomoyuki

2018-06-01

Candidate drugs that can be metabolically transformed into reactive electrophilic products, such as epoxides, quinones, and nitroso compounds, are of special concern because subsequent covalent binding to bio-macromolecules can cause adverse drug reactions, such as allergic reactions, hepatotoxicity, and genotoxicity. Several strategies have been reported for screening reactive metabolites, such as a covalent binding assay with radioisotope-labeled drugs and a trapping method followed by LC-MS/MS analyses. Of these, a trapping method using glutathione is the most common, especially at the early stage of drug development. However, the cysteine of glutathione is not the only nucleophilic site in vivo; lysine, histidine, arginine, and DNA bases are also nucleophilic. Indeed, the glutathione trapping method tends to overlook several types of reactive metabolites, such as aldehydes, acylglucuronides, and nitroso compounds. Here, we introduce an alternate way for screening reactive metabolites as follows: A mixture of the light and heavy isotopes of simplified amino acid motifs and a DNA motif is used as a biomimetic trapping cocktail. This mixture consists of [ 2 H 0 ]/[ 2 H 3 ]-1-methylguanidine (arginine motif, Δ 3 Da), [ 2 H 0 ]/[ 2 H 4 ]-2-mercaptoethanol (cysteine motif, Δ 4 Da), [ 2 H 0 ]/[ 2 H 5 ]-4-methylimidazole (histidine motif, Δ 5 Da), [ 2 H 0 ]/[ 2 H 9 ]-n-butylamine (lysine motif, Δ 9 Da), and [ 13 C 0 , 15 N 0 ]/[ 13 C 1 , 15 N 2 ]-2'-deoxyguanosine (DNA motif, Δ 3 Da). Mass tag triggered data-dependent acquisition is used to find the characteristic doublet peaks, followed by specific identification of the light isotope peak using MS/MS. Forty-two model drugs were examined using an in vitro microsome experiment to validate the strategy. Graphical abstract Biomimetic trapping cocktail to screen reactive metabolites.

Cytotoxic, mutagenicity, and genotoxicity effects of guanylhydrazone derivatives.

PubMed

Pinhatti, Valéria Rodrigues; da Silva, Juliana; Martins, Tales Leandro Costa; Moura, Dinara Jaqueline; Rosa, Renato Moreira; Villela, Izabel; Stopiglia, Cheila Denise Ottonelli; da Silva Santos, Selma; Scroferneker, Maria Lúcia; Machado, Carlos Renato; Saffi, Jenifer; Henriques, João Antonio Pêgas

2016-08-01

Several studies have reported that guanylhydrazones display a variety of desirable biological properties, such as antihypertensive, antibacterial, and antimalarial behaviour. They furthermore promote anti-pneumocystosis and anti-trypanosomiasis, exhibit antitumor activity, and show significant cytotoxicity against cancer cell lines. In this work, we have evaluated the cytotoxicity, mutagenicity, and genotoxicity of two guanylhydrazones derivatives, (E)-2-[(2,3-dimethoxyphenyl) methylene] hydrazine carboxymidamide hydrochloride (2,3-DMeB) and (E)-2-[(3,4-dimethoxyphenyl) methylene] hydrazine carboxymidamide hydrochloride (3,4-DMeB), in different biological models. Both 2,3-DMeB and 3,4-DMeB induce weak cytotoxic and mutagenic effects in bacteria and yeast. The genotoxicity of these compounds was determined in a fibroblast cell line (V79) using alkaline comet assay, as well as a modified comet assay with bacterial enzymes formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (EndoIII). Both guanylhydrazone derivatives induced DNA damage. Treatment of V79 cells with EndoIII and FPG proteins demonstrated a significant effect of 2,3-DMeB and 3,4-DMeB with respect to oxidized bases. In addition, the derivatives induced a significant increase in the frequency of micronucleated cells at high doses. The antifungal and anti-trypanosomal properties of these guanylhydrazone derivatives were also evaluated, and the obtained results suggest that 2,3-DMeB is more effective than 3,4-DMeB. The biological activity of 2,3-DMeB and 3,4-DMeB may thus be related, at least in part, to their oxidative potential, as well as to their ability to interact with DNA. Considering the previously reported in vitro antitumor activity of guanylhydrazone derivatives in combination with the lack of acute toxicity and the fact that DNA damage is only observed at high doses should render both compounds good candidates for in vivo studies on antitumor activity. Copyright © 2016 Elsevier B.V. All rights reserved.

Aqueous extract from pecan nut [Carya illinoinensis (Wangenh) C. Koch] shell show activity against breast cancer cell line MCF-7 and Ehrlich ascites tumor in Balb-C mice.

PubMed

Hilbig, Josiane; Policarpi, Priscila de Britto; Grinevicius, Valdelúcia Maria Alves de Souza; Mota, Nádia Sandrine Ramos Santos; Toaldo, Isabela Maia; Luiz, Marilde Terezinha Bordignon; Pedrosa, Rozangela Curi; Block, Jane Mara

2018-01-30

In Brazil many health disorders are treated with the consumption of different varieties of tea. Shell extracts of pecan nut (Carya illinoinensis), which have significant amounts of phenolic compounds in their composition, are popularly taken as tea to prevent diverse pathologies. Phenolic compounds from pecan nut shell extract have been associated with diverse biological effects but the effect on tumor cells has not been reported yet. The aim of the current work was to evaluate the relationship between DNA fragmentation, cell cycle arrest and apoptosis induced by pecan nut shell extract and its antitumor activity. Cytotoxicity, proliferation, cell death and cell cycle were evaluated in MCF-7 cells by MTT, colony assay, differential coloring and flow cytometry assays, respectively. DNA damage effects were evaluated through intercalation into CT-DNA and plasmid DNA cleavage. Tumor growth inhibition, survival time increase, apoptosis and cell cycle arrest were assessed in Ehrlich ascites tumor in Balb/C mice. The cytotoxic effect of pecan nut shell extracts, the induction of cell death by apoptosis and also the cell cycle arrest in MCF-7 cells have been demonstrated. The survival time in mice with Ehrlich ascites tumor increased by 67%. DNA damage was observed in the CT-DNA, plasmid DNA and comet assays. The mechanism involved in the antitumor effect of pecan nut shell extracts may be related to the activation of key proteins involved in apoptosis cell death (Bcl-XL, Bax and p53) and on the cell cycle regulation (cyclin A, cyclin B and CDK2). These results were attributed to the phenolic profile of the extract, which presented compounds such as gallic, 4-hydroxybenzoic, chlorogenic, vanillic, caffeic and ellagic acid, and catechin, epicatechin, epigallocatechin and epicatechin gallate. The results indicated that pecan nut shell extracts are effective against tumor cells growth and may be considered as an alternative to the treatment of cancer. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Shi-Wei; Wu, Chun-Ying; Wang, Yen-Ting

Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53more » status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cell's p53 status. - Highlights: ► Compound C caused more apoptosis in p53 wildtype than p53-mutant skin cancer cells. ► Compound C can upregulate p53 expression and induce p53 activation. ► Compound C induced p53 effects were dependent on ROS induced DNA damage pathway. ► p53-knockdown attenuated compound C-induced apoptosis but not autophagy. ► Compound C-induced apoptosis in skin cancer cells was dependent on p53 status.« less

The Flaxseed-Derived Lignan Phenolic Secoisolariciresinol Diglucoside (SDG) Protects Non-Malignant Lung Cells from Radiation Damage

PubMed Central

Velalopoulou, Anastasia; Tyagi, Sonia; Pietrofesa, Ralph A.; Arguiri, Evguenia; Christofidou-Solomidou, Melpo

2015-01-01

Plant phenolic compounds are common dietary antioxidants that possess antioxidant and anti-inflammatory properties. Flaxseed (FS) has been reported to be radioprotective in murine models of oxidative lung damage. Flaxseed’s protective properties are attributed to its main biphenolic lignan, secoisolariciresinol diglucoside (SDG). SDG is a free radical scavenger, shown in cell free systems to protect DNA from radiation-induced damage. The objective of this study was to investigate the in vitro radioprotective efficacy of SDG in murine lung cells. Protection against irradiation (IR)-induced DNA double and single strand breaks was assessed by γ-H2AX labeling and alkaline comet assay, respectively. The role of SDG in modulating the levels of cytoprotective enzymes was evaluated by qPCR and confirmed by Western blotting. Additionally, effects of SDG on clonogenic survival of irradiated cells were evaluated. SDG protected cells from IR-induced death and ameliorated DNA damage by reducing mean comet tail length and percentage of γ-H2AX positive cells. Importantly, SDG significantly increased gene and protein levels of antioxidant HO-1, GSTM1 and NQO1. Our results identify the potent radioprotective properties of the synthetic biphenolic SDG, preventing DNA damage and enhancing the antioxidant capacity of normal lung cells; thus, rendering SDG a potential radioprotector against radiation exposure. PMID:26703588

The Flaxseed-Derived Lignan Phenolic Secoisolariciresinol Diglucoside (SDG) Protects Non-Malignant Lung Cells from Radiation Damage.

PubMed

Velalopoulou, Anastasia; Tyagi, Sonia; Pietrofesa, Ralph A; Arguiri, Evguenia; Christofidou-Solomidou, Melpo

2015-12-22

Plant phenolic compounds are common dietary antioxidants that possess antioxidant and anti-inflammatory properties. Flaxseed (FS) has been reported to be radioprotective in murine models of oxidative lung damage. Flaxseed's protective properties are attributed to its main biphenolic lignan, secoisolariciresinol diglucoside (SDG). SDG is a free radical scavenger, shown in cell free systems to protect DNA from radiation-induced damage. The objective of this study was to investigate the in vitro radioprotective efficacy of SDG in murine lung cells. Protection against irradiation (IR)-induced DNA double and single strand breaks was assessed by γ-H2AX labeling and alkaline comet assay, respectively. The role of SDG in modulating the levels of cytoprotective enzymes was evaluated by qPCR and confirmed by Western blotting. Additionally, effects of SDG on clonogenic survival of irradiated cells were evaluated. SDG protected cells from IR-induced death and ameliorated DNA damage by reducing mean comet tail length and percentage of γ-H2AX positive cells. Importantly, SDG significantly increased gene and protein levels of antioxidant HO-1, GSTM1 and NQO1. Our results identify the potent radioprotective properties of the synthetic biphenolic SDG, preventing DNA damage and enhancing the antioxidant capacity of normal lung cells; thus, rendering SDG a potential radioprotector against radiation exposure.

Microfluidic Bead Suspension Hopper

PubMed Central

2014-01-01

Many high-throughput analytical platforms, from next-generation DNA sequencing to drug discovery, rely on beads as carriers of molecular diversity. Microfluidic systems are ideally suited to handle and analyze such bead libraries with high precision and at minute volume scales; however, the challenge of introducing bead suspensions into devices before they sediment usually confounds microfluidic handling and analysis. We developed a bead suspension hopper that exploits sedimentation to load beads into a microfluidic droplet generator. A suspension hopper continuously delivered synthesis resin beads (17 μm diameter, 112,000 over 2.67 h) functionalized with a photolabile linker and pepstatin A into picoliter-scale droplets of an HIV-1 protease activity assay to model ultraminiaturized compound screening. Likewise, trypsinogen template DNA-coated magnetic beads (2.8 μm diameter, 176,000 over 5.5 h) were loaded into droplets of an in vitro transcription/translation system to model a protein evolution experiment. The suspension hopper should effectively remove any barriers to using suspensions as sample inputs, paving the way for microfluidic automation to replace robotic library distribution. PMID:24761972

Increased N7-methyldeoxyguanosine DNA adducts after occupational exposure to pesticides and influence of genetic polymorphisms of paraoxonase-1 and glutathione S-transferase M1 and T1.

PubMed

Gómez-Martín, Antonio; Altakroni, Bashar; Lozano-Paniagua, David; Margison, Geoffrey P; de Vocht, Frank; Povey, Andrew C; Hernández, Antonio F

2015-06-01

There are concerns about genetic risks associated with long-term exposure to pesticides as these compounds may damage DNA, resulting in mutations that eventually lead to cancer, neurological, and reproductive adverse health effects. This study assessed DNA damage in intensive agricultural workers exposed to pesticides by determining the levels of N7-methyldeoxyguanosine (N7-MedG), an adduct known to be a robust biomarker of recent exposure to chemical methylating agents. A cohort of 39 plastic greenhouse workers was assessed for changes in lymphocyte DNA N7-MedG levels between low level and high level exposures during the course of a spraying season. The contributions of genetic polymorphisms of the pesticide-metabolizing enzymes paraoxonase-1 (PON1) and the glutathione S-transferases, GSTM1 and GSTT1, on N7-MedG levels and other potential confounders were also assessed. N7-MedG increased in the period of high pesticide exposure as compared to the low exposure period (0.23 and 0.18 µmol N7-MedG/mol dG for the unadjusted and adjusted linear mixed models, P = 0.02 and 0.08, respectively). Significant decreased levels of erythrocyte acetylcholinesterase and plasma cholinesterase were observed in the high versus low exposure period in both the unadjusted (2.85 U/g hemoglobin and 213.13 U/L, respectively) and adjusted linear mixed models (2.99 U/g hemoglobin and 230.77 U/L, respectively), indicating pesticide intake. In intensive agriculture workers, higher pesticide exposure increased DNA alkylation levels, further demonstrating the genotoxicity of pesticides in man. In addition, pesticide-exposed individuals with inherited susceptible metabolic genotypes (particularly, null genotype for GSTM1 and the PON1 192R allele) appear to have an increased risk of genotoxic DNA damage. Environ. Mol. Mutagen. 56:437-445, 2015. © 2014 Wiley Periodicals, Inc. © 2014 Wiley Periodicals, Inc.

Twenty-five Years of DNA-Encoded Chemical Libraries.

PubMed

Neri, Dario

2017-05-04

Reference library: The availability of DNA-encoded chemical libraries containing billions of compounds facilitates the discovery of binding molecules for pharmaceutical applications and for investigating biological processes. This Special Issue highlights the use of this library technology and some of the latest developments in the field. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeting SOD1 induces synthetic lethal killing in BLM- and CHEK2-deficient colorectal cancer cells

PubMed Central

Sajesh, Babu V.; McManus, Kirk J.

2015-01-01

Cancer is a major cause of death throughout the world, and there is a large need for better and more personalized approaches to combat the disease. Over the past decade, synthetic lethal approaches have been developed that are designed to exploit the aberrant molecular origins (i.e. defective genes) that underlie tumorigenesis. BLM and CHEK2 are two evolutionarily conserved genes that are somatically altered in a number of tumor types. Both proteins normally function in preserving genome stability through facilitating the accurate repair of DNA double strand breaks. Thus, uncovering synthetic lethal interactors of BLM and CHEK2 will identify novel candidate drug targets and lead chemical compounds. Here we identify an evolutionarily conserved synthetic lethal interaction between SOD1 and both BLM and CHEK2 in two distinct cell models. Using quantitative imaging microscopy, real-time cellular analyses, colony formation and tumor spheroid models we show that SOD1 silencing and inhibition (ATTM and LCS-1 treatments), or the induction of reactive oxygen species (2ME2 treatment) induces selective killing within BLM- and CHEK2-deficient cells relative to controls. We further show that increases in reactive oxygen species follow SOD1 silencing and inhibition that are associated with the persistence of DNA double strand breaks, and increases in apoptosis. Collectively, these data identify SOD1 as a novel candidate drug target in BLM and CHEK2 cancer contexts, and further suggest that 2ME2, ATTM and LCS-1 are lead therapeutic compounds warranting further pre-clinical study. PMID:26318585

Targeting SOD1 induces synthetic lethal killing in BLM- and CHEK2-deficient colorectal cancer cells.

PubMed

Sajesh, Babu V; McManus, Kirk J

2015-09-29

Cancer is a major cause of death throughout the world, and there is a large need for better and more personalized approaches to combat the disease. Over the past decade, synthetic lethal approaches have been developed that are designed to exploit the aberrant molecular origins (i.e. defective genes) that underlie tumorigenesis. BLM and CHEK2 are two evolutionarily conserved genes that are somatically altered in a number of tumor types. Both proteins normally function in preserving genome stability through facilitating the accurate repair of DNA double strand breaks. Thus, uncovering synthetic lethal interactors of BLM and CHEK2 will identify novel candidate drug targets and lead chemical compounds. Here we identify an evolutionarily conserved synthetic lethal interaction between SOD1 and both BLM and CHEK2 in two distinct cell models. Using quantitative imaging microscopy, real-time cellular analyses, colony formation and tumor spheroid models we show that SOD1 silencing and inhibition (ATTM and LCS-1 treatments), or the induction of reactive oxygen species (2ME2 treatment) induces selective killing within BLM- and CHEK2-deficient cells relative to controls. We further show that increases in reactive oxygen species follow SOD1 silencing and inhibition that are associated with the persistence of DNA double strand breaks, and increases in apoptosis. Collectively, these data identify SOD1 as a novel candidate drug target in BLM and CHEK2 cancer contexts, and further suggest that 2ME2, ATTM and LCS-1 are lead therapeutic compounds warranting further pre-clinical study.

Metabolic Profiles and cDNA-AFLP Analysis of Salvia miltiorrhiza and Salvia castanea Diel f. tomentosa Stib

PubMed Central

Liang, Xiao; Liang, Zongsuo; Zhang, Meixiang; Shen, Shuang; Liu, Hongyun; Liu, Yan

2012-01-01

Plants of the genus Salvia produce various types of phenolic compounds and tanshinones which are effective for treatment of coronary heart disease. Salvia miltiorrhiza and S. castanea Diels f. tomentosa Stib are two important members of the genus. In this study, metabolic profiles and cDNA-AFLP analysis of four samples were employed to identify novel genes potentially involved in phenolic compounds and tanshinones biosynthesis, including the red roots from the two species and two tanshinone-free roots from S. miltiorrhiza. The results showed that the red roots of S. castanea Diels f. tomentosa Stib produced high contents of rosmarinic acid (21.77 mg/g) and tanshinone IIA (12.60 mg/g), but low content of salvianolic acid B (1.45 mg/g). The red roots of S. miltiorrhiza produced high content of salvianolic acid B (18.69 mg/g), while tanshinones accumulation in this sample was much less than that in S. castanea Diels f. tomentosa Stib. Tanshinones were not detected in the two tanshinone-free samples, which produced high contents of phenolic compounds. A cDNA-AFLP analysis with 128 primer pairs revealed that 2300 transcript derived fragments (TDFs) were differentially expressed among the four samples. About 323 TDFs were sequenced, of which 78 TDFs were annotated with known functions through BLASTX searching the Genbank database and 14 annotated TDFs were assigned into secondary metabolic pathways through searching the KEGGPATHWAY database. The quantitative real-time PCR analysis indicated that the expression of 9 TDFs was positively correlated with accumulation of phenolic compounds and tanshinones. These TDFs additionally showed coordinated transcriptional response with 6 previously-identified genes involved in biosynthesis of tanshinones and phenolic compounds in S. miltiorrhiza hairy roots treated with yeast extract. The sequence data in the present work not only provided us candidate genes involved in phenolic compounds and tanshinones biosynthesis but also gave us further insight into secondary metabolism in Salvia. PMID:22303439

A pharmacological screen for compounds that rescue the developmental lethality of a Drosophila ATM mutant.

PubMed

Rimkus, Stacey A; Wassarman, David A

2018-01-01

Ataxia-telangiectasia (A-T) is a neurodegenerative disease caused by mutation of the A-T mutated (ATM) gene. ATM encodes a protein kinase that is activated by DNA damage and phosphorylates many proteins, including those involved in DNA repair, cell cycle control, and apoptosis. Characteristic biological and molecular functions of ATM observed in mammals are conserved in Drosophila melanogaster. As an example, conditional loss-of-function ATM alleles in flies cause progressive neurodegeneration through activation of the innate immune response. However, unlike in mammals, null alleles of ATM in flies cause lethality during development. With the goals of understanding biological and molecular roles of ATM in a whole animal and identifying candidate therapeutics for A-T, we performed a screen of 2400 compounds, including FDA-approved drugs, natural products, and bioactive compounds, for modifiers of the developmental lethality caused by a temperature-sensitive ATM allele (ATM8) that has reduced kinase activity at non-permissive temperatures. Ten compounds reproducibly suppressed the developmental lethality of ATM8 flies, including Ronnel, which is an organophosphate. Ronnel and other suppressor compounds are known to cause mitochondrial dysfunction or to inhibit the enzyme acetylcholinesterase, which controls the levels of the neurotransmitter acetylcholine, suggesting that detrimental consequences of reduced ATM kinase activity can be rescued by inhibiting the function of mitochondria or increasing acetylcholine levels. We carried out further studies of Ronnel because, unlike the other compounds that suppressed the developmental lethality of homozygous ATM8 flies, Ronnel was toxic to the development of heterozygous ATM8 flies. Ronnel did not affect the innate immune response of ATM8 flies, and it further increased the already high levels of DNA damage in brains of ATM8 flies, but its effects were not harmful to the lifespan of rescued ATM8 flies. These results provide new leads for understanding the biological and molecular roles of ATM and for the treatment of A-T.

Antineoplastic and cytogenetic effects of complexes of Pd (II) with 4N-substituted derivatives of 2-acetyl-pyridine-thiosemicarbazone.

PubMed

Papageorgiou, A; Iakovidou, Z; Mourelatos, D; Mioglou, E; Boutis, L; Kotsis, A; Kovala-Demertzi, D; Domopoulou, A; West, D X; Dermetzis, M A

1997-01-01

The effect of novel Pd(II) complexes with derivatives of 2-acetyl-pyridinethisemicarbazone, N4-ethyl (HAc4Et) and 3-hexamethyleneiminylthiosemicarbazone (HAchexim), on Sister Chromatid Exchange (SCE) rates and human lymphocyte proliferation kinetics was studied. Also, the effect of Pd(II) complexes on DNA synthesis of P388 and L1210 cell cultures and against Leukemia P388 was investigated. Among these compounds, the compound Bis(3-hexamethyleneiminyl-2-acetylpyridine-thisemicarbazonato++ +) palladium (II) was found to be distinctly effective against Leukemia P388, in inhibiting incorporation of 3H-thymidine into DNA and in inducing SCEs and cell division delays.

Isolation of bioactive and other oxoaporphine alkaloids from two annonaceous plants, Xylopia aethiopica and Miliusa cf. banacea.

PubMed

Harrigan, G G; Gunatilaka, A A; Kingston, D G; Chan, G W; Johnson, R K

1994-01-01

The oxoaporphine alkaloids oxophoebine [1] and liriodenine [2] have been isolated from Xylopia aethiopica (Annonaceae). Both showed selective toxicity against DNA repair and recombination deficient mutants of the yeast Saccharomyces cerevisae. Three related but inactive compounds, oxoglaucine [3], O-methylmoschatoline [4], and lysicamine [5], were also isolated from this plant. Selective toxicity was also observed for 10-methoxyliriodenine (lauterine) [6] and 10-hydroxyliriodenine [7], two oxoaporphine alkaloids isolated from Miliusa cf. banacea (Annonaceae). The structure of 10-hydroxyliriodenine [7], a novel oxoaporphine, was determined by spectroscopic methods and chemical conversion to compound 6. The role of the bioactive oxoaporphine alkaloids as DNA topoisomerase inhibitors is discussed.

DNA stable-isotope probing (DNA-SIP).

PubMed

Dunford, Eric A; Neufeld, Josh D

2010-08-02

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

Helicases as Prospective Targets for Anti-Cancer Therapy

PubMed Central

Gupta, Rigu; Brosh, Robert M.

2008-01-01

It has been proposed that selective inactivation of a DNA repair pathway may enhance anti-cancer therapies that eliminate cancerous cells through the cytotoxic effects of DNA damaging agents or radiation. Given the unique and critically important roles of DNA helicases in the DNA damage response, DNA repair, and maintenance of genomic stability, a number of strategies currently being explored or in use to combat cancer may be either mediated or enhanced through the modulation of helicase function. The focus of this review will be to examine the roles of helicases in DNA repair that might be suitably targeted by cancer therapeutic approaches. Treatment of cancers with anti-cancer drugs such as small molecule compounds that modulate helicase expression or function is a viable approach to selectively kill cancer cells through the inactivation of helicase-dependent DNA repair pathways, particularly those associated with DNA recombination, replication restart, and cell cycle checkpoint. PMID:18473724

Inhibition of DNA polymerase λ and associated inflammatory activities of extracts from steamed germinated soybeans.

PubMed

Mizushina, Yoshiyuki; Kuriyama, Isoko; Yoshida, Hiromi

2014-04-01

During the screening of selective DNA polymerase (pol) inhibitors from more than 50 plant food materials, we found that the extract from steamed germinated soybeans (Glycine max L.) inhibited human pol λ activity. Among the three processed soybean samples tested (boiled soybeans, steamed soybeans, and steamed germinated soybeans), both the hot water extract and organic solvent extract from the steamed germinated soybeans had the strongest pol λ inhibition. We previously isolated two glucosyl compounds, a cerebroside (glucosyl ceramide, AS-1-4, compound ) and a steroidal glycoside (eleutheroside A, compound ), from dried soybean, and these compounds were prevalent in the extracts of the steamed germinated soybeans as pol inhibitors. The hot water and organic solvent extracts of the steamed germinated soybeans and compounds and selectively inhibited the activity of eukaryotic pol λ in vitro but did not influence the activities of other eukaryotic pols, including those from the A-family (pol γ), B-family (pols α, δ, and ε), and Y-family (pols η, ι, and κ), and also showed no effect on the activity of pol β, which is of the same family (X) as pol λ. The tendency for in vitro pol λ inhibition by these extracts and compounds showed a positive correlation with the in vivo suppression of TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation in mouse ear. These results suggest that steamed germinated soybeans, especially the glucosyl compound components, may be useful for their anti-inflammatory properties.

Antiviral lead compounds from marine sponges.

PubMed

Sagar, Sunil; Kaur, Mandeep; Minneman, Kenneth P

2010-10-11

Marine sponges are currently one of the richest sources of pharmacologically active compounds found in the marine environment. These bioactive molecules are often secondary metabolites, whose main function is to enable and/or modulate cellular communication and defense. They are usually produced by functional enzyme clusters in sponges and/or their associated symbiotic microorganisms. Natural product lead compounds from sponges have often been found to be promising pharmaceutical agents. Several of them have successfully been approved as antiviral agents for clinical use or have been advanced to the late stages of clinical trials. Most of these drugs are used for the treatment of human immunodeficiency virus (HIV) and herpes simplex virus (HSV). The most important antiviral lead of marine origin reported thus far is nucleoside Ara-A (vidarabine) isolated from sponge Tethya crypta. It inhibits viral DNA polymerase and DNA synthesis of herpes, vaccinica and varicella zoster viruses. However due to the discovery of new types of viruses and emergence of drug resistant strains, it is necessary to develop new antiviral lead compounds continuously. Several sponge derived antiviral lead compounds which are hoped to be developed as future drugs are discussed in this review. Supply problems are usually the major bottleneck to the development of these compounds as drugs during clinical trials. However advances in the field of metagenomics and high throughput microbial cultivation has raised the possibility that these techniques could lead to the cost-effective large scale production of such compounds. Perspectives on biotechnological methods with respect to marine drug development are also discussed.

Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase.

PubMed

Chan, Pan F; Germe, Thomas; Bax, Benjamin D; Huang, Jianzhong; Thalji, Reema K; Bacqué, Eric; Checchia, Anna; Chen, Dongzhao; Cui, Haifeng; Ding, Xiao; Ingraham, Karen; McCloskey, Lynn; Raha, Kaushik; Srikannathasan, Velupillai; Maxwell, Anthony; Stavenger, Robert A

2017-05-30

A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase-DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development.

Evaluation of genotoxic potential of avarol, avarone, and its methoxy and methylamino derivatives in prokaryotic and eukaryotic test models.

PubMed

Kolarević, Stoimir; Milovanović, Dragana; Kračun-Kolarević, Margareta; Kostić, Jovana; Sunjog, Karolina; Martinović, Rajko; Đorđević, Jelena; Novaković, Irena; Sladić, Dušan; Vuković-Gačić, Branka

2018-01-04

In this study, mutagenic and genotoxic potential of anti-tumor compounds avarol, avarone, and its derivatives 3'-methoxyavarone, 4'-(methylamino)avarone and 3'-(methylamino)avarone was evaluated and compared to cytostatics commonly used in chemotherapy (5-fluorouracil, etoposid, and cisplatin). Mutagenic potential of selected hydroquinone and quinones was assessed in prokaryotic model by the SOS/umuC assay in Salmonella typhimurium TA1535/pSK1002. Genotoxic potential was also assessed in eukaryotic models using comet assay in human fetal lung cell line (MRC-5), human adenocarcinoma epithelial cell line (A549), and in human peripheral blood cells (HPBC). The results indicated that avarol and avarone do not exert mutagenic/genotoxic potential. Among the studied avarone derivatives, mutagenic potential was detected by SOS/umuC test for 3'-(methylamino)avarone, but only after metabolic activation. The results of comet assay indicated that 3'-methoxyavarone and 3'-(methylamino)avarone have a significant impact on the level of DNA damage in the MRC-5 cell line. Genotoxic potential was not observed in A549 cells or HPBC probably due to a different uptake rate for the compounds and lower in metabolism rate within these cells.

DNA Polymerase Eta and Chemotherapeutic Agents

PubMed Central

2011-01-01

Abstract The discovery of human DNA polymerase eta (pol η) has a major impact on the fields of DNA replication/repair fields. Since the discovery of human pol η, a number of new DNA polymerases with the ability to bypass various DNA lesions have been discovered. Among these polymerases, pol η is the most extensively studied lesion bypass polymerase with a defined major biological function, that is, to replicate across the cyclobutane pyrimidine dimers introduced by UV irradiation. Cyclobutane pyrimidine dimer is a major DNA lesion that causes distortion of DNA structure and block the replicative DNA polymerases during DNA replication process. Genetic defects in the pol η gene, Rad30, results in a disease called xeroderma pigmentosum variant. This review focuses on the overall properties of pol η and the mechanism that involved in regulating its activity in cells. In addition, the role of pol η in the action of DNA-targeting anticancer compounds is also discussed. Antioxid. Redox Signal. 14, 2521–2529. PMID:21050139

Induction of DNA-protein cross-links by platinum compounds.

PubMed

Woźniak, K; Walter, Z

2000-01-01

The differences between cis- and trans-diamminedichloroplatinum II (DDP) in forming DNA-protein cross-links in isolated human lymphocytes were investigated. Both cis- and trans-DDP can induce DNA-protein cross-links. We show that cis-DDP forms complexes between DNA and proteins faster than trans-DDP. This results from an increase in the quantity of DNA and platinum together with an increase in drug concentration. Under the same conditions trans-DDP causes a decrease in DNA-forming complexes with proteins. After a 12 h incubation of lymphocytes we observe a similar level of DNA in DNA-protein cross-links induced by DDP isomers, but more platinum appears in complexes induced by trans-DDP. The results obtained demonstrate that the antitumor drug - cis-DDP and the clinically ineffective trans-DDP induce links between DNA and proteins in a different manner. We suggest that the therapeutic activity of cis-DDP can in part arise from rapidly forming DNA-protein complexes which can destroy the most important cellular processes, such as replication and transcription.

Design, Synthesis and Characterisation of New Chimeric Ruthenium(II)-Gold(I) Complexes as Improved Cytotoxic Agents

PubMed Central

Massai, Lara; Fernández-Gallardo, Jacob; Guerri, Annalisa; Arcangeli, Annarosa; Pillozzi, Serena

2015-01-01

Two heterobimetallic complexes, i.e. [RuCl2(p-cymene)(µ-dppm)AuC] (1) and [RuCl2(p-cymene)(µ-dppm)Au S(thiazoline)] (3), based on known cytotoxic [Ru(p-cymene)Cl2(PR3)] and [AuX(PR3)] (X = Cl, SR) molecular scaffolds, with the diphosphane linker 1,1-bis(diphenylphosphino) methane, dppm, were conveniently prepared and characterised. Remarkably, the new compounds manifested a more favourable in vitro pharmacological profile toward cancer cells than individual ruthenium and gold species being either more cytotoxic or more selective. The interactions of the study compounds with (pBR322) DNA and their inhibitory effects on cathepsin B were also assessed. In addition, their reactivity toward suitable models of protein targets was explored and clear evidence gained for disruption of the bimetallic motif and for protein binding of monometallic fragments. Overall, the data reported here strongly support the concept of multifunctional heterometallic compounds as “improved” candidate agents for cancer treatment. The mechanistic and pharmacological implications of the present findings are discussed. PMID:25996553

Design, synthesis and characterisation of new chimeric ruthenium(II)-gold(I) complexes as improved cytotoxic agents.

PubMed

Massai, Lara; Fernández-Gallardo, Jacob; Guerri, Annalisa; Arcangeli, Annarosa; Pillozzi, Serena; Contel, María; Messori, Luigi

2015-06-28

Two heterobimetallic complexes, i.e. [RuCl2(p-cymene)(μ-dppm)AuC] (1) and [RuCl2(p-cymene)(μ-dppm)Au(S-thiazoline)] (3), based on known cytotoxic [Ru(p-cymene)Cl2(PR3)] and [AuX(PR3)] (X = Cl, SR) molecular scaffolds, with the diphosphane linker 1,1-bis(diphenylphosphino)methane, dppm, were conveniently prepared and characterised. Remarkably, the new compounds manifested a more favourable in vitro pharmacological profile toward cancer cells than individual ruthenium and gold species being either more cytotoxic or more selective. The interactions of the studied compounds with (pBR322) DNA and their inhibitory effects on cathepsin B were also assessed. In addition, their reactivity toward suitable models of protein targets was explored and clear evidence gained for disruption of the bimetallic motif and for protein binding of monometallic fragments. Overall, the data reported here strongly support the concept of multifunctional heterometallic compounds as "improved" candidate agents for cancer treatment. The mechanistic and pharmacological implications of the present findings are discussed.

Metabolic Design and Control for Production in Prokaryotes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chhabra, Swapnil R.; Keasling, J.D.

2010-11-10

Prokaryotic life on earth is manifested by its diversity and omnipresence. These microbes serve as natural sources of a large variety of compounds with the potential to serve the ever growing, medicinal, chemical and transportation needs of the human population. However, commercially viable production of these compounds can be realized only through significant improvement of the native production capacity of natural isolates. The most favorable way to achieve this goal is through the genetic manipulation of metabolic pathways that direct the production of these molecules. While random mutagenesis and screening have dominated the industrial production of such compounds in themore » past our increased understanding of microbial physiology over the last five decades has shifted this trend towards rational approaches for metabolic design. Major drivers of this trend include recombinant DNA technology, high throughput characterization of macromolecular cellular components, quantitative modeling for metabolic engine ring, targeted combinatorial engineering and synthetic biology. In this chapter we track the evolution of microbial engineering technologies from the black box era of random mutagenesis to the science and engineering-driven era of metabolic design.« less

Furry pet allergens, fungal DNA and microbial volatile organic compounds (MVOCs) in the commercial aircraft cabin environment.

PubMed

Fu, Xi; Lindgren, Torsten; Guo, Moran; Cai, Gui-Hong; Lundgren, Håkan; Norbäck, Dan

2013-06-01

There has been concern about the cabin environment in commercial aircraft. We measured cat, dog and horse allergens and fungal DNA in cabin dust and microbial volatile organic compounds (MVOCs) in cabin air. Samples were collected from two European airline companies, one with cabins having textile seats (TSC) and the other with cabins having leather seats (LSC), 9 airplanes from each company. Dust was vacuumed from seats and floors in the flight deck and different parts of the cabin. Cat (Fel d1), dog (Can f1) and horse allergens (Equ cx) were analyzed by ELISA. Five sequences of fungal DNA were analyzed by quantitative PCR. MVOCs were sampled on charcoal tubes in 42 TSC flights, and 17 compounds were analyzed by gas chromatography mass spectrometry (GC-MS) with selective ion monitoring (SIM). MVOC levels were compared with levels in homes from Nordic countries. The weight of dust was 1.8 times larger in TSC cabins as compared to LSC cabins (p < 0.001). In cabins with textile seats, the geometric mean (GM) concentrations of Fel d1, Can f1 and Equ cx were 5359 ng g(-1), 6067 ng g(-1), and 13 703 ng g(-1) (GM) respectively. Levels of Fel d1, Can f1 and Equ cx were 50 times, 27 times and 75 times higher respectively, in TSC cabins as compared to LSC cabins (p < 0.001). GM levels of Aspergillus/Penicillium DNA, Aspergillus versicolor DNA, Stachybotrys chartarum DNA and Streptomyces DNA were all higher in TSC as compared to LSC (p < 0.05). The sum of MVOCs in cabin air (excluding butanols) was 3192 ng m(-3) (GM), 3.7 times higher than in homes (p < 0.001) and 2-methyl-1-butanol and 3-methyl-1-butanol concentrations were 15-17 times higher as compared to homes (p < 0.001). Concentrations of isobutanol, 1-butanol, dimethyldisulfide, 2-hexanone, 2-heptanone, 3-octanone, isobutyl acetate and ethyl-2-methylbutyrate were lower in cabin air as compared to homes (p < 0.05). In conclusion, textile seats are much more contaminated by pet allergens and fungal DNA than leather seats. The use of seats with smooth surfaces should be encouraged. The MVOC levels differed between cabin air and homes.

Release of the cyano moiety in the crystal structure of N-cyanomethyl-N-(2-methoxyethyl)-daunomycin complexed with d(CGATCG).

PubMed

Saminadin, P; Dautant, A; Mondon, M; Langlois D'estaintot, B; Courseille, C; Précigoux, G

2000-01-01

Doxorubicin is among the most widely used anthracycline in cancer chemotherapy. In an attempt to avoid the cardiotoxicity and drug resistance of doxorubicin therapy, several analogues were synthesized. The cyanomorpholinyl derivative is the most cytotoxic. They differ greatly from their parent compound in their biological and pharmacological properties, inducing cross-links in drug DNA complexes. The present study concerns N-cyanomethyl-N-(2-methoxyethyl)-daunomycin (CMDa), a synthetic analogue of cyanomorpholino-daunomycin. Compared to doxorubicin, CMDa displays a cytotoxic activity on L1210 leukemia cells at higher concentration but is effective on doxorubicin resistant cells. The results of fluorescence quenching experiments as well as the melting temperature (DeltaTm = 7.5 degrees C) studies are consistent with a drug molecule which intercalates between the DNA base pairs and stabilizes the DNA double helix. The crystal structure of CMDa complexed to the hexanucleotide d(CGATCG) has been determined at 1.5 A resolution. The complex crystallizes in the space group P41212 and is similar to other anthracycline-hexanucleotide complexes. In the crystal state, the observed densities indicate the formation of N-hydroxymethyl-N-(2-methoxyethyl)-daunomycin (HMDa) with the release of the cyano moiety without DNA alkylation. The formation of this degradation compound is discussed in relation with other drug modifications when binding to DNA. Comparison with two other drug-DNA crystal structures suggests a correlation between a slight change in DNA conformation and the nature of the amino sugar substituents at the N3' position located in the minor groove.

Prototype Systems Containing Human Cytochrome P450 for High-Throughput Real-Time Detection of DNA Damage by Compounds That Form DNA-Reactive Metabolites.

PubMed

Brito Palma, Bernardo; Fisher, Charles W; Rueff, José; Kranendonk, Michel

2016-05-16

The formation of reactive metabolites through biotransformation is the suspected cause of many adverse drug reactions. Testing for the propensity of a drug to form reactive metabolites has increasingly become an integral part of lead-optimization strategy in drug discovery. DNA reactivity is one undesirable facet of a drug or its metabolites and can lead to increased risk of cancer and reproductive toxicity. Many drugs are metabolized by cytochromes P450 in the liver and other tissues, and these reactions can generate hard electrophiles. These hard electrophilic reactive metabolites may react with DNA and may be detected in standard in vitro genotoxicity assays; however, the majority of these assays fall short due to the use of animal-derived organ extracts that inadequately represent human metabolism. The current study describes the development of bacterial systems that efficiently detect DNA-damaging electrophilic reactive metabolites generated by human P450 biotransformation. These assays use a GFP reporter system that detects DNA damage through induction of the SOS response and a GFP reporter to control for cytotoxicity. Two human CYP1A2-competent prototypes presented here have appropriate characteristics for the detection of DNA-damaging reactive metabolites in a high-throughput manner. The advantages of this approach include a short assay time (120-180 min) with real-time measurement, sensitivity to small amounts of compound, and adaptability to a microplate format. These systems are suitable for high-throughput assays and can serve as prototypes for the development of future enhanced versions.

Genotoxicity Revaluation of Three Commercial Nitroheterocyclic Drugs: Nifurtimox, Benznidazole, and Metronidazole

PubMed Central

Buschini, Annamaria; Ferrarini, Lisa; Franzoni, Susanna; Galati, Serena; Lazzaretti, Mirca; Mussi, Francesca; Northfleet de Albuquerque, Cristina; Maria Araújo Domingues Zucchi, Tânia; Poli, Paola

2009-01-01

Nitroheterocyclic compounds are widely used as therapeutic agents against a variety of protozoan and bacterial infections. However, the literature on these compounds, suspected of being carcinogens, is widely controversial. In this study, cytotoxic and genotoxic potential of three drugs, Nifurtimox (NFX), Benznidazole (BNZ), and Metronidazole (MTZ) was re-evaluated by different assays. Only NFX reduces survival rate in actively proliferating cells. The compounds are more active for base-pair substitution than frameshift induction in Salmonella; NFX and BNZ are more mutagenic than MTZ; they are widely dependent from nitroreduction whereas microsomal fraction S9 weakly affects the mutagenic potential. Comet assay detects BNZ- and NFX-induced DNA damage at doses in the range of therapeutically treated patient plasma concentration; BNZ seems to mainly act through ROS generation whereas a dose-dependent mechanism of DNA damaging is suggested for NFX. The lack of effects on mammalian cells for MTZ is confirmed also in MN assay whereas MN induction is observed for NFX and BNZ. The effects of MTZ, that shows comparatively low reduction potential, seem to be strictly dependent on anaerobic/hypoxic conditions. Both NFX and BNZ may not only lead to cellular damage of the infective agent but also interact with the DNA of mammalian cells. PMID:20981287

Genotoxicity revaluation of three commercial nitroheterocyclic drugs: nifurtimox, benznidazole, and metronidazole.

PubMed

Buschini, Annamaria; Ferrarini, Lisa; Franzoni, Susanna; Galati, Serena; Lazzaretti, Mirca; Mussi, Francesca; Northfleet de Albuquerque, Cristina; Maria Araújo Domingues Zucchi, Tânia; Poli, Paola

2009-01-01

Nitroheterocyclic compounds are widely used as therapeutic agents against a variety of protozoan and bacterial infections. However, the literature on these compounds, suspected of being carcinogens, is widely controversial. In this study, cytotoxic and genotoxic potential of three drugs, Nifurtimox (NFX), Benznidazole (BNZ), and Metronidazole (MTZ) was re-evaluated by different assays. Only NFX reduces survival rate in actively proliferating cells. The compounds are more active for base-pair substitution than frameshift induction in Salmonella; NFX and BNZ are more mutagenic than MTZ; they are widely dependent from nitroreduction whereas microsomal fraction S9 weakly affects the mutagenic potential. Comet assay detects BNZ- and NFX-induced DNA damage at doses in the range of therapeutically treated patient plasma concentration; BNZ seems to mainly act through ROS generation whereas a dose-dependent mechanism of DNA damaging is suggested for NFX. The lack of effects on mammalian cells for MTZ is confirmed also in MN assay whereas MN induction is observed for NFX and BNZ. The effects of MTZ, that shows comparatively low reduction potential, seem to be strictly dependent on anaerobic/hypoxic conditions. Both NFX and BNZ may not only lead to cellular damage of the infective agent but also interact with the DNA of mammalian cells.

Human Immunodeficiency Virus Type 1 cDNA Integration: New Aromatic Hydroxylated Inhibitors and Studies of the Inhibition Mechanism

PubMed Central

Farnet, C. M.; Wang, B.; Hansen, M.; Lipford, J. R.; Zalkow, L.; Robinson, W. E.; Siegel, J.; Bushman, F.

1998-01-01

Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA is a required step for viral replication. Integrase, the virus-encoded enzyme important for integration, has not yet been exploited as a target for clinically useful inhibitors. Here we report on the identification of new polyhydroxylated aromatic inhibitors of integrase including ellagic acid, purpurogallin, 4,8,12-trioxatricornan, and hypericin, the last of which is known to inhibit viral replication. These compounds and others were characterized in assays with subviral preintegration complexes (PICs) isolated from HIV-1-infected cells. Hypericin was found to inhibit PIC assays, while the other compounds tested were inactive. Counterscreening of these and other integrase inhibitors against additional DNA-modifying enzymes revealed that none of the polyhydroxylated aromatic compounds are active against enzymes that do not require metals (methylases, a pox virus topoisomerase). However, all were cross-reactive with metal-requiring enzymes (restriction enzymes, a reverse transcriptase), implicating metal atoms in the inhibitory mechanism. In mechanistic studies, we localized binding of some inhibitors to the catalytic domain of integrase by assaying competition of binding by labeled nucleotides. These findings help elucidate the mechanism of action of the polyhydroxylated aromatic inhibitors and provide practical guidance for further inhibitor development. PMID:9736543

Identification of Bisindolylmaleimide IX as a potential agent to treat drug-resistant BCR-ABL positive leukemia

PubMed Central

Liu, Huijuan; Zang, Yi; Azam, Mohammad; Habib, Samy L.; Li, Jia; Ruan, Xinsen; Jia, Hao; Wang, Xueying; Li, Baojie

2016-01-01

Chronic myeloid leukemia (CML) treatment with BCR-ABL inhibitors is often hampered by development of drug resistance. In a screen for novel chemotherapeutic drug candidates with genotoxic activity, we identified a bisindolylmaleimide derivative, IX, as a small molecule compound with therapeutic potential against CML including drug-resistant CML. We show that Bisindolylmaleimide IX inhibits DNA topoisomerase, generates DNA breaks, activates the Atm-p53 and Atm-Chk2 pathways, and induces cell cycle arrest and cell death. Interestingly, Bisindolylmaleimide IX is highly effective in targeting cells positive for BCR-ABL. BCR-ABL positive cells display enhanced DNA damage and increased cell cycle arrest in response to Bisindolylmaleimide IX due to decreased expression of topoisomerases. Cells positive for BCR-ABL or drug-resistant T315I BCR-ABL also display increased cytotoxicity since Bisindolylmaleimide IX inhibits B-Raf and the downstream oncogene addiction pathway. Mouse cancer model experiments showed that Bisindolylmaleimide IX, at doses that show little side effect, was effective in treating leukemia-like disorders induced by BCR-ABL or T315I BCR-ABL, and prolonged the lifespan of these model mice. Thus, Bisindolylmaleimide IX presents a novel drug candidate to treat drug-resistant CML via activating BCR-ABL-dependent genotoxic stress response and inhibiting the oncogene addiction pathway activated by BCR-ABL. PMID:27564101

Microbial Toluene Removal in Hypoxic Model Constructed Wetlands Occurs Predominantly via the Ring Monooxygenation Pathway.

PubMed

Martínez-Lavanchy, P M; Chen, Z; Lünsmann, V; Marin-Cevada, V; Vilchez-Vargas, R; Pieper, D H; Reiche, N; Kappelmeyer, U; Imparato, V; Junca, H; Nijenhuis, I; Müller, J A; Kuschk, P; Heipieper, H J

2015-09-01

In the present study, microbial toluene degradation in controlled constructed wetland model systems, planted fixed-bed reactors (PFRs), was queried with DNA-based methods in combination with stable isotope fractionation analysis and characterization of toluene-degrading microbial isolates. Two PFR replicates were operated with toluene as the sole external carbon and electron source for 2 years. The bulk redox conditions in these systems were hypoxic to anoxic. The autochthonous bacterial communities, as analyzed by Illumina sequencing of 16S rRNA gene amplicons, were mainly comprised of the families Xanthomonadaceae, Comamonadaceae, and Burkholderiaceae, plus Rhodospirillaceae in one of the PFR replicates. DNA microarray analyses of the catabolic potentials for aromatic compound degradation suggested the presence of the ring monooxygenation pathway in both systems, as well as the anaerobic toluene pathway in the PFR replicate with a high abundance of Rhodospirillaceae. The presence of catabolic genes encoding the ring monooxygenation pathway was verified by quantitative PCR analysis, utilizing the obtained toluene-degrading isolates as references. Stable isotope fractionation analysis showed low-level of carbon fractionation and only minimal hydrogen fractionation in both PFRs, which matches the fractionation signatures of monooxygenation and dioxygenation. In combination with the results of the DNA-based analyses, this suggests that toluene degradation occurs predominantly via ring monooxygenation in the PFRs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Radiolabeled Rhein as Small-Molecule Necrosis Avid Agents for Imaging of Necrotic Myocardium.

PubMed

Luo, Qi; Jin, Qiaomei; Su, Chang; Zhang, Dongjian; Jiang, Cuihua; Fish, Anne Folta; Feng, Yuanbo; Ni, Yicheng; Zhang, Jian; Yin, Zhiqi

2017-01-17

A rapid and accurate identification of necrotic myocardium is of great importance for diagnosis, risk stratification, clinical decision-making, and prognosis evaluation of myocardial infarction. Here, we explored technetium-99m labeled rhein derivatives for rapid imaging of the necrotic myocardium. Three hydrazinonicotinic acid-linker-rhein (HYNIC-linker-rhein) derivatives were synthesized, and then, these synthetic compounds were labeled with technetium-99m using ethylenediaminediacetic acid (EDDA) and tricine as coligands [ 99m Tc(EDDA)-HYNIC-linker-rhein]. The necrosis avidity of the three 99m Tc-labeled rhein derivatives was tested in a mouse model of ethanol-induced muscular necrosis by gamma counting, histochemical staining, and autoradiography. A lead tracer for visualization of necrotic myocardium was assessed by single photon emission computed tomography/computed tomography (SPECT/CT) imaging in a rat model with reperfused myocardial infarction. The necrosis avidity mechanism of the tracer was explored by DNA binding studies in vitro and blocking experiments in vivo. Results showed that the uptake in necrotic muscles of the three 99m Tc-compounds was higher than that in viable muscles (P < 0.001). Autoradiography and histochemical staining results were consistent with selective uptake of the radiotracer in the necrotic regions. Among the these tracers, 99m Tc(EDDA)-HYNIC-ethylenediamine-rhein [ 99m Tc(EDDA)-HYNIC-2C-rhein] displayed the best distribution profiles for imaging. The necrotic myocardium lesions were clearly visualized by SPECT/CT using 99m Tc(EDDA)-HYNIC-2C-rhein at 1 h after injection. The necrotic-to-viable myocardium and necrotic myocardium-to-blood uptake ratios of 99m Tc(EDDA)-HYNIC-2C-rhein were 4.79 and 3.02 at 1 h after injection. DNA binding studies suggested HYNIC-linker-rhein bound to DNA through intercalation. The uptake of 99m Tc(EDDA)-HYNIC-2C-rhein in necrotic muscle was significantly blocked by excessive unlabeled rhein, with 77.61% decline at 1 h after coinjection. These findings suggested 99m Tc(EDDA)-HYNIC-2C-rhein emerged as a "hot spot" imaging probe that has a potential for rapid imaging of necrotic myocardium. The necrosis avidity mechanism of 99m Tc(EDDA)-HYNIC-linker-rhein may be due to its interaction with exposed DNA in necrotic tissues.

The DNA-PK Inhibitor VX-984 Enhances the Radiosensitivity of Glioblastoma Cells Grown In Vitro and as Orthotopic Xenografts.

PubMed

Timme, Cindy R; Rath, Barbara H; O'Neill, John W; Camphausen, Kevin; Tofilon, Philip J

2018-06-01

Radiotherapy is a primary treatment modality for glioblastomas (GBM). Because DNA-PKcs is a critical factor in the repair of radiation-induced double strand breaks (DSB), this study evaluated the potential of VX-984, a new DNA-PKcs inhibitor, to enhance the radiosensitivity of GBM cells. Treatment of the established GBM cell line U251 and the GBM stem-like cell (GSC) line NSC11 with VX-984 under in vitro conditions resulted in a concentration-dependent inhibition of radiation-induced DNA-PKcs phosphorylation. In a similar concentration-dependent manner, VX-984 treatment enhanced the radiosensitivity of each GBM cell line as defined by clonogenic analysis. As determined by γH2AX expression and neutral comet analyses, VX-984 inhibited the repair of radiation-induced DNA double-strand break in U251 and NSC11 GBM cells, suggesting that the VX-984-induced radiosensitization is mediated by an inhibition of DNA repair. Extending these results to an in vivo model, treatment of mice with VX-984 inhibited radiation-induced DNA-PKcs phosphorylation in orthotopic brain tumor xenografts, indicating that this compound crosses the blood-brain tumor barrier at sufficient concentrations. For mice bearing U251 or NSC11 brain tumors, VX-984 treatment alone had no significant effect on overall survival; radiation alone increased survival. The survival of mice receiving the combination protocol was significantly increased as compared with control and as compared with radiation alone. These results indicate that VX-984 enhances the radiosensitivity of brain tumor xenografts and suggest that it may be of benefit in the therapeutic management of GBM. Mol Cancer Ther; 17(6); 1207-16. ©2018 AACR . ©2018 American Association for Cancer Research.

Induction of antibodies to nuclear antigens in rabbits by immunization with hydralazine-human serum albumin conjugates.

PubMed Central

Yamauchi, Y; Litwin, A; Adams, L; Zimmer, H; Hess, E V

1975-01-01

The antihypertensive drug hydralazine can induce in man a syndrome similar to spontaneous systemic lupus erythematosus (SLE). The pathogenesis of this drug-induced syndrome is not understood. In this investigation, five groups of rabbits were studied: group I, 10 rabbits hyperimmunized with hydralazine conjugated to human serum albumin (HSA) in complete Freund's adjuvant (CFA); group II, four rabbits with HSA in CFA; group III, four rabbits with CFA alone; group IV, five rabbits with hydralazine conjugated to rabbit serum albumin (RSA); and group V, four rabbits with a major metabolite of hydralazine conjugated to HSA. The rabbits immunized with hydralazine-HSA developed rising titers of antibodies to hydralazine and progressively increasing amounts of antibodies to both single-stranded and native DNA. The antibodies to DNA were cross-reactive with hydralazine as determined by inhibition of DNA binding and DNA hemagglutination tests. Similar results were obtained in rabbits immunized with the metabolite-HSA compound except the major hapten antibody response was to the metabolite. The DNA antibodies in this group were also capable of being absorbed by metabolite-HSA as well as hydralazine-HSA, indicative of the cross-reactivity between hydralazine and its metabolite. Immunization with hydralazine-RSA caused rabbits to produce antibodies to hydralazine but not to DNA, indicating the requirement for an immune response to the carrier protein in order for antibodies reactive with DNA to be produced. Thus, hyperimmunization of rabbits with hydralazine-protein conjugates may provide a useful animal model of SLE. The data suggests that an immune response to hydralazine may be important in human hydralazine-induced SLE. Images PMID:808562

Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients.

PubMed

Lueders, Tillmann; Manefield, Mike; Friedrich, Michael W

2004-01-01

Stable isotope probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of 'light' and 'heavy' nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems.

T-705 (favipiravir) and related compounds: Novel broad-spectrum inhibitors of RNA viral infections.

PubMed

Furuta, Yousuke; Takahashi, Kazumi; Shiraki, Kimiyasu; Sakamoto, Kenichi; Smee, Donald F; Barnard, Dale L; Gowen, Brian B; Julander, Justin G; Morrey, John D

2009-06-01

A series of pyrazinecarboxamide derivatives T-705 (favipiravir), T-1105 and T-1106 were discovered to be candidate antiviral drugs. These compounds have demonstrated good activity in treating viral infections in laboratory animals caused by various RNA viruses, including influenza virus, arenaviruses, bunyaviruses, West Nile virus (WNV), yellow fever virus (YFV), and foot-and-mouth disease virus (FMDV). Treatment has in some cases been effective when initiated up to 5-7 days after virus infection, when the animals already showed signs of illness. Studies on the mechanism of action of T-705 have shown that this compound is converted to the ribofuranosyltriphosphate derivative by host enzymes, and this metabolite selectively inhibits the influenza viral RNA-dependent RNA polymerase without cytotoxicity to mammalian cells. Interestingly, these compounds do not inhibit host DNA and RNA synthesis and inosine 5'-monophosphate dehydrogenase (IMPDH) activity. From in vivo studies using several animal models, the pyrazinecarboxamide derivatives were found to be effective in protecting animals from death, reducing viral burden, and limiting disease manifestations, even when treatment was initiated after virus inoculation. Importantly, T-705 imparts its beneficial antiviral effects without significant toxicity to the host. Prompt development of these compounds is expected to provide effective countermeasures against pandemic influenza virus and several bioweapon threats, all of which are of great global public health concern given the current paucity of highly effective broad-spectrum drugs.

Repair of DNA-polypeptide crosslinks by human excision nuclease

NASA Astrophysics Data System (ADS)

Reardon, Joyce T.; Sancar, Aziz

2006-03-01

DNA-protein crosslinks are relatively common DNA lesions that form during the physiological processing of DNA by replication and recombination proteins, by side reactions of base excision repair enzymes, and by cellular exposure to bifunctional DNA-damaging agents such as platinum compounds. The mechanism by which pathological DNA-protein crosslinks are repaired in humans is not known. In this study, we investigated the mechanism of recognition and repair of protein-DNA and oligopeptide-DNA crosslinks by the human excision nuclease. Under our assay conditions, the human nucleotide excision repair system did not remove a 16-kDa protein crosslinked to DNA at a detectable level. However, 4- and 12-aa-long oligopeptides crosslinked to the DNA backbone were recognized by some of the damage recognition factors of the human excision nuclease with moderate selectivity and were excised from DNA at relatively efficient rates. Our data suggest that, if coupled with proteolytic degradation of the crosslinked protein, the human excision nuclease may be the major enzyme system for eliminating protein-DNA crosslinks from the genome. damage recognition | nucleotide excision repair

The effects of estrogen receptors α- and β-specific agonists and antagonists on cell proliferation and energy metabolism in human bone cell line.

PubMed

Somjen, D; Katzburg, S; Sharon, O; Grafi-Cohen, M; Knoll, E; Stern, N

2011-02-01

In cultured human osteoblasts estradiol-17β (E2) modulated DNA synthesis, the specific activity of creatine kinase BB (CK), 12 and 15 lipoxygenase (LO) mRNA expression and formation of 12- and 15-hydroxyeicosatetraenoic acid (HETE). We now investigate the response of human bone cell line (SaOS2) to phytoestrogens and estrogen receptors (ER)-specific agonists and antagonists. Treatment of SaSO2 with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ-specific agonist), 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα-specific agonist), biochainin A (BA), daidzein (D), genistein (G) and raloxifene (Ral) showed increased DNA synthesis and CK. Ral inhibited completely all stimulations except DPN and to some extent D. The ERα-specific antagonist methyl-piperidino-pyrazole (MPP) and the ERβ-specific antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a]pyrimidin-3-yl] phenol (PTHPP) inhibited DNA synthesis, CK and reactive oxygen species (ROS) formation induced by estrogens according to their receptors affinity. The LO inhibitor baicaleine inhibited only E2, DPN and G's effects. E2 and Ral unlike all other compounds had no effect on ERα mRNA expression, while ERβ mRNA expression was stimulated by all compounds. All compounds modulated the expression of 12LO and 15LO mRNA, except E2, PPT and Ral for 12LO, and 12- and 15-HETE productions and stimulated ROS formation which was inhibited by NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and N-acetyl cysteine and the estrogen inhibitor ICI. DPI did not affect hormonal-induced DNA and CK. In conclusion, we provide evidence for the separation of mediation via ERα and ERβ pathways in the effects of estrogenic compounds on osteoblasts, but the role of LO/HETE/ROS is unclear. Copyright © 2010 Wiley-Liss, Inc.

Dose-Response Assessment of Four Genotoxic Chemicals in a Combined Mouse and Rat Micronucleus and Comet Assay Protocol

PubMed Central

Recio, Leslie; Hobbs, Cheryl; Caspary, William; Witt, Kristine L.

2012-01-01

The in vivo micronucleus (MN) assay has proven to be an effective measure of genotoxicity potential. However, sampling a single tissue (bone marrow) for a single indicator of genetic damage using the MN assay provides a limited genotoxicity profile. The in vivo alkaline (pH>13) Comet assay, which detects a broad spectrum of DNA damage, can be applied to a variety of rodent tissues following administration of test agents. To determine if the Comet assay is a useful supplement to the in vivo MN assay, a combined test protocol (MN/Comet assay) was conducted in male B6C3F1 mice and F344/N rats using four model genotoxicants: ethyl methanesulfonate (EMS), acrylamide (ACM), cyclophosphamide (CP), and vincristine sulfate (VS). Test compounds were administered on 4 consecutive days at 24-hour intervals (VS was administered to rats for 3 days); animals were euthanized 4 hours after the last administration. All compounds induced significant increases in micronucleated reticulocytes (MN-RET) in the peripheral blood of mice, and all but ACM induced MN-RET in rats. EMS and ACM induced significant increases in DNA damage, measured by the Comet assay, in multiple tissues of mice and rats. CP-induced DNA damage was detected in leukocytes and duodenum cells. VS, a spindle fiber disrupting agent, was negative in the Comet assay. Based on these results, the MN/Comet assay holds promise for providing more comprehensive assessments of potential genotoxicants, and the National Toxicology Program is presently using this combined protocol in its overall evaluation of the genotoxicity of substances of public health concern. PMID:20371966

Bisphenol A Exposure Disrupts Genomic Imprinting in the Mouse

PubMed Central

Susiarjo, Martha; Sasson, Isaac; Mesaros, Clementina; Bartolomei, Marisa S.

2013-01-01

Exposure to endocrine disruptors is associated with developmental defects. One compound of concern, to which humans are widely exposed, is bisphenol A (BPA). In model organisms, BPA exposure is linked to metabolic disorders, infertility, cancer, and behavior anomalies. Recently, BPA exposure has been linked to DNA methylation changes, indicating that epigenetic mechanisms may be relevant. We investigated effects of exposure on genomic imprinting in the mouse as imprinted genes are regulated by differential DNA methylation and aberrant imprinting disrupts fetal, placental, and postnatal development. Through allele-specific and quantitative real-time PCR analysis, we demonstrated that maternal BPA exposure during late stages of oocyte development and early stages of embryonic development significantly disrupted imprinted gene expression in embryonic day (E) 9.5 and 12.5 embryos and placentas. The affected genes included Snrpn, Ube3a, Igf2, Kcnq1ot1, Cdkn1c, and Ascl2; mutations and aberrant regulation of these genes are associated with imprinting disorders in humans. Furthermore, the majority of affected genes were expressed abnormally in the placenta. DNA methylation studies showed that BPA exposure significantly altered the methylation levels of differentially methylated regions (DMRs) including the Snrpn imprinting control region (ICR) and Igf2 DMR1. Moreover, exposure significantly reduced genome-wide methylation levels in the placenta, but not the embryo. Histological and immunohistochemical examinations revealed that these epigenetic defects were associated with abnormal placental development. In contrast to this early exposure paradigm, exposure outside of the epigenetic reprogramming window did not cause significant imprinting perturbations. Our data suggest that early exposure to common environmental compounds has the potential to disrupt fetal and postnatal health through epigenetic changes in the embryo and abnormal development of the placenta. PMID:23593014

DNA Binding, Cleavage and Antibacterial Activity of Mononuclear Cu(II), Ni(II) and Co(II) Complexes Derived from Novel Benzothiazole Schiff Bases.

PubMed

Vamsikrishna, Narendrula; Kumar, Marri Pradeep; Tejaswi, Somapangu; Rambabu, Aveli; Shivaraj

2016-07-01

A series of novel bivalent metal complexes M(L1)2 and M(L2)2 where M = Cu(II), Ni(II), Co(II) and L1 = 2-((benzo [d] thiazol-6-ylimino)methyl)-4-bromophenol [BTEMBP], L2 = 1-((benzo [d] thiazol-6-ylimino)methyl) naphthalen-2-ol [BTEMNAPP] were synthesized. All the compounds have been characterized by elemental analysis, SEM, Mass, (1)H NMR, (13)C NMR, UV-Vis, IR, ESR, spectral data and magnetic susceptibility measurements. Based on the analytical and spectral data four-coordinated square planar geometry is assigned to all the complexes. DNA binding properties of these complexes have been investigated by electronic absorption spectroscopy, fluorescence and viscosity measurements. It is observed that these binary complexes strongly bind to calf thymus DNA by an intercalation mode. DNA cleavage efficacy of these complexes was tested in presence of H2O2 and UV light by gel electrophoresis and found that all the complexes showed better nuclease activity. Finally the compounds were screened for antibacterial activity against few pathogens and found that the complexes have potent biocidal activity than their free ligands.

CpG methylation increases the DNA binding of 9-aminoacridine carboxamide Pt analogues.

PubMed

Kava, Hieronimus W; Murray, Vincent

2016-10-01

This study investigated the effect of CpG methylation on the DNA binding of cisplatin analogues with an attached aminoacridine intercalator. DNA-targeted 9-aminoacridine carboxamide Pt complexes are known to bind at 5'-CpG sequences. Their binding to methylated and non-methylated 5'-CpG sequences was determined and compared with cisplatin. The damage profiles of each platinum compound were quantified via a polymerase stop assay with fluorescently labelled primers and capillary electrophoresis. Methylation at 5'-CpG was shown to significantly increase the binding intensity for the 9-aminoacridine carboxamide compounds, whereas no significant increase was found for cisplatin. 5'-CpG methylation had the largest effect on the 9-ethanolamine-acridine carboxamide Pt complex, followed by the 9-aminoacridine carboxamide Pt complex and the 7-fluoro complex. The methylation state of a cell's genome is important in maintaining normal gene expression, and is often aberrantly altered in cancer cells. An analogue of cisplatin which differentially targets methylated DNA may be able to improve its therapeutic activity, or alter its range of targets and evade the chemoresistance which hampers cisplatin efficacy in clinical use. Copyright © 2016 Elsevier Ltd. All rights reserved.

Therapeutic nucleic acids: current clinical status

PubMed Central

Sridharan, Kannan

2016-01-01

Abstract Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are simple linear polymers that have been the subject of considerable research in the last two decades and have now moved into the realm of being stand‐alone therapeutic agents. Much of this has stemmed from the appreciation that they carry out myriad functions that go beyond mere storage of genetic information and protein synthesis. Therapy with nucleic acids either uses unmodified DNA or RNA or closely related compounds. From both a development and regulatory perspective, they fall somewhere between small molecules and biologics. Several of these compounds are in clinical development and many have received regulatory approval for human use. This review addresses therapeutic uses of DNA based on antisense oligonucleotides, DNA aptamers and gene therapy; and therapeutic uses of RNA including micro RNAs, short interfering RNAs, ribozymes, RNA decoys and circular RNAs. With their specificity, functional diversity and limited toxicity, therapeutic nucleic acids hold enormous promise. However, challenges that need to be addressed include targeted delivery, mass production at low cost, sustaining efficacy and minimizing off‐target toxicity. Technological developments will hold the key to this and help accelerate drug approvals in the years to come. PMID:27111518

Epigenetic mechanisms in anti-cancer actions of bioactive food components – the implications in cancer prevention

PubMed Central

Stefanska, B; Karlic, H; Varga, F; Fabianowska-Majewska, K; Haslberger, AG

2012-01-01

The hallmarks of carcinogenesis are aberrations in gene expression and protein function caused by both genetic and epigenetic modifications. Epigenetics refers to the changes in gene expression programming that alter the phenotype in the absence of a change in DNA sequence. Epigenetic modifications, which include amongst others DNA methylation, covalent modifications of histone tails and regulation by non-coding RNAs, play a significant role in normal development and genome stability. The changes are dynamic and serve as an adaptation mechanism to a wide variety of environmental and social factors including diet. A number of studies have provided evidence that some natural bioactive compounds found in food and herbs can modulate gene expression by targeting different elements of the epigenetic machinery. Nutrients that are components of one-carbon metabolism, such as folate, riboflavin, pyridoxine, cobalamin, choline, betaine and methionine, affect DNA methylation by regulating the levels of S-adenosyl-L-methionine, a methyl group donor, and S-adenosyl-L-homocysteine, which is an inhibitor of enzymes catalyzing the DNA methylation reaction. Other natural compounds target histone modifications and levels of non-coding RNAs such as vitamin D, which recruits histone acetylases, or resveratrol, which activates the deacetylase sirtuin and regulates oncogenic and tumour suppressor micro-RNAs. As epigenetic abnormalities have been shown to be both causative and contributing factors in different health conditions including cancer, natural compounds that are direct or indirect regulators of the epigenome constitute an excellent approach in cancer prevention and potentially in anti-cancer therapy. PMID:22536923

Synthesis and structure-activity relationship of amidine derivatives of 3,4-ethylenedioxythiophene as novel antibacterial agents.

PubMed

Stolić, Ivana; Čipčić Paljetak, Hana; Perić, Mihaela; Matijašić, Mario; Stepanić, Višnja; Verbanac, Donatella; Bajić, Miroslav

2015-01-27

Current antibacterial chemotherapeutics are facing an alarming increase in bacterial resistance pressuring the search for novel agents that would expand the available therapeutic arsenal against resistant bacterial pathogens. In line with these efforts, a series of 9 amidine derivatives of 3,4-ethylenedioxythiophene were synthesized and, together with 18 previously synthesized analogs, evaluated for their relative DNA binding affinity, in vitro antibacterial activities and preliminary in vitro safety profile. Encouraging antibacterial activity of several subclasses of tested amidine derivatives against Gram-positive (including resistant MRSA, MRSE, VRE strains) and Gram-negative bacterial strains was observed. The bis-phenyl derivatives were the most antibacterially active, while compound 19 from bis-benzimidazole class exhibited the widest spectrum of activity (with MIC of 4, 2, 0.5 and ≤0.25 μg/ml against laboratory strains of Staphyloccocus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Moraxella catarrhalis, respectively and 4-32 μg/ml against clinical isolates of sensitive and resistant S. aureus, Staphylococcus epidermidis and Enterococcus faecium) and also demonstrated the strongest DNA binding affinity (ΔTm of 15.4 °C). Asymmetrically designed compounds and carboxamide-amidines were, in general, less active. Molecular docking indicated that the shape of the 3,4-ethylenedioxythiophene derivatives and their ability to form multiple electrostatic and hydrogen bonds with DNA, corresponds to the binding modes of other minor-groove binders. Herein reported results encourage further investigation of this class of compounds as novel antibacterial DNA binding agents. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

DNA damage evaluation of hydroxyapatite on fibroblast cell L929 using the single cell gel electrophoresis assay.

PubMed

Rajab, N F; Yaakob, T A; Ong, B Y; Hamid, M; Ali, A M; Annuar, B O; Inayat-Hussain, S H

2004-05-01

Hydroxyapatite is the main component of the bone which is a potential biomaterial substance that can be applied in orthopaedics. In this study, the biocompatibility of this biomaterial was assessed using an in vitro technique. The cytotoxicity and genotoxicity effect of HA2 and HA3 against L929 fibroblast cell was evaluated using the MTT Assay and Alkaline Comet Assay respectively. Both HA2 and HA3 compound showed low cytotoxicity effect as determined using MTT Assay. Cells viability following 72 hours incubation at maximum concentration of both HA2 and HA3 (200 mg/ml) were 75.3 +/- 8.8% and 86.7 +/- 13.1% respectively. However, the cytotoxicity effect of ZnSO4.7H2O as a positive control showed an IC50 values of 46 mg/ml (160 microM). On the other hand, both HA2 and HA3 compound showed a slight genotoxicity effect as determined using the Alkaline Comet Assay following incubation at the concentration 200 mg/ml for 72 hours. This assay has been widely used in genetic toxicology to detect DNA strand breaks and alkali-labile site. The percentage of the cells with DNA damage for both substance was 27.7 +/- 1.3% and 15.6 +/- 1.0% for HA2 and HA3 respectively. Incubation of the cells for 24 hours with 38 microg/ml (IC25) of positive control showed an increase in percentage of cells with DNA damage (67.5 +/- 0.7%). In conclusion, our study indicated that both hydroxyapatite compounds showed a good biocompatibility in fibroblast cells.

Antioxidative Dietary Compounds Modulate Gene Expression Associated with Apoptosis, DNA Repair, Inhibition of Cell Proliferation and Migration

PubMed Central

Wang, Likui; Gao, Shijuan; Jiang, Wei; Luo, Cheng; Xu, Maonian; Bohlin, Lars; Rosendahl, Markus; Huang, Wenlin

2014-01-01

Many dietary compounds are known to have health benefits owing to their antioxidative and anti-inflammatory properties. To determine the molecular mechanism of these food-derived compounds, we analyzed their effect on various genes related to cell apoptosis, DNA damage and repair, oxidation and inflammation using in vitro cell culture assays. This review further tests the hypothesis proposed previously that downstream products of COX-2 (cyclooxygenase-2) called electrophilic oxo-derivatives induce antioxidant responsive elements (ARE), which leads to cell proliferation under antioxidative conditions. Our findings support this hypothesis and show that cell proliferation was inhibited when COX-2 was down-regulated by polyphenols and polysaccharides. Flattened macrophage morphology was also observed following the induction of cytokine production by polysaccharides extracted from viili, a traditional Nordic fermented dairy product. Coix lacryma-jobi (coix) polysaccharides were found to reduce mitochondrial membrane potential and induce caspase-3- and 9-mediated apoptosis. In contrast, polyphenols from blueberries were involved in the ultraviolet-activated p53/Gadd45/MDM2 DNA repair system by restoring the cell membrane potential. Inhibition of hypoxia-inducible factor-1 by saponin extracts of ginsenoside (Ginsen) and Gynostemma and inhibition of S100A4 by coix polysaccharides inhibited cancer cell migration and invasion. These observations suggest that antioxidants and changes in cell membrane potential are the major driving forces that transfer signals through the cell membrane into the cytosol and nucleus, triggering gene expression, changes in cell proliferation and the induction of apoptosis or DNA repair. PMID:25226533

Biofilm prevention by dicephalic cationic surfactants and their interactions with DNA.

PubMed

Piecuch, A; Lamch, Ł; Paluch, E; Obłąk, E; Wilk, K A

2016-09-01

The studies were aimed to contribute to the elucidation of the relationships between structure of the double-headed cationic surfactants-N,N-bis[3,3'-(dimethylamine)- propyl]alkylamide dihydrochlorides and N,N-bis[3,3'-(trimethylammonio)propyl]alkylamide dibromides (alkyl: n-C9 H19 , n-C11 H23 , n-C13 H27 , n-C15 H31 ) and their antibacterial and biofilm preventing activity. The minimal inhibitory and bactericidal concentrations (MIC and MBC) of dicephalic surfactants against Staphylococcus epidermidis and Pseudomonas aeruginosa were tested using standard methods. Pseudomonas aeruginosa was resistant to studied compounds but MBC values against Staph. epidermidis reached 0·48-0·01 mmol l(-1) . The influence of dicephalic surfactants on bacterial biofilm and adhesion to the various surfaces was investigated with crystal violet staining or colony counting. The reduction in bacterial adhesion was observed, especially in the case of glass and stainless steel. The condensation of the DNA was shown in the ethidium bromide intercalation assay. Dicephalic surfactants exhibited antibacterial activity against Staph. epidermidis. The activity of studied compounds depended on the hydrocarbon chain length and the counterion. Surfactants deposited on different materials reduced Staph. epidermidis adhesion, dependently on the surfactant structure and the substratum. Dicephalic surfactants showed the ability of DNA compaction. This study points the possibility of application of dicephalic surfactants as the surface-coating agents to prevent biofilm formation. These compounds efficiently condensed DNA and are potential candidates for further studies towards the transfection. © 2016 The Society for Applied Microbiology.

Exploring the DNA binding/cleavage, cellular accumulation and topoisomerase inhibition of 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinone Mannich bases and their platinum(II) complexes.

PubMed

Neves, Amanda P; Pereira, Michelle X G; Peterson, Erica J; Kipping, Ralph; Vargas, Maria D; Silva, Floriano P; Carneiro, J Walkimar M; Farrell, Nicholas P

2013-02-01

Several chlorido and amino Pt(2+) complexes of 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinone Mannich bases HL exhibiting moderate to high cytotoxicity against cancer cell lines were studied in order to investigate their modes of DNA binding, in vitro DNA strand breaks, mechanism of topoisomerase (Topo I) inhibition and cellular accumulation. DNA model base studies have shown that complex 1a [Pt(HL1)Cl(2)] was capable of binding covalently to 9-ethylguanine (9-EtG) and 5'-GMP. (1)H NMR and mass spectrometry studies have shown that both chlorides were substituted by 9-EtG ligands, whereas 5'-GMP was able to replace only one chlorido ligand, due to steric hindrance. The chlorido Pt(2+) complexes [Pt(HL)Cl(2)] highly accumulate in prostate (PC-3) and melanoma (MDA-MB-435) cell lines, being able to induce DNA strand breaks in vitro and inhibit Topo I by a catalytic mode. On the other hand, the free 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinones HL and the amino Pt(2+) complexes [Pt(L(-))(NH(3))(2)]NO(3) neither cause DNA strand breakage nor exhibit strong DNA interaction, nevertheless the latter were also found to be catalytic inhibitors of Topo I at 100μM. Thus, coordination of the Mannich bases HL to the "PtCl(2)" fragment substantially affects the chemical and biophysical properties of the pro-ligands, leading to an improvement of their DNA binding properties and generating compounds that cleave DNA and catalytically inhibit Topo I. Finally, the high cytotoxicity exhibited by the free (uncomplexed) 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinones might be associated with their decomposition in solution, which is not observed for the Pt(2+) complexes. Copyright © 2012 Elsevier Inc. All rights reserved.

Photo-triggered recognition between host and guest compounds in a giant vesicle encapsulating photo-pierceable vesicles.

PubMed

Suzuki, Kentaro; Machida, Kotaro; Yamaguchi, Kazuo; Sugawara, Tadashi

2018-01-01

Here, we used centrifugal precipitation to construct a giant vesicle (GV) encapsulating smaller giant vesicles (GV-in-GV) which comprises a photo-resistant outer GV and a photo-pierceable inner GV; the outer GV contained a fluorescent probe (SYBR Green I) in its inner water pool, and the inner GV contained double-stranded DNA (dsDNA) in its inner water pool. The phospholipid membrane of the inner GV was made photo-pierceable by inclusion of ca. 15mol% of a caged phospholipid in its membrane. Immediately after exposure of the GV-in-GVs to UV irradiation, strong fluorescence was detected in the inner water pool of the outer GV, indicating that dsDNA had been released from the inner GV and had complexed with the fluorescent probe. These dynamics can be recognized as a macroscopic representation of the molecular level function of a caged compound. Copyright © 2017 Elsevier B.V. All rights reserved.

Laboratory and clinical studies of cancer chemoprevention by antioxidants in berries.

PubMed

Stoner, Gary David; Wang, Li-Shu; Casto, Bruce Cordell

2008-09-01

Reactive oxygen species (ROS) are a major cause of cellular injury in an increasing number of diseases, including cancer. Most ROS are created in the cell through normal cellular metabolism. They can be produced by environmental insults such as ultraviolet light and toxic chemicals, as well as by the inflammatory process. Interception of ROS or limiting their cellular effects is a major role of antioxidants. Due to their content of phenolic and flavonoid compounds, berries exhibit high antioxidant potential, exceeding that of many other foodstuffs. Through their ability to scavenge ROS and reduce oxidative DNA damage, stimulate antioxidant enzymes, inhibit carcinogen-induced DNA adduct formation and enhance DNA repair, berry compounds have been shown to inhibit mutagenesis and cancer initiation. Berry constituents also influence cellular processes associated with cancer progression including signaling pathways associated with cell proliferation, differentiation, apoptosis and angiogenesis. This review article summarizes laboratory and human studies, demonstrating the protective effects of berries and berry constituents on oxidative and other cellular processes leading to cancer development.

Laboratory and clinical studies of cancer chemoprevention by antioxidants in berries

PubMed Central

Stoner, Gary David; Wang, Li-Shu; Casto, Bruce Cordell

2008-01-01

Reactive oxygen species (ROS) are a major cause of cellular injury in an increasing number of diseases, including cancer. Most ROS are created in the cell through normal cellular metabolism. They can be produced by environmental insults such as ultraviolet light and toxic chemicals, as well as by the inflammatory process. Interception of ROS or limiting their cellular effects is a major role of antioxidants. Due to their content of phenolic and flavonoid compounds, berries exhibit high antioxidant potential, exceeding that of many other foodstuffs. Through their ability to scavenge ROS and reduce oxidative DNA damage, stimulate antioxidant enzymes, inhibit carcinogen-induced DNA adduct formation and enhance DNA repair, berry compounds have been shown to inhibit mutagenesis and cancer initiation. Berry constituents also influence cellular processes associated with cancer progression including signaling pathways associated with cell proliferation, differentiation, apoptosis and angiogenesis. This review article summarizes laboratory and human studies, demonstrating the protective effects of berries and berry constituents on oxidative and other cellular processes leading to cancer development. PMID:18544560

Focus on Fotemustine.

PubMed

De Rossi, A; Rossi, L; Laudisi, A; Sini, V; Toppo, L; Marchesi, F; Tortorelli, G; Leti, M; Turriziani, M; Aquino, A; Bonmassar, E; De Vecchis, L; Torino, F

2006-12-01

Fotemustine is a cytotoxic alkylating agent, belonging to the group of nitrosourea family. Its mechanism of action is similar to that of other nitrosoureas, characterized by a mono-functional/bi-functional alkylating activity. Worth of consideration is the finding that the presence of high levels of the DNA repair enzyme O6-methylguanine-DNA-methyltransferase (MGMT) in cancer cells confers drug resistance. In different clinical trials Fotemustine showed a remarkable antitumor activity as single agent, and in association with other antineoplastic compounds or treatment modalities. Moreover, its toxicity is generally considered acceptable. The drug has been employed in the treatment of metastatic melanoma, and, on the basis of its pharmacokinetic properties, in brain tumors, either primitive or metastatic. Moreover, Fotemustine shows pharmacodynamic properties similar to those of mono-functional alkylating compounds (e.g. DNA methylating drugs, such as Temozolomide), that have been recently considered for the management of acute refractory leukaemia. Therefore, it is reasonable to assume that this agent could be a good candidate to play a potential role in haematological malignancies.

Cerium chloride stimulated controlled conversion of B-to-Z DNA in self-assembled nanostructures.

PubMed

Bhanjadeo, Madhabi M; Nayak, Ashok K; Subudhi, Umakanta

2017-01-22

DNA adopts different conformation not only because of novel base pairs but also while interacting with inorganic or organic compounds. Self-assembled branched DNA (bDNA) structures or DNA origami that change conformation in response to environmental cues hold great promises in sensing and actuation at the nanoscale. Recently, the B-Z transition in DNA is being explored to design various nanomechanical devices. In this communication we have demonstrated that Cerium chloride binds to the phosphate backbone of self-assembled bDNA structure and induce B-to-Z transition at physiological concentration. The mechanism of controlled conversion from right-handed to left-handed has been assayed by various dye binding studies using CD and fluorescence spectroscopy. Three different bDNA structures have been identified to display B-Z transition. This approach provides a rapid and reversible means to change bDNA conformation, which can be used for dynamic and progressive control at the nanoscale. Copyright © 2016 Elsevier Inc. All rights reserved.

Inhibition of herpes simplex virus DNA polymerase by purine ribonucleoside monophosphates.

PubMed

Frank, K B; Cheng, Y C

1986-02-05

Purine ribonucleoside monophosphates were found to inhibit chain elongation catalyzed by herpes simplex virus (HSV) DNA polymerase when DNA template-primer concentrations were rate-limiting. Inhibition was fully competitive with DNA template-primer during chain elongation; however, DNA polymerase-associated exonuclease activity was inhibited noncompetitively with respect to DNA. Combinations of 5'-GMP and phosphonoformate were kinetically mutually exclusive in dual inhibitor studies. Pyrimidine nucleoside monophosphates and deoxynucleoside monophosphates were less inhibitory than purine riboside monophosphates. The monophosphates of 9-beta-D-arabinofuranosyladenine, Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), 9-(2-hydroxyethoxymethyl)guanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine exerted little or no inhibition. In contrast to HSV DNA polymerase, human DNA polymerase alpha was not inhibited by purine ribonucleoside monophosphates. These studies suggest the possibility of a physiological role of purine ribonucleoside monophosphates as regulators of herpesvirus DNA synthesis and a new approach to developing selective anti-herpesvirus compounds.

The prebiotic synthesis of oligonucleotides

NASA Technical Reports Server (NTRS)

Oro, J.; Stephen-Sherwood, E.

1974-01-01

This paper is primarily a review of recent developments in the abiotic synthesis of nucleotides, short chain oligonucleotides, and their mode of replication in solution. It also presents preliminary results from this laboratory on the prebiotic synthesis of thymidine oligodeoxynucleotides. A discussion, based on the physicochemical properties of RNA and DNA oligomers, relevant to the molecular evolution of these compounds leads to the tentative hypothesis that oligodeoxyribonucleotides of about 12 units may have been of sufficient length to initiate a self replicating coding system. Two models are suggested to account for the synthesis of high molecular weight oligomers using short chain templates and primers.

One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

PubMed Central

Nitsche, Andreas; Kurth, Andreas; Dunkhorst, Anna; Pänke, Oliver; Sielaff, Hendrik; Junge, Wolfgang; Muth, Doreen; Scheller, Frieder; Stöcklein, Walter; Dahmen, Claudia; Pauli, Georg; Kage, Andreas

2007-01-01

Background As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method. PMID:17697378

DNA Damage Potential of Engine Emissions Measured In Vitro by Micronucleus Test in Human Bronchial Epithelial Cells.

PubMed

Cervena, Tereza; Rossnerova, Andrea; Sikorova, Jitka; Beranek, Vit; Vojtisek-Lom, Michal; Ciganek, Miroslav; Topinka, Jan; Rossner, Pavel

2017-09-01

Internal combustion engine emissions belong among the major anthropogenic sources of air pollution in urban areas. According to the International Agency for Research on Cancer, there is sufficient evidence of the carcinogenicity of diesel exhaust in human beings. Although alternative fuels, mainly biodiesel, have recently become popular, little is still known about the genotoxicity of emissions from these fuels. We analysed DNA damage expressed as the frequency of micronuclei (MN) in human bronchial epithelial cells (BEAS-2B), induced by extractable organic matter (EOM; tested concentrations: 1, 10 and 25 μg/ml) obtained from particle emissions from various blends of biodiesel with diesel fuels (including neat diesel fuel (B0), a blend of 70% B0 and 30% biodiesel (B30) and neat biodiesel (B100)). We also tested the effect of selected diesel exhaust organic/genotoxic components [benzo[a]pyrene (B[a]P) concentrations: 25, 100 and 200 μM; 1-nitropyrene (1-NP) concentrations: 1, 5 and 10 μM; 3-nitrobenzanthrone (3-NBA) concentrations: 1, 5 and 50 μM]. The cells were treated with the compounds for 28 and 48 hr. Our results showed that most of the tested compounds (except for the 25 μM B[a]P, 28-hr treatment) significantly increased MN frequency. The genotoxicity of EOMs from the engine emissions of diesel and biodiesel engines was comparable. Both nitro-PAH compounds demonstrated higher genotoxic potential in comparison with B[a]P. Considering our results and due to increasing popularity of alternative fuels, it is prudent that the potential genotoxic effects of various fuels are investigated across engine technologies and operating conditions in a relevant model system. © 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

Molecular docking and inhibition studies on the interactions of Bacopa monnieri's potent phytochemicals against pathogenic Staphylococcus aureus.

PubMed

Emran, Talha Bin; Rahman, Md Atiar; Uddin, Mir Muhammad Nasir; Dash, Raju; Hossen, Md Firoz; Mohiuddin, Mohammad; Alam, Md Rashadul

2015-04-17

Bacopa monnieri Linn. (Plantaginaceae), a well-known medicinal plant, is widely used in traditional medicine system. It has long been used in gastrointestinal discomfort, skin diseases, epilepsy and analgesia. This research investigated the in vitro antimicrobial activity of Bacopa monnieri leaf extract against Staphylococcus aureus and the interaction of possible compounds involved in this antimicrobial action. Non-edible plant parts were extracted with ethanol and evaporated in vacuo to obtain the crude extract. A zone of inhibition studies and the minimum inhibitory concentration (MIC) of plant extracts were evaluated against clinical isolates by the microbroth dilution method. Docking study was performed to analyze and identify the interactions of possible antimicrobial compounds of Bacopa monnieri in the active site of penicillin binding protein and DNA gyrase through GOLD 4.12 software. A zone of inhibition studies showed significant (p < 0.05) inhibition capacity of different concentrations of Bacopa monnieri's extract against Staphylococcus aureus. The extract also displayed very remarkable minimum inhibitory concentrations (≥16 μg/ml) which was significant compared to that (≥75 μg/ml) of the reference antibiotic against the experimental strain Staphylococcus aureus. Docking studies recommended that luteolin, an existing phytochemical of Bacopa monnieri, has the highest fitness score and more specificity towards the DNA gyrase binding site rather than penicillin binding protein. Bacopa monnieri extract and its compound luteolin have a significant antimicrobial activity against Staphylococcus aureus. Molecular binding interaction of an in silico data demonstrated that luteolin has more specificity towards the DNA gyrase binding site and could be a potent antimicrobial compound.

Soil organic phosphorus flows to water via critical and non-critical hydrological source areas

NASA Astrophysics Data System (ADS)

Wang, Ying; Surridge, Ben; Haygarth, Phil

2015-04-01

Soil organic phosphorus flows to water via critical and non-critical hydrological source areas Ying Wang, Ben W.J. Surridge, Philip M. Haygarth Lancaster Environment Centre, Lancaster University, LA1 4YQ, UK Critical source areas (CSAs) are zones in the landscape where easily connected hydrology coincides with a phosphorus (P) sources in the soil. The P export risks in CSAs are hypothesised to be higher compared with non-critical source areas (Non-CSAs) and specifically that the magnitudes of P forms in CSA areas were higher than Non-CSAs. Past research on CSAs has often neglected forms of organic P, such as DNA and phospholipids which are among the most potentially biodegradable organic P compounds. The objectives of this study were i) to quantify the magnitude of organic P compounds in agricultural soils and specifically determine whether these magnitudes differed significantly between CSAs and Non-CSAs; ii) determine the variation of P magnitude between and within individual fields; iii) identify the P delivery concentrations in soil solution after raining events in CSAs. The study focussed on soils collected from the Morland sub-catchment of the River Eden catchment in Cumbria, northern England. CSA and Non-CSA pairs were identified using the SCIMAP modelling and field assessment providing five CSA - Non-CSA pairs in total. The results showed that there are significant differences in the total P (TP) concentrations, the proportions of DNA-P, WETP (water extractable total P), WERP (water extractable reactive P) and WEUP (water extractable unreactive P) between CSA and Non-CSA. We also found that the concentrations of all the P forms showed distribution variation between fields or even within the same field. Liable organic P such as DNA-P and PLD-P was presented considerable proportions of total P in soil, especially DNA-P which had a good correlation with TP. DNA-P in the ten areas accounted for a considerable proportion of soil TP (4.9 to 16.6%). Given the potential lability and bioavailability of DNA and phospholipids, our data demonstrate that these soil organic P could be a potential pool to support plant nutrition and a potential contributor to water pollution problems. Furthermore, For a number of soil samples, comparisons were made between extraction of P within phospholipids and DNA and the results of 31 PNMR analyses, to provide further characterisation of the organic P fractions within these soils.Data of this study will also be presented concerning the forms and magnitudes of P fractions in runoff pathways across an agricultural catchment, including the prevalence of organic P in these pathways. Keywords: Organic P; Soil; CSA; DNA; Phospholipids

Genotoxicity testing of two lead-compounds in somatic cells of Drosophila melanogaster.

PubMed

Carmona, Erico R; Creus, Amadeu; Marcos, Ricard

2011-09-18

The in vivo genotoxic activity of two inorganic lead compounds was studied in Drosophila melanogaster by measurement of two different genetic endpoints. We used the wing-spot test and the comet assay. The comet assay was conducted with larval haemocytes. The results from the wing-spot test showed that neither lead chloride, PbCl(2), nor lead nitrate, Pb(NO(3))(2), were able to induce significant increases in the frequency of mutant spots. In addition, the combined treatments with gamma-radiation and PbCl(2) or Pb(NO(3))(2) did not show significant variations in the frequency of the three categories of mutant spots recorded, compared with the frequency induced by gamma-radiation alone. This seems to indicate that the lead compounds tested do not interact with the repair of the genetic damage induced by ionizing radiation. When the lead compounds were evaluated in the in vivo comet assay with haemocytes, Pb(NO(3))(2) was effective in inducing significant increases of DNA damage with a direct dose-response pattern. These results confirm the usefulness of the comet assay with haemocytes as an in vivo model and support the assumption that there is a genotoxic risk associated with lead exposure. Copyright © 2011 Elsevier B.V. All rights reserved.

Bio-important antipyrine derived Schiff bases and their transition metal complexes: Synthesis, spectroscopic characterization, antimicrobial, anthelmintic and DNA cleavage investigation

NASA Astrophysics Data System (ADS)

Manjunath, M.; Kulkarni, Ajaykumar D.; Bagihalli, Gangadhar B.; Malladi, Shridhar; Patil, Sangamesh A.

2017-01-01

Spectroscopic (IR, NMR, UV-vis, ESR, ESI-mass), magnetic and TGA studies suggests octahedral geometry for all the CoII, NiII and CuII complexes of the Schiff bases, derived from 4-aminoantipyrine and 8-formyl-7-Hydroxy-4-methylcoumarin/5-formyl-6-hydroxycoumarin, coordinated through ONO donor sites. Antibacterial (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi), antifungal (Aspergillus niger, Aspergillus flavus and Cladosporium) and DNA cleavage properties of the metal complexes are investigated. The results suggested that some of the synthesized compounds are potential antimicrobials. The synthesized compounds tested for their anthelmintic activities and it was found that CoII and NiII complexes exhibited good anthelmintic properties.

Development of programmable small DNA-binding molecules with epigenetic activity for induction of core pluripotency genes.

PubMed

Pandian, Ganesh N; Ohtsuki, Akimichi; Bando, Toshikazu; Sato, Shinsuke; Hashiya, Kaori; Sugiyama, Hiroshi

2012-04-15

Epigenetic modifications that govern the gene expression are often overlooked with the design of artificial genetic switches. N-Methylpyrrole-N-methylimidazole (PI) hairpin polyamides are programmable small DNA binding molecules that have been studied in the context of gene regulation. Recently, we synthesized a library of compounds by conjugating PI polyamides with SAHA, a chromatin-modifier. Among these novel compounds, PI polyamide-SAHA conjugate 1 was shown to epigenetically activate pluripotency genes in mouse embryonic fibroblasts. Here, we report the synthesis of the derivatives of conjugate 1 and demonstrate that these epigenetically active molecules could be developed to improve the induction of pluripotency factors. Copyright © 2012 Elsevier Ltd. All rights reserved.

Chemical Space of DNA-Encoded Libraries.

PubMed

Franzini, Raphael M; Randolph, Cassie

2016-07-28

In recent years, DNA-encoded chemical libraries (DECLs) have attracted considerable attention as a potential discovery tool in drug development. Screening encoded libraries may offer advantages over conventional hit discovery approaches and has the potential to complement such methods in pharmaceutical research. As a result of the increased application of encoded libraries in drug discovery, a growing number of hit compounds are emerging in scientific literature. In this review we evaluate reported encoded library-derived structures and identify general trends of these compounds in relation to library design parameters. We in particular emphasize the combinatorial nature of these libraries. Generally, the reported molecules demonstrate the ability of this technology to afford hits suitable for further lead development, and on the basis of them, we derive guidelines for DECL design.

Isolation and expression of three gibberellin 20-oxidase cDNA clones from Arabidopsis.

PubMed

Phillips, A L; Ward, D A; Uknes, S; Appleford, N E; Lange, T; Huttly, A K; Gaskin, P; Graebe, J E; Hedden, P

1995-07-01

Using degenerate oligonucleotide primers based on a pumpkin (Cucurbita maxima) gibberellin (GA) 20-oxidase sequence, six different fragments of dioxygenase genes were amplified by polymerase chain reaction from arabidopsis thaliana genomic DNA. One of these was used to isolate two different full-length cDNA clones, At2301 and At2353, from shoots of the GA-deficient Arabidopsis mutant ga1-2. A third, related clone, YAP169, was identified in the Database of Expressed Sequence Tags. The cDNA clones were expressed in Escherichia coli as fusion proteins, each of which oxidized GA12 at C-20 to GA15, GA24, and the C19 compound GA9, a precursor of bioactive GAs; the C20 tricarboxylic acid compound GA25 was formed as a minor product. The expression products also oxidized the 13-hydroxylated substrate GA53, but less effectively than GA12. The three cDNAs hybridized to mRNA species with tissue-specific patterns of accumulation, with At2301 being expressed in stems and inflorescences, At2353 in inflorescences and developing siliques, and YAP169 in siliques only. In the floral shoots of the ga1-2 mutant, transcript levels corresponding to each cDNA decreased dramatically after GA3 application, suggesting that GA biosynthesis may be controlled, at least in part, through down-regulation of the expression of the 20-oxidase genes.

Metal based biologically active compounds: Design, synthesis, DNA binding and antidiabetic activity of 6-methyl-3-formyl chromone derived hydrazones and their metal (II) complexes.

PubMed

Philip, Jessica Elizabeth; Shahid, Muhammad; Prathapachandra Kurup, M R; Velayudhan, Mohanan Puzhavoorparambil

2017-10-01

Two chromone hydrazone ligands HL 1 and HL 2 were synthesized and characterized by elemental analyses, IR, 1 H NMR & 13 C NMR, electronic absorption and mass spectra. The reactions of the chromone hydrazones with transition metals such as Ni, Cu, and Zn (II) salts of acetate afforded mononuclear metal complexes. Characterization and structure elucidation of the prepared chromone hydrazone metal (II) complexes were done by elemental, IR, electronic, EPR spectra and thermo gravimetric analyses as well as conductivity and magnetic susceptibility measurements. The spectroscopic data showed that the ligand acts as a mono basic bidentate with coordination sites are azomethine nitrogen and hydrazonic oxygen, and they exhibited distorted geometry. The biological studies involved antidiabetic activity i.e. enzyme inhibition of α-amylase and α-glucosidase, Calf Thymus - DNA (CT-DNA) interaction and molecular docking. Potential capacity of synthesized compounds to inhibit the α-amylase and α-glucosidase activity was assayed whereas DNA interaction studies were carried out with the help UV-Vis absorption titration and viscosity method. The docking studies of chromone hydrazones show that they are minor groove binders. Complexes were found to be good DNA - intercalates. Chromone hydrazones and its transition metal complexes have shown comparable antidiabetic activity with a standard drug acarbose. Copyright © 2017 Elsevier B.V. All rights reserved.

[Design, synthesis and biological evaluation of novel para-substituted 1-benzyl-quinazoline-2, 4 (1H, 3H)-diones as human PARP-1 inhibitors].

PubMed

Yao, Hai-Ping; Zhu, Zhi-Xiang; Ji, Ming; Chen, Xiao-Guang; Xu, Bai-Ling

2014-04-01

Poly(ADP-ribose) polymerase-1 (PARP-1) has emerged as a promising anticancer drug target due to its key role in the DNA repair process. It can polymerize ADP-ribose units on its substrate proteins which are involved in the regulation of DNA repair. In this work, a novel series of para-substituted 1-benzyl-quinazoline-2, 4 (1H, 3H)-diones was designed and synthesized, and the inhibitory activities against PARP-1 of compounds 7a-7e, 8a-8f, 9a-9c and 10a-10c were evaluated. Of all the tested compounds, nine compounds displayed inhibitory activities with IC50 values ranging from 4.6 to 39.2 micromol x L(-1). In order to predict the binding modes of the potent molecules, molecular docking was performed using CDOCKER algorithm, and that will facilitate to further develop more potent PARP-1 inhibitors with a quinazolinedione scaffold.

Small-Molecule Inhibitors of the SOX18 Transcription Factor.

PubMed

Fontaine, Frank; Overman, Jeroen; Moustaqil, Mehdi; Mamidyala, Sreeman; Salim, Angela; Narasimhan, Kamesh; Prokoph, Nina; Robertson, Avril A B; Lua, Linda; Alexandrov, Kirill; Koopman, Peter; Capon, Robert J; Sierecki, Emma; Gambin, Yann; Jauch, Ralf; Cooper, Matthew A; Zuegg, Johannes; Francois, Mathias

2017-03-16

Pharmacological modulation of transcription factors (TFs) has only met little success over the past four decades. This is mostly due to standard drug discovery approaches centered on blocking protein/DNA binding or interfering with post-translational modifications. Recent advances in the field of TF biology have revealed a central role of protein-protein interaction in their mode of action. In an attempt to modulate the activity of SOX18 TF, a known regulator of vascular growth in development and disease, we screened a marine extract library for potential small-molecule inhibitors. We identified two compounds, which inspired a series of synthetic SOX18 inhibitors, able to interfere with the SOX18 HMG DNA-binding domain, and to disrupt HMG-dependent protein-protein interaction with RBPJ. These compounds also perturbed SOX18 transcriptional activity in a cell-based reporter gene system. This approach may prove useful in developing a new class of anti-angiogenic compounds based on the inhibition of TF activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

CC-1065 functional analogues possessing different electron-withdrawing substituents and leaving groups: synthesis, kinetics, and sequence specificity of reaction with DNA and biological evaluation.

PubMed

Wang, Y; Gupta, R; Huang, L; Lown, J W

1993-12-24

Antitumor agent CC-1065 functional analogues possessing different electron-withdrawing substituents and leaving groups have been synthesized. The extent and the relative rates of DNA cleavage following alkylation by these CPI structures and thermal treatment were determined independently by an ethidium binding assay and by agarose gel electrophoresis experiments. The anticipated preferential covalent binding to adenine sites within the minor groove was confirmed by sequencing determination of selected agents on high-resolution gels. Certain of the synthetic agents, unlike CC-1065, also bind covalently to G sites with weaker intensity. The cytotoxicities of these compounds were also determined against KB cells in vitro. Compounds bearing a bromo or nitro group in the benzene ring and a methylsulfonyl as a leaving group are 10 and 5 times more potent than their unsubstituted counterparts, respectively. Compounds bearing a methylsulfonyl as a leaving group are more potent than those bearing a chlorine.

Evaluation of the efficacy of radiation-modifying compounds using γH2AX as a molecular marker of DNA double-strand breaks.

PubMed

Mah, Li-Jeen; Orlowski, Christian; Ververis, Katherine; Vasireddy, Raja S; El-Osta, Assam; Karagiannis, Tom C

2011-01-25

Radiation therapy is a widely used therapeutic approach for cancer. To improve the efficacy of radiotherapy there is an intense interest in combining this modality with two broad classes of compounds, radiosensitizers and radioprotectors. These either enhance tumour-killing efficacy or mitigate damage to surrounding non-malignant tissue, respectively. Radiation exposure often results in the formation of DNA double-strand breaks, which are marked by the induction of H2AX phosphorylation to generate γH2AX. In addition to its essential role in DDR signalling and coordination of double-strand break repair, the ability to visualize and quantitate γH2AX foci using immunofluorescence microscopy techniques enables it to be exploited as an indicator of therapeutic efficacy in a range of cell types and tissues. This review will explore the emerging applicability of γH2AX as a marker for monitoring the effectiveness of radiation-modifying compounds.

Evaluation of the efficacy of radiation-modifying compounds using γH2AX as a molecular marker of DNA double-strand breaks

PubMed Central

2011-01-01

Radiation therapy is a widely used therapeutic approach for cancer. To improve the efficacy of radiotherapy there is an intense interest in combining this modality with two broad classes of compounds, radiosensitizers and radioprotectors. These either enhance tumour-killing efficacy or mitigate damage to surrounding non-malignant tissue, respectively. Radiation exposure often results in the formation of DNA double-strand breaks, which are marked by the induction of H2AX phosphorylation to generate γH2AX. In addition to its essential role in DDR signalling and coordination of double-strand break repair, the ability to visualize and quantitate γH2AX foci using immunofluorescence microscopy techniques enables it to be exploited as an indicator of therapeutic efficacy in a range of cell types and tissues. This review will explore the emerging applicability of γH2AX as a marker for monitoring the effectiveness of radiation-modifying compounds. PMID:21261999

Spermatogenesis is not impaired in a nucleotide excision repair-deficient min mouse model with or without neonatal mutagen treatment.

PubMed

Andreassen, Ashild; Steffensen, Inger-Lise; Olsen, Ann-Karin; Tanaka, Kiyoji; Wiger, Richard

2011-01-01

Mice deficient in the xeroderma pigmentosum group A gene (Xpa) exhibit impaired nucleotide excision repair (NER) and are expected to accumulate bulky DNA adducts when subjected to certain compounds (eg, heterocyclic amines). Multiple intestinal neoplasia (Min) mice (B6(Min)(/+)) are particularly sensitive to low concentrations of mutagenic compounds in food. They develop intestinal tumors spontaneously, and the number and size of the tumors increase following exposure to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which humans are exposed to via fried food. We previously reported that NER is inefficient in adult testicular cells. Reduced NER (genetic deficiency; Xpa(-/-)) is expected to represent risk factors for PhIP-induced genotoxicity and could possibly disturb spermatogenesis, particularly in B6(Min)(/+) mice. We therefore studied spermatogenesis in mice with combinations of Xpa and Min or wild-type genotypes 11 weeks after exposure to PhIP on days 3 to 6. Fewer offspring were obtained from B6(Min)(/+)Xpa(-/-) than from B6(Min)(/+)Xpa(+/+) or B6(Min)(/+)Xpa(+/-). Distributions of the different testicular cell types, indicative of normal spermatogenesis and relative testes weights, did not differ significantly in PhIP-exposed or unexposed mice regardless of their genotypes. We conclude that the removal of bulky DNA adducts does not seem to be essential for normal spermatogenesis.

Metformin improves defective hematopoiesis and delays tumor formation in Fanconi anemia mice.

PubMed

Zhang, Qing-Shuo; Tang, Weiliang; Deater, Matthew; Phan, Ngoc; Marcogliese, Andrea N; Li, Hui; Al-Dhalimy, Muhsen; Major, Angela; Olson, Susan; Monnat, Raymond J; Grompe, Markus

2016-12-15

Fanconi anemia (FA) is an inherited bone marrow failure disorder associated with a high incidence of leukemia and solid tumors. Bone marrow transplantation is currently the only curative therapy for the hematopoietic complications of this disorder. However, long-term morbidity and mortality remain very high, and new therapeutics are badly needed. Here we show that the widely used diabetes drug metformin improves hematopoiesis and delays tumor formation in Fancd2 -/- mice. Metformin is the first compound reported to improve both of these FA phenotypes. Importantly, the beneficial effects are specific to FA mice and are not seen in the wild-type controls. In this preclinical model of FA, metformin outperformed the current standard of care, oxymetholone, by improving peripheral blood counts in Fancd2 -/- mice significantly faster. Metformin increased the size of the hematopoietic stem cell compartment and enhanced quiescence in hematopoietic stem and progenitor cells. In tumor-prone Fancd2 -/- Trp53 +/- mice, metformin delayed the onset of tumors and significantly extended the tumor-free survival time. In addition, we found that metformin and the structurally related compound aminoguanidine reduced DNA damage and ameliorated spontaneous chromosome breakage and radials in human FA patient-derived cells. Our results also indicate that aldehyde detoxification might be one of the mechanisms by which metformin reduces DNA damage in FA cells. © 2016 by The American Society of Hematology.