Characterization of ribonuclease III from Brucella.

Science.gov (United States)

Wu, Chang-Xian; Xu, Xian-Jin; Zheng, Ke; Liu, Fang; Yang, Xu-Dong; Chen, Chuang-Fu; Chen, Huan-Chun; Liu, Zheng-Fei

2016-04-01

Bacterial ribonuclease III (RNase III) is a highly conserved endonuclease, which plays pivotal roles in RNA maturation and decay pathways by cleaving double-stranded structure of RNAs. Here we cloned rncS gene from the genomic DNA of Brucella melitensis, and analyzed the cleavage properties of RNase III from Brucella. We identified Brucella-encoding small RNA (sRNA) by high-throughput sequencing and northern blot, and found that sRNA of Brucella and Homo miRNA precursor (pre-miRNA) can be bound and cleaved by B.melitensis ribonuclease III (Bm-RNase III). Cleavage activity of Bm-RNase III is bivalent metal cations- and alkaline buffer-dependent. We constructed several point mutations in Bm-RNase III, whose cleavage activity indicated that the 133th Glutamic acid residue was required for catalytic activity. Western blot revealed that Bm-RNase III was differently expressed in Brucella virulence strain 027 and vaccine strain M5-90. Collectively, our data suggest that Brucella RNase III can efficiently bind and cleave stem-loop structure of small RNA, and might participate in regulation of virulence in Brucella. Copyright © 2016 Elsevier B.V. All rights reserved.

A Phosphate Starvation-Inducible Ribonuclease of Bacillus licheniformis.

Science.gov (United States)

Nguyen, Thanh Trung; Nguyen, Minh Hung; Nguyen, Huy Thuan; Nguyen, Hoang Anh; Le, Thi Hoi; Schweder, Thomas; Jürgen, Britta

2016-08-28

The BLi03719 protein of Bacillus licheniformis DSM13 belongs to the most abundant extracellular proteins under phosphate starvation conditions. In this study, the function of this phosphate starvation inducible protein was determined. An amino-acid sequence analysis of the BLi03719-encoding gene showed a high similarity with genes encoding the barnase of Bacillus amyloliquefaciens FZB42 and binase-like RNase of Bacillus pumilus SARF-032. The comparison of the control strain and a BLi03719-deficient strain revealed a strongly reduced extracellular ribonuclease activity of the mutant. Furthermore, this knockout mutant exhibited delayed growth with yeast RNA as an alternative phosphate and carbon source. These results suggest that BLi03719 is an extracellular ribonuclease expressed in B. licheniformis under phosphate starvation conditions. Finally, a BLi03719 mutant showed an advantageous effect on the overexpression of the heterologous amyE gene under phosphate-limited growth conditions.

A Hydrogen Exchange Method Using Tritium and Sephadex: Its Application to Ribonuclease*

Science.gov (United States)

Englander, S. Walter

2012-01-01

A new method for measuring the hydrogen exchange of macromolecules in solution is described. The method uses tritium to trace the movement of hydrogen, and utilizes Sephadex columns to effect, in about 2 minutes, a separation between tritiated macromolecule and tritiated solvent great enough to allow the measurement of bound tritium. High sensitivity and freedom from artifact is demonstrated and the possible value of the technique for investigation of other kinds of colloid-small molecule interaction is indicated. Competition experiments involving tritium, hydrogen, and deuterium indicate the absence of any equilibrium isotope effect in the ribonuclease-hydrogen isotope system, though a secondary kinetic isotope effect is apparent when ribonuclease is largely deuterated. Ribonuclease shows four clearly distinguishable kinetic classes of exchangeable hydrogens. Evidence is marshaled to suggest the independently measurable classes II, III, and IV (in order of decreasing rate of exchange) to represent “random-chain” peptides, peptides involved in α-helix, and otherwise shielded side-chain and peptide hydrogens, respectively. PMID:14075117

Structure-Activity Analysis of Vinylogous Urea Inhibitors of Human Immunodeficiency Virus-Encoded Ribonuclease H ▿

OpenAIRE

Chung, Suhman; Wendeler, Michaela; Rausch, Jason W.; Beilhartz, Greg; Gotte, Matthias; O'Keefe, Barry R.; Bermingham, Alun; Beutler, John A.; Liu, Shixin; Zhuang, Xiaowei; Le Grice, Stuart F. J.

2010-01-01

Vinylogous ureas 2-amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide and N-[3-(aminocarbonyl)-4,5-dimethyl-2-thienyl]-2-furancarboxamide (compounds 1 and 2, respectively) were recently identified to be modestly potent inhibitors of the RNase H activity of HIV-1 and HIV-2 reverse transcriptase (RT). Both compounds shared a 3-CONH2-substituted thiophene ring but were otherwise structurally unrelated, which prevented a precise definition of the pharmacophore. We have therefore exa...

Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork.

Science.gov (United States)

Parajuli, Shankar; Teasley, Daniel C; Murali, Bhavna; Jackson, Jessica; Vindigni, Alessandro; Stewart, Sheila A

2017-09-15

Faithful DNA replication is essential for genome stability. To ensure accurate replication, numerous complex and redundant replication and repair mechanisms function in tandem with the core replication proteins to ensure DNA replication continues even when replication challenges are present that could impede progression of the replication fork. A unique topological challenge to the replication machinery is posed by RNA-DNA hybrids, commonly referred to as R-loops. Although R-loops play important roles in gene expression and recombination at immunoglobulin sites, their persistence is thought to interfere with DNA replication by slowing or impeding replication fork progression. Therefore, it is of interest to identify DNA-associated enzymes that help resolve replication-impeding R-loops. Here, using DNA fiber analysis, we demonstrate that human ribonuclease H1 (RNH1) plays an important role in replication fork movement in the mammalian nucleus by resolving R-loops. We found that RNH1 depletion results in accumulation of RNA-DNA hybrids, slowing of replication forks, and increased DNA damage. Our data uncovered a role for RNH1 in global DNA replication in the mammalian nucleus. Because accumulation of RNA-DNA hybrids is linked to various human cancers and neurodegenerative disorders, our study raises the possibility that replication fork progression might be impeded, adding to increased genomic instability and contributing to disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Interplay between intergrin-linked kinase and ribonuclease inhibitor affects growth and metastasis of bladder cancer through signaling ILK pathways.

Science.gov (United States)

Zhuang, Xiang; Lv, Mengxin; Zhong, Zhenyu; Zhang, Luyu; Jiang, Rong; Chen, Junxia

2016-08-30

Integrin-linked kinase (ILK) is a multifunctional adaptor protein which is involved with protein signalling within cells to modulate malignant (cancer) cell movement, cell cycle, metastasis and epithelial-mesenchymal transition (EMT). Our previous experiment demonstrated that ILK siRNA inhibited the growth and induced apoptosis of bladder cancer cells as well as increased the expression of Ribonuclease inhibitor (RI), an important cytoplasmic protein with many functions. We also reported that RI overexpression inhibited ILK and phosphorylation of AKT and GSK3β. ILK and RI gene both locate on chromosome 11p15 and the two genes are always at the adjacent position of same chromosome during evolution, which suggest that ILK and RI could have some relationship. However, underlying interacting mechanisms remain unclear between them. Here, we postulate that RI might regulate ILK signaling pathway via interacting with ILK. Co-immunoprecipitation, GST pull-down and co-localization under laser confocal microscope assay were used to determine the interaction between ILK and RI exogenously and endogenously. Furthermore, we further verified that there is a direct binding between the two proteins by fluorescence resonance energy transfer (FRET) in cells. Next, The effects of interplay between ILK and RI on the key target protein expressions of PI3K/AKT/mTOR signaling pathway were determined by western blot, immunohistochemistry and immunofluorescence assay in vivo and in vitro. Finally, the interaction was assessed using nude mice xenograft model. We first found that ILK could combine with RI both in vivo and in vitro by GST pull-down, co-immunoprecipitation (Co-IP) and FRET. The protein levels of ILK and RI revealed a significant inverse correlation in vivo and in vitro. Subsequently, The results showed that up-regulating ILK could increase cell proliferation, change cell morphology and regulate cell cycle. We also demonstrated that the overexpression of ILK remarkably

Plasma autoantibodies against heat shock protein 70, enolase 1 and ribonuclease/angiogenin inhibitor 1 as potential biomarkers for cholangiocarcinoma.

Directory of Open Access Journals (Sweden)

Rucksak Rucksaken

Full Text Available The diagnosis of cholangiocarcinoma (CCA is often challenging, leading to poor prognosis. CCA arises via chronic inflammation which may be associated with autoantibodies production. This study aims to identify IgG antibodies directed at self-proteins and tumor-associated antigens. Proteins derived from immortalized cholangiocyte cell line (MMNK1 and CCA cell lines (M055, M214 and M139 were separated using 2-dimensional electrophoresis and incubated with pooled plasma of patients with CCA and non-neoplastic controls by immunoblotting. Twenty five immunoreactive spots against all cell lines-derived proteins were observed on stained gels and studied by LC-MS/MS. Among these, heat shock protein 70 (HSP70, enolase 1 (ENO1 and ribonuclease/angiogenin inhibitor 1 (RNH1 obtained the highest matching scores and were thus selected for further validation. Western blot revealed immunoreactivity against HSP70 and RNH1 in the majority of CCA cases and weakly in healthy individuals. Further, ELISA showed that plasma HSP70 autoantibody level in CCA was significantly capable to discriminate CCA from healthy individuals with an area under the receiver operating characteristic curve of 0.9158 (cut-off 0.2630, 93.55% sensitivity and 73.91% specificity. Plasma levels of IgG autoantibodies against HSP70 were correlated with progression from healthy individuals to cholangitis to CCA (r = 0.679, P<0.001. In addition, circulating ENO1 and RNH1 autoantibodies levels were also significantly higher in cholangitis and CCA compared to healthy controls (P<0.05. Moreover, the combinations of HSP70, ENO1 or RNH1 autoantibodies positivity rates improved specificity to over 78%. In conclusion, plasma IgG autoantibodies against HSP70, ENO1 and RNH1 may represent new diagnostic markers for CCA.

THE AMINO-ACID-SEQUENCE OF IGUANA (IGUANA-IGUANA) PANCREATIC RIBONUCLEASE

NARCIS (Netherlands)

ZHAO, W; BEINTEMA, JJ; HOFSTEENGE, J

1994-01-01

The pyrimidine-specific ribonuclease superfamily constitutes a group of homologous proteins so far found only in higher vertebrates. Four separate families are found in mammals, which have resulted from gene duplications in mammalian ancestors. To learn more about the evolutionary history of this

Mitochondrial tRNA cleavage by tRNA-targeting ribonuclease causes mitochondrial dysfunction observed in mitochondrial disease

Energy Technology Data Exchange (ETDEWEB)

Ogawa, Tetsuhiro, E-mail: atetsu@mail.ecc.u-tokyo.ac.jp; Shimizu, Ayano; Takahashi, Kazutoshi; Hidaka, Makoto; Masaki, Haruhiko, E-mail: amasaki@mail.ecc.u-tokyo.ac.jp

2014-08-15

Highlights: • MTS-tagged ribonuclease was translocated successfully to the mitochondrial matrix. • MTS-tagged ribonuclease cleaved mt tRNA and reduced COX activity. • Easy and reproducible method of inducing mt tRNA dysfunction. - Abstract: Mitochondrial DNA (mtDNA) is a genome possessed by mitochondria. Since reactive oxygen species (ROS) are generated during aerobic respiration in mitochondria, mtDNA is commonly exposed to the risk of DNA damage. Mitochondrial disease is caused by mitochondrial dysfunction, and mutations or deletions on mitochondrial tRNA (mt tRNA) genes are often observed in mtDNA of patients with the disease. Hence, the correlation between mt tRNA activity and mitochondrial dysfunction has been assessed. Then, cybrid cells, which are constructed by the fusion of an enucleated cell harboring altered mtDNA with a ρ{sup 0} cell, have long been used for the analysis due to difficulty in mtDNA manipulation. Here, we propose a new method that involves mt tRNA cleavage by a bacterial tRNA-specific ribonuclease. The ribonuclease tagged with a mitochondrial-targeting sequence (MTS) was successfully translocated to the mitochondrial matrix. Additionally, mt tRNA cleavage, which resulted in the decrease of cytochrome c oxidase (COX) activity, was observed.

Secondary structure of prokaryotic 5S ribosomal ribonucleic acids: a study with ribonucleases

DEFF Research Database (Denmark)

Douthwaite, S; Garrett, R A

1981-01-01

The structures of 5S ribosomal RNAs from Escherichia coli and Bacillus stearothermophilus were examined by using ribonucleases A, T1, and T2 and a double helix specific cobra venom ribonuclease. By using both 5' and 3'-32P-end labeling methods and selecting for digested but intact 5S RNA molecules...... evidence for three of the helical regions of the Fox and Woese model of 5S RNA [Fox, G. E., & Woese, C. (1975) Nature (London) 256, 505] and support other important structural features which include a nucleotide looped out from a helical region which has been proposed as a recognition site for protein L18....

Insulin and the PI3K/AKT Signaling Pathway Regulate Ribonuclease 7 Expression in the Human Urinary Tract

Science.gov (United States)

Eichler, Tad; Becknell, Brian; Easterling, Robert S.; Ingraham, Susan E.; Cohen, Daniel M.; Schwaderer, Andrew; Hains, David S.; Li, Birong; Cohen, Ariel; Metheny, Jackie; Trindandapani, Susheela; Spencer, John David

2017-01-01

Diabetes mellitus is a systemic disease associated with a deficiency of insulin production or action. Diabetic patients have an increased susceptibility to infection with the urinary tract being the most common site of infection. Recent studies suggest that Ribonuclease 7 (RNase 7) is a potent antimicrobial peptide that plays an important role in protecting the urinary tract from bacterial insult. The impact of diabetes on RNase 7 expression and function are unknown. Here, we investigate the effects of insulin on RNase 7. Using human urine specimens, we measured urinary RNase 7 concentrations in healthy control patients and insulin-deficient type 1 diabetics before and after starting insulin therapy. Compared to controls, diabetic patients had suppressed urinary RNase 7 concentrations, which increased with insulin. Using primary human urothelial cells, we explored the mechanisms by which insulin induces RNase 7. Insulin induces RNase 7 production via the phosphatidylinositide 3-kinase signaling pathway (PI3K/AKT) to shield urothelial cells from uropathogenic E. coli. In contrast, we show that uropathogenic E. coli suppresses PI3K/AKT and RNase 7. Together, these results indicate that insulin and PI3K/AKT signaling are essential for RNase 7 expression. They also suggest that increased infection risks in diabetic patients may be secondary to suppressed RNase 7 production. These data may provide unique insight into novel UTI therapeutic strategies in at risk populations. PMID:27401534

Influence of the solubilization of ribonuclease and of its hydrophobic derivatives on water-in-oil microemulsions

International Nuclear Information System (INIS)

Michel, Fabienne

1993-01-01

This research thesis addresses the study of the structural disruption of a water-in-oil microemulsion during the solubilization of an enzyme. More precisely, the microemulsion is the water/isooctane system, stabilised by the commonly named AOT anionic surfactant, and the disrupting agent is an enzymatic protein, ribonuclease A. The author addresses the following topics: interactions in microemulsion, percolation in microemulsion, use of microemulsions as micro-reactor, chemical modification of enzymes, and reactivity of enzymes. After a recall of the main results concerning AOT inverse micelles, the author addresses the influence of ribonuclease solubilisation on the micellar system. The micellar environment is then used as a micro-reactor in order to fix hydrophobic molecules in a covalent way onto the ribonuclease A, and then to promote the percolation process. The author then studies the structure of the microemulsion in presence of modified enzymes [fr

Antitumour activity and other biological actions of oligomers of ribonuclease A

Czech Academy of Sciences Publication Activity Database

Matoušek, Josef; Gottes, G.; Poučková, P.; Souček, J.; Slavík, Tomáš; Vottariello, F.; Libonati, M.

2003-01-01

Roč. 278, č. 26 (2003), s. 23817-23822 ISSN 0021-9258 R&D Projects: GA ČR GA523/01/0114 Institutional research plan: CEZ:AV0Z5045916 Keywords : ribonuclease A Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.482, year: 2003

Ribonuclease 7, an antimicrobial peptide up-regulated during infection, contributes to microbial defense of the human urinary tract

Science.gov (United States)

Spencer, John David; Schwaderer, Andrew L.; Wang, Huanyu; Bartz, Julianne; Kline, Jennifer; Eichler, Tad; DeSouza, Kristin R.; Sims-Lucas, Sunder; Baker, Peter; Hains, David S.

2012-01-01

The mechanisms that maintain sterility in the urinary tract are incompletely understood; however, recent studies stress the importance of antimicrobial peptides in protecting the urinary tract from infection. Ribonuclease 7 (RNase 7), a potent antimicrobial peptide contributing to urinary tract sterility, is expressed by intercalated cells in the renal collecting tubules and is present in the urine at levels sufficient to kill bacteria at baseline. Here, we characterize the expression and function of RNase 7 in the human urinary tract during infection. Both quantitative real-time PCR and ELISA assays demonstrated increases in RNASE7 expression in the kidney along with kidney and urinary RNase 7 peptide concentrations with infection. While immunostaining localized RNase 7 production to the intercalated cells of the collecting tubule during sterility, its expression during pyelonephritis was found to increase throughout the nephron but not in glomeruli or the interstitium. Recombinant RNase 7 exhibited antimicrobial activity against uropathogens at low micromolar concentrations by disrupting the microbial membrane as determined by atomic force microscopy. Thus, RNase 7 expression is increased in the urinary tract with infection, and has antibacterial activity against uropathogens at micromolar concentrations. PMID:23302724

A ribonuclease from the wild mushroom Boletus griseus.

Science.gov (United States)

Wang, Hexiang; Ng, T B

2006-10-01

A ribonuclease (RNase) with a molecular mass of 29 kDa and cospecific for poly A and poly U was isolated from fruiting bodies of the mushroom Boletus griseus. Its N-terminal sequence exhibited some similarity to those of RNases from the mushrooms Irpex lacteus and Lentinus edodes. The RNase was adsorbed on diethylaminoethyl-cellulose, Q-Sepharose, and Affi-gel blue gel and was unadsorbed on CM-cellulose. The enzyme exhibited a temperature optimum between 60 and 70 degrees C and a pH optimum at 3.5.

Substrate recognition by ribonucleoprotein ribonuclease MRP.

Science.gov (United States)

Esakova, Olga; Perederina, Anna; Quan, Chao; Berezin, Igor; Krasilnikov, Andrey S

2011-02-01

The ribonucleoprotein complex ribonuclease (RNase) MRP is a site-specific endoribonuclease essential for the survival of the eukaryotic cell. RNase MRP closely resembles RNase P (a universal endoribonuclease responsible for the maturation of the 5' ends of tRNA) but recognizes distinct substrates including pre-rRNA and mRNA. Here we report the results of an in vitro selection of Saccharomyces cerevisiae RNase MRP substrates starting from a pool of random sequences. The results indicate that RNase MRP cleaves single-stranded RNA and is sensitive to sequences in the immediate vicinity of the cleavage site requiring a cytosine at the position +4 relative to the cleavage site. Structural implications of the differences in substrate recognition by RNases P and MRP are discussed.

Studies on protein synthesis by protoplasts of saccharomyces carlsbergensis III. Studies on the specificity and the mechanism of the action of ribonuclease on protein synthesis

NARCIS (Netherlands)

Kloet, S.R. de; Dam, G.J.W. van; Koningsberger, V.V.

1962-01-01

In this paper, the experimental results are presented of a continued study on the specificity and the mechanism of the inhibition by ribonuclease of protein synthesis in protoplasts of Saccharomyces carlsbergensis. By comparing the effects of native pancreatic ribonuclease with those of

A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25

DEFF Research Database (Denmark)

Douthwaite, S; Garrett, R A; Wagner, R

1979-01-01

An RNA fragment, constituting three subfragments of nucleotide sequences 1-11, 69-87 and 89-120, is the most ribonuclease-resistant part of the native 5S RNA of Escherichia coli, at 0 degrees C. A smaller fragment of nucleotide sequence 69-87 and 90-110 is ribonuclease-resistant at 25 degrees....... Degradation of the L25-5S RNA complex with ribonuclease A or T2 yielded RNA fragments similar to those of the free 5S RNA at 0 degrees C and 25 degrees C; moreover L25 remained strongly bound to both RNA fragments and also produced some opening of the RNA structure in at least two positions. Protein L18...... initially protected most of the 5S RNA against ribonuclease digestion, at 0 degrees C, but was then gradually released prior to the formation of the larger RNA fragment. It cannot be concluded, therefore, as it was earlier (Gray et al., 1973), that this RNA fragment contains the primary binding site of L18....

Novel Ribonuclease Activity Differs between Fibrillarins from Arabidopsis thaliana

Czech Academy of Sciences Publication Activity Database

Rodriguez-Corona, U.; Pereira-Santana, A.; Sobol, Margaryta; Rodriguez-Zapata, L.C.; Hozák, Pavel; Castano, E.

2017-01-01

Roč. 8, podzim (2017), č. článku 1878. ISSN 1664-462X R&D Projects: GA ČR GAP305/11/2232; GA ČR GA16-03346S; GA ČR GA15-08738S; GA TA ČR(CZ) TE01020118; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:68378050 Keywords : nucleoli * fibrillarin * ribonuclease * phosphoinositides * phosphatidic acid * glycinearginine rich domain Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 4.298, year: 2016

Lansoprazole and carbonic anhydrase IX inhibitors sinergize against human melanoma cells.

Science.gov (United States)

Federici, Cristina; Lugini, Luana; Marino, Maria Lucia; Carta, Fabrizio; Iessi, Elisabetta; Azzarito, Tommaso; Supuran, Claudiu T; Fais, Stefano

2016-01-01

Proton Pump Inhibitors (PPIs) reduce tumor acidity and therefore resistance of tumors to drugs. Carbonic Anhydrase IX (CA IX) inhibitors have proven to be effective against tumors, while tumor acidity might impair their full effectiveness. To analyze the effect of PPI/CA IX inhibitors combined treatment against human melanoma cells. The combination of Lansoprazole (LAN) and CA IX inhibitors (FC9-399A and S4) has been investigated in terms of cell proliferation inhibition and cell death in human melanoma cells. The combination of these inhibitors was more effective than the single treatments in both inhibiting cell proliferation and in inducing cell death in human melanoma cells. These results represent the first successful attempt in combining two different proton exchanger inhibitors. This is the first evidence on the effectiveness of a new approach against tumors based on the combination of PPI and CA IX inhibitors, thus providing an alternative strategy against tumors.

Crystallization and preliminary X-ray diffraction analysis of the P3 RNA domain of yeast ribonuclease MRP in a complex with RNase P/MRP protein components Pop6 and Pop7

International Nuclear Information System (INIS)

Perederina, Anna; Esakova, Olga; Quan, Chao; Khanova, Elena; Krasilnikov, Andrey S.

2009-01-01

This article describes the first successful crystallization of components of eukaryotic ribonucleases P/MRP. Yeast RNase MRP RNA domain P3 was crystallized in a complex with the proteins Pop6 and Pop7; the crystals diffracted to 3.25 Å resolution. Eukaryotic ribonucleases P and MRP are closely related RNA-based enzymes which contain a catalytic RNA component and several protein subunits. The roles of the protein subunits in the structure and function of eukaryotic ribonucleases P and MRP are not clear. Crystals of a complex that included a circularly permuted 46-nucleotide-long P3 domain of the RNA component of Saccharomyces cerevisiae ribonuclease MRP and selenomethionine derivatives of the shared ribonuclease P/MRP protein components Pop6 (18.2 kDa) and Pop7 (15.8 kDa) were obtained using the sitting-drop vapour-diffusion method. The crystals belonged to space group P4 2 22 (unit-cell parameters a = b = 127.2, c = 76.8 Å, α = β = γ = 90°) and diffracted to 3.25 Å resolution

Crystallization and preliminary X-ray diffraction analysis of the P3 RNA domain of yeast ribonuclease MRP in a complex with RNase P/MRP protein components Pop6 and Pop7.

Science.gov (United States)

Perederina, Anna; Esakova, Olga; Quan, Chao; Khanova, Elena; Krasilnikov, Andrey S

2010-01-01

Eukaryotic ribonucleases P and MRP are closely related RNA-based enzymes which contain a catalytic RNA component and several protein subunits. The roles of the protein subunits in the structure and function of eukaryotic ribonucleases P and MRP are not clear. Crystals of a complex that included a circularly permuted 46-nucleotide-long P3 domain of the RNA component of Saccharomyces cerevisiae ribonuclease MRP and selenomethionine derivatives of the shared ribonuclease P/MRP protein components Pop6 (18.2 kDa) and Pop7 (15.8 kDa) were obtained using the sitting-drop vapour-diffusion method. The crystals belonged to space group P4(2)22 (unit-cell parameters a = b = 127.2, c = 76.8 A, alpha = beta = gamma = 90 degrees ) and diffracted to 3.25 A resolution.

Structural investigation of ribonuclease A conformational preferences using high pressure protein crystallography

Energy Technology Data Exchange (ETDEWEB)

Kurpiewska, Katarzyna, E-mail: kurpiews@chemia.uj.edu.pl [Jagiellonian University, Faculty of Chemistry, Department of Crystal Chemistry and Crystal Physics, Protein Crystallography Group, Ingardena 3, 30-060 Kraków (Poland); Dziubek, Kamil; Katrusiak, Andrzej [Adam Mickiewicz University, Faculty of Chemistry, Department of Materials Chemistry, Umultowska 89b, 61-61 Poznań (Poland); Font, Josep [School of Medical Science, University of Sydney, NSW 2006 (Australia); Ribò, Marc; Vilanova, Maria [Universitat de Girona, Laboratorid’Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Campus de Montilivi, 17071 Girona (Spain); Lewiński, Krzysztof [Jagiellonian University, Faculty of Chemistry, Department of Crystal Chemistry and Crystal Physics, Protein Crystallography Group, Ingardena 3, 30-060 Kraków (Poland)

2016-04-01

Highlights: • A unique crystallographic studies of wild-type and mutated form of the same protein under high pressure. • Compressibility of RNase A molecule is significantly affected by a single amino acid substitution. • High pressure protein crystallography helps understanding protein flexibility and identify conformational substrates. - Abstract: Hydrostatic pressure in range 0.1–1.5 GPa is used to modify biological system behaviour mostly in biophysical studies of proteins in solution. Due to specific influence on the system equilibrium high pressure can act as a filter that enables to identify and investigate higher energy protein conformers. The idea of the presented experiments is to examine the behaviour of RNase A molecule under high pressure before and after introduction of destabilizing mutation. For the first time crystal structures of wild-type bovine pancreatic ribonuclease A and its markedly less stable variant modified at position Ile106 were determined at different pressures. X-ray diffraction experiments at high pressure showed that the secondary structure of RNase A is well preserved even beyond 0.67 GPa at room temperature. Detailed structural analysis of ribonuclease A conformation observed under high pressure revealed that pressure influences hydrogen bonds pattern, cavity size and packing of molecule.

Structural investigation of ribonuclease A conformational preferences using high pressure protein crystallography

International Nuclear Information System (INIS)

Kurpiewska, Katarzyna; Dziubek, Kamil; Katrusiak, Andrzej; Font, Josep; Ribò, Marc; Vilanova, Maria; Lewiński, Krzysztof

2016-01-01

Highlights: • A unique crystallographic studies of wild-type and mutated form of the same protein under high pressure. • Compressibility of RNase A molecule is significantly affected by a single amino acid substitution. • High pressure protein crystallography helps understanding protein flexibility and identify conformational substrates. - Abstract: Hydrostatic pressure in range 0.1–1.5 GPa is used to modify biological system behaviour mostly in biophysical studies of proteins in solution. Due to specific influence on the system equilibrium high pressure can act as a filter that enables to identify and investigate higher energy protein conformers. The idea of the presented experiments is to examine the behaviour of RNase A molecule under high pressure before and after introduction of destabilizing mutation. For the first time crystal structures of wild-type bovine pancreatic ribonuclease A and its markedly less stable variant modified at position Ile106 were determined at different pressures. X-ray diffraction experiments at high pressure showed that the secondary structure of RNase A is well preserved even beyond 0.67 GPa at room temperature. Detailed structural analysis of ribonuclease A conformation observed under high pressure revealed that pressure influences hydrogen bonds pattern, cavity size and packing of molecule.

Is zucchini a phosphodiesterase or a ribonuclease?

Directory of Open Access Journals (Sweden)

Osamu Nureki

2014-12-01

Full Text Available Zucchini (Zuc, a member of the phospholipase D (PLD superfamily, is essential for the primary PIWI-interacting RNA (piRNA biogenesis and the suppression of transposon expression, which are crucial for the genome integrity of germline cells. However, it has been ambiguous whether Zuc acts as a phosphodiesterase to produce phosphatidic acid (PA, the lipid signaling molecule, or as a nuclease. The recent three papers describing the crystal structures and functional analyses of fly and mouse Zuc proteins have elucidated that Zuc is a PLD family single-strand ribonuclease, not a phosphodiesterase, and functions in the maturation of primary piRNAs. This review will discuss in detail how the crystal structures clearly predict the function of Zuc, which is subsequently demonstrated by biochemical analysis to conclude the previous controversial discussion on the real function of Zuc.

Organization of the gene coding for human protein C inhibitor (plasminogen activator inhibitor-3). Assignment of the gene to chromosome 14

NARCIS (Netherlands)

Meijers, J. C.; Chung, D. W.

1991-01-01

Protein C inhibitor (plasminogen activator inhibitor-3) is a plasma glycoprotein and a member of the serine proteinase inhibitor superfamily. In the present study, the human gene for protein C inhibitor was isolated and characterized from three independent phage that contained overlapping inserts

How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing.

Science.gov (United States)

Koralewska, Natalia; Hoffmann, Weronika; Pokornowska, Maria; Milewski, Marek; Lipinska, Andrea; Bienkowska-Szewczyk, Krystyna; Figlerowicz, Marek; Kurzynska-Kokorniak, Anna

2016-01-01

Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by a variety of proteins are being elucidated, less is known about non-protein factors, e.g. RNA molecules, that may influence this enzyme's activity. Therefore, we decided to investigate the question of whether the RNA molecules can function not only as Dicer substrates but also as its regulators. Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript, we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs that are longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding to this enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by participating in regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.

2-Arylbenzo[b]furan derivatives as potent human lipoxygenase inhibitors.

Science.gov (United States)

Lang, Li; Dong, Ningning; Wu, Deyan; Yao, Xue; Lu, Weiqiang; Zhang, Chen; Ouyang, Ping; Zhu, Jin; Tang, Yun; Wang, Wei; Li, Jian; Huang, Jin

2016-01-01

Human lipoxygenases (LOXs) have been emerging as effective therapeutic targets for inflammatory diseases. In this study, we found that four natural 2-arylbenzo[b]furan derivatives isolated from Artocarpus heterophyllus exhibited potent inhibitory activities against human LOXs, including moracin C (1), artoindonesianin B-1 (2), moracin D (3), moracin M (4). In our in vitro experiments, compound 1 was identified as the most potent LOX inhibitor and the moderate subtype selective inhibitor of 12-LOX. Compounds 1 and 2 act as competitive inhibitors of LOXs. Moreover, 1 significantly inhibits LTB4 production and chemotactic capacity of neutrophils, and is capable of protecting vascular barrier from plasma leakage in vivo. In addition, the preliminary structure-activity relationship analysis was performed based on the above four naturally occurring (1-4) and six additional synthetic 2-arylbenzo[b]furan derivatives. Taken together, these 2-arylbenzo[b]furan derivatives, as LOXs inhibitors, could represent valuable leads for the future development of therapeutic agents for inflammatory diseases.

A nonradioactive assay for poly(a)-specific ribonuclease activity by methylene blue colorimetry.

Science.gov (United States)

Cheng, Yuan; Liu, Wei-Feng; Yan, Yong-Bin; Zhou, Hai-Meng

2006-01-01

A simple nonradioactive assay, which was based on the specific shift of the absorbance maximum of methylene blue induced by its intercalation into poly(A) molecules, was developed for poly(A)-specific ribonuclease (PARN). A good linear relationship was found between the absorbance at 662 nm and the poly(A) concentration. The assay conditions, including the concentration of methylene blue, the incubation temperature and time, and the poly(A) concentration were evaluated and optimized.

A dual surface plasmon resonance assay for the determination of ribonuclease H activity

Czech Academy of Sciences Publication Activity Database

Šípová, Hana; Vaisocherová, Hana; Štepánek, J.; Homola, Jiří

2010-01-01

Roč. 26, č. 4 (2010), s. 1605-1611 ISSN 0956-5663 R&D Projects: GA AV ČR KAN200670701; GA MŠk OC09058; GA ČR GA202/09/0193 Grant - others:Univerzita Karlova(CZ) SVV-2010-261 304 Institutional research plan: CEZ:AV0Z20670512 Keywords : Surface plasmon resonance * Enzyme activity assay * Ribonuclease H * Biosensor Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 5.361, year: 2010

Barnase as a new therapeutic agent triggering apoptosis in human cancer cells.

Directory of Open Access Journals (Sweden)

Evelina Edelweiss

Full Text Available BACKGROUND: RNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI presented in the cytoplasm of mammalian cells. METHODOLOGY/PRINCIPAL FINDINGS: In search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC(50 ranging from 0.2 to 13 microM and to leukemia cell lines with IC(50 values ranging from 2.4 to 82 microM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC(50 = 1.8 nM was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells.

Typing of multiple single-nucleotide polymorphisms using ribonuclease cleavage of DNA/RNA chimeric single-base extension primers and detection by MALDI-TOF mass spectrometry

DEFF Research Database (Denmark)

Mengel-From, Jonas; Sanchez Sanchez, Juan Jose; Børsting, Claus

2005-01-01

A novel single-base extension (SBE) assay using cleavable and noncleavable SBE primers in the same reaction mix is described. The cleavable SBE primers consisted of deoxyribonucleotides and one ribonucleotide (hereafter denoted chimeric primers), whereas the noncleavable SBE primers consisted....... A ribonuclease mix was developed to specifically cleave the chimeric primers, irrespective of the base of the ribonucleotide, whereas standard primers without a ribonucleotide were unaffected by the ribonuclease treatment. The SBE products were analyzed in linear mode using a matrix-assisted laser desorption...... containing 9 chimeric primers and 8 standard primers....

Crystal structure of a complex of human chymase with its benzimidazole derived inhibitor

International Nuclear Information System (INIS)

Matsumoto, Yoshiyuki; Kakuda, Shinji; Koizumi, Masahiro; Mizuno, Tsuyoshi; Muroga, Yumiko; Kawamura, Takashi; Takimoto-Kamimura, Midori

2013-01-01

The crystal structure of human chymase complexed with a novel benzimidazole inhibitor, TJK002, was determined at 2.8 Å resolution. The present study shows that the benzimidazole ring of the inhibitor takes the stable stacking interaction with the protonated His57 in the catalytic domain of human chymase. The crystal structure of human chymase complexed with a novel benzimidazole inhibitor, TJK002, was determined at 2.8 Å resolution. The X-ray crystallographic study shows that the benzimidazole inhibitor forms a non-covalent interaction with the catalytic domain of human chymase. The hydrophobic fragment of the inhibitor occupies the S1 pocket. The carboxylic acid group of the inhibitor forms hydrogen bonds with the imidazole N(∊) atom of His57 and/or the O(γ) atom of Ser195 which are members of the catalytic triad. This imidazole ring of His57 induces π–π stacking to the benzene ring of the benzimidazole scaffold as P2 moiety. Fragment molecular orbital calculation of the atomic coordinates by X-ray crystallography shows that this imidazole ring of His57 could be protonated with the carboxyl group of Asp102 or hydroxyl group of Ser195 and the stacking interaction is stabilized. A new drug design strategy is proposed where the stacking to the protonated imidazole of the drug target protein with the benzimidazole scaffold inhibitor causes unpredicted potent inhibitory activity for some enzymes

Potent selective nonpeptidic inhibitors of human lung tryptase

OpenAIRE

Burgess, Laurence E.; Newhouse, Bradley J.; Ibrahim, Prabha; Rizzi, James; Kashem, Mohammed A.; Hartman, Ann; Brandhuber, Barbara J.; Wright, Clifford D.; Thomson, David S.; Vigers, Guy P. A.; Koch, Kevin

1999-01-01

Human lung tryptase, a homotetrameric serine protease unique to mast cell secretory granules, has been implicated in the pathogenesis of asthma. A hypothesis that tethered symmetrical inhibitors might bridge two adjacent active sites was explored via a rationally designed series of bisbenzamidines. These compounds demonstrated a remarkable distanced-defined structure–activity relationship against human tryptase with one series possessing subnanomolar potencies. Additional evidence supporting ...

Active Site Mapping of Human Cathepsin F with Dipeptide Nitrile Inhibitors.

Science.gov (United States)

Schmitz, Janina; Furtmann, Norbert; Ponert, Moritz; Frizler, Maxim; Löser, Reik; Bartz, Ulrike; Bajorath, Jürgen; Gütschow, Michael

2015-08-01

Cleavage of the invariant chain is the key event in the trafficking pathway of major histocompatibility complex class II. Cathepsin S is the major processing enzyme of the invariant chain, but cathepsin F acts in macrophages as its functional synergist which is as potent as cathepsin S in invariant chain cleavage. Dedicated low-molecular-weight inhibitors for cathepsin F have not yet been developed. An active site mapping with 52 dipeptide nitriles, reacting as covalent-reversible inhibitors, was performed to draw structure-activity relationships for the non-primed binding region of human cathepsin F. In a stepwise process, new compounds with optimized fragment combinations were designed and synthesized. These dipeptide nitriles were evaluated on human cysteine cathepsins F, B, L, K and S. Compounds 10 (N-(4-phenylbenzoyl)-leucylglycine nitrile) and 12 (N-(4-phenylbenzoyl)leucylmethionine nitrile) were found to be potent inhibitors of human cathepsin F, with Ki values nitriles from our study, a 3D activity landscape was generated to visualize structure-activity relationships for this series of cathepsin F inhibitors. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of amino acids and amino acid derivatives on crystallization of hemoglobin and ribonuclease A

International Nuclear Information System (INIS)

Ito, Len; Kobayashi, Toyoaki; Shiraki, Kentaro; Yamaguchi, Hiroshi

2008-01-01

The effect of the addition of amino acids and amino acid derivatives on the crystallization of hemoglobin and ribonuclease A has been evaluated. The results showed that certain types of additives expand the concentration conditions in which crystals are formed. Determination of the appropriate conditions for protein crystallization remains a highly empirical process. Preventing protein aggregation is necessary for the formation of single crystals under aggregation-prone solution conditions. Because many amino acids and amino acid derivatives offer a unique combination of solubility and stabilizing properties, they open new avenues into the field of protein aggregation research. The use of amino acids and amino acid derivatives can potentially influence processes such as heat treatment and refolding reactions. The effect of the addition of several amino acids, such as lysine, and several amino acid derivatives, such as glycine ethyl ester and glycine amide, on the crystallization of equine hemoglobin and bovine pancreatic ribonuclease A has been examined. The addition of these amino acids and amino acid derivatives expanded the range of precipitant concentration in which crystals formed without aggregation. The addition of such additives appears to promote the crystallization of proteins

Effect of amino acids and amino acid derivatives on crystallization of hemoglobin and ribonuclease A

Energy Technology Data Exchange (ETDEWEB)

Ito, Len, E-mail: len@ksc.kwansei.ac.jp; Kobayashi, Toyoaki [School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan); Shiraki, Kentaro [Institute of Applied Physics, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573 (Japan); Yamaguchi, Hiroshi [School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan)

2008-05-01

The effect of the addition of amino acids and amino acid derivatives on the crystallization of hemoglobin and ribonuclease A has been evaluated. The results showed that certain types of additives expand the concentration conditions in which crystals are formed. Determination of the appropriate conditions for protein crystallization remains a highly empirical process. Preventing protein aggregation is necessary for the formation of single crystals under aggregation-prone solution conditions. Because many amino acids and amino acid derivatives offer a unique combination of solubility and stabilizing properties, they open new avenues into the field of protein aggregation research. The use of amino acids and amino acid derivatives can potentially influence processes such as heat treatment and refolding reactions. The effect of the addition of several amino acids, such as lysine, and several amino acid derivatives, such as glycine ethyl ester and glycine amide, on the crystallization of equine hemoglobin and bovine pancreatic ribonuclease A has been examined. The addition of these amino acids and amino acid derivatives expanded the range of precipitant concentration in which crystals formed without aggregation. The addition of such additives appears to promote the crystallization of proteins.

Studies on protein synthesis by protoplasts of Saccharomyces carlsbergensis I. The effect of ribonuclease on protein synthesis

NARCIS (Netherlands)

Kloet, S.R. de; Wermeskerken, R.K.A. van; Koningsberger, V.V.

1961-01-01

Ribonuclease was found to inhibit the protein synthesis in the naked yeast protoplast for nearly 100%. Even small concentrations (5 μg/ml) were found inhibitory. The cause of this inhibition can be attributed at least in part to a 90% inhibition of the respiration. Amino acid uptake was found to

An ibuprofen-antagonized plasmin inhibitor released by human endothelial cells.

Science.gov (United States)

Rockwell, W B; Ehrlich, H P

1991-02-01

Serum-free culture medium harvested from endothelial cell monolayer cultures derived from human scars and dermis was examined for inhibition of fibrinolysis using a fibrin plate assay. Human cultured fibroblasts and smooth muscle cells did not produce any detectable inhibitory activity. The inhibitor is spontaneously released from the cultured endothelial cells over time. In the fibrin plate assay of plasmin-induced fibrinolysis, one nonsteroidal antiinflammatory (NSAI) drug, ibuprofen, was demonstrated to antagonize the inhibition of fibrinolysis. The antagonistic activity of ibuprofen appears unrelated to its NSAI drug activity because other NSAI drugs such as indomethacin and tolmetin have minimal antagonistic activity. Heating the cultured endothelial cells to 42 degrees C stimulates greater release of the inhibitor in a shorter period of time. This plasmin inhibitor, which is produced by endothelial cells, may contribute to postburn vascular occlusion, leading to secondary progressive necrosis in burn-traumatized patients.

Identification by virtual screening and in vitro testing of human DOPA decarboxylase inhibitors.

Directory of Open Access Journals (Sweden)

Frederick Daidone

Full Text Available Dopa decarboxylase (DDC, a pyridoxal 5'-phosphate (PLP enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinson's disease (PD. PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a to use virtual screening to identify potential human DDC inhibitors and (b to evaluate the reliability of our virtual-screening (VS protocol by experimentally testing the "in vitro" activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with K(i values ≥10 µM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with K(i values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a K(i value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery.

Potent selective nonpeptidic inhibitors of human lung tryptase

Science.gov (United States)

Burgess, Laurence E.; Newhouse, Bradley J.; Ibrahim, Prabha; Rizzi, James; Kashem, Mohammed A.; Hartman, Ann; Brandhuber, Barbara J.; Wright, Clifford D.; Thomson, David S.; Vigers, Guy P. A.; Koch, Kevin

1999-01-01

Human lung tryptase, a homotetrameric serine protease unique to mast cell secretory granules, has been implicated in the pathogenesis of asthma. A hypothesis that tethered symmetrical inhibitors might bridge two adjacent active sites was explored via a rationally designed series of bisbenzamidines. These compounds demonstrated a remarkable distanced-defined structure–activity relationship against human tryptase with one series possessing subnanomolar potencies. Additional evidence supporting the concept of active-site bridging is also presented. PMID:10411878

A serine palmitoyltransferase inhibitor blocks hepatitis C virus replication in human hepatocytes.

Science.gov (United States)

Katsume, Asao; Tokunaga, Yuko; Hirata, Yuichi; Munakata, Tsubasa; Saito, Makoto; Hayashi, Hitohisa; Okamoto, Koichi; Ohmori, Yusuke; Kusanagi, Isamu; Fujiwara, Shinya; Tsukuda, Takuo; Aoki, Yuko; Klumpp, Klaus; Tsukiyama-Kohara, Kyoko; El-Gohary, Ahmed; Sudoh, Masayuki; Kohara, Michinori

2013-10-01

Host cell lipid rafts form a scaffold required for replication of hepatitis C virus (HCV). Serine palmitoyltransferases (SPTs) produce sphingolipids, which are essential components of the lipid rafts that associate with HCV nonstructural proteins. Prevention of the de novo synthesis of sphingolipids by an SPT inhibitor disrupts the HCV replication complex and thereby inhibits HCV replication. We investigated the ability of the SPT inhibitor NA808 to prevent HCV replication in cells and mice. We tested the ability of NA808 to inhibit SPT's enzymatic activity in FLR3-1 replicon cells. We used a replicon system to select for HCV variants that became resistant to NA808 at concentrations 4- to 6-fold the 50% inhibitory concentration, after 14 rounds of cell passage. We assessed the ability of NA808 or telaprevir to inhibit replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in mice with humanized livers (transplanted with human hepatocytes). NA808 was injected intravenously, with or without pegylated interferon alfa-2a and HCV polymerase and/or protease inhibitors. NA808 prevented HCV replication via noncompetitive inhibition of SPT; no resistance mutations developed. NA808 prevented replication of all HCV genotypes tested in mice with humanized livers. Intravenous NA808 significantly reduced viral load in the mice and had synergistic effects with pegylated interferon alfa-2a and HCV polymerase and protease inhibitors. The SPT inhibitor NA808 prevents replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in cultured hepatocytes and in mice with humanized livers. It might be developed for treatment of HCV infection or used in combination with pegylated interferon alfa-2a or HCV polymerase or protease inhibitors. Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

Archaeal ribonuclease P proteins have potential for biotechnological applications where precise hybridization of nucleic acids is needed.

Science.gov (United States)

Miyanoshita, Mitsuru; Nakashima, Takashi; Kakuta, Yoshimitsu; Kimura, Makoto

2015-01-01

Fluorescence resonance energy transfer-based assay showed that archaeal ribonuclease P (RNase P) proteins significantly promoted DNA annealing and strand displacement. Moreover, we found that archaeal RNase P proteins could discriminate nucleotide exchanges in DNA chains via their activity accelerating DNA strand displacement, suggesting that they have potential for biotechnological application to genetic diagnosis.

Assay of hybrid ribonuclease using a membrane filter-immobilized synthetic hybrid: application to the human leukemic cell

International Nuclear Information System (INIS)

Papaphilis, A.D.; Kamper, E.F.

1985-01-01

A method for assaying hybrid ribonuclease has been devised which utilizes as substrate the synthetic hybrid [ 3 H]polyriboadenylic acid [poly(rA)]:polydeoxythymidylic acid [poly(dT)] immobilized on the solid matrix of nitrocellulose filters. The hybridization on filter of [ 3 H]poly(rA) to poly(dT) has been explored in terms of efficacy of the process and the response of the product to RNase H. A pulse of uv irradiation of poly(dT) while in dry state on the filter increased its firm binding to the filter in a concentration-dependent manner, resulting in a concomitant increase of the yield of hybrid formation. The filter-immobilized hybrid was 95% resistant to RNase A but sensitive to RNase H. When stored in toluene in the cold the hybrid maintained its stability for over 6 months, as judged by its resistance to RNase A. The method offers a number of advantages over assays that use solution hybrids as substrates and was readily applicable in the screening of leukemic patients, in the leukocytes of which it has demonstrated increased RNase H levels

Escape from Human Immunodeficiency Virus Type 1 (HIV-1 Entry Inhibitors

Directory of Open Access Journals (Sweden)

Carol D. Weiss

2012-12-01

Full Text Available The human immunodeficiency virus (HIV enters cells through a series of molecular interactions between the HIV envelope protein and cellular receptors, thus providing many opportunities to block infection. Entry inhibitors are currently being used in the clinic, and many more are under development. Unfortunately, as is the case for other classes of antiretroviral drugs that target later steps in the viral life cycle, HIV can become resistant to entry inhibitors. In contrast to inhibitors that block viral enzymes in intracellular compartments, entry inhibitors interfere with the function of the highly variable envelope glycoprotein as it continuously adapts to changing immune pressure and available target cells in the extracellular environment. Consequently, pathways and mechanisms of resistance for entry inhibitors are varied and often involve mutations across the envelope gene. This review provides a broad overview of entry inhibitor resistance mechanisms that inform our understanding of HIV entry and the design of new inhibitors and vaccines.

Poly(A-Specific Ribonuclease Mediates 3′-End Trimming of Argonaute2-Cleaved Precursor MicroRNAs

Directory of Open Access Journals (Sweden)

Mayuko Yoda

2013-11-01

Full Text Available MicroRNAs (miRNAs are typically generated as ∼22-nucleotide double-stranded RNAs via the processing of precursor hairpins by the ribonuclease III enzyme Dicer, after which they are loaded into Argonaute (Ago proteins to form an RNA-induced silencing complex (RISC. However, the biogenesis of miR-451, an erythropoietic miRNA conserved in vertebrates, occurs independently of Dicer and instead requires cleavage of the 3′ arm of the pre-miR-451 precursor hairpin by Ago2. The 3′ end of the Ago2-cleaved pre-miR-451 intermediate is then trimmed to the mature length by an unknown nuclease. Here, using a classical chromatographic approach, we identified poly(A-specific ribonuclease (PARN as the enzyme responsible for the 3′–5′ exonucleolytic trimming of Ago2-cleaved pre-miR-451. Surprisingly, our data show that trimming of Ago2-cleaved precursor miRNAs is not essential for target silencing, indicating that RISC is functional with miRNAs longer than the mature length. Our findings define the maturation step in the miRNA biogenesis pathway that depends on Ago2-mediated cleavage.

Effect of phosphodiesterase inhibitors on human arteries in vitro

NARCIS (Netherlands)

Vroom, M. B.; Pfaffendorf, M.; van Wezel, H. B.; van Zwieten, P. A.

1996-01-01

In the present study, we investigated if the relaxant effects of phosphodiesterase (PDE) III inhibitors on human vessels could be inhibited by a nitric oxide synthase blocker, L-NAME, or by a blocker of ATP-sensitive potassium channels (KATP), glibenclamide. The experiments were performed using an

A Ribonuclease Isolated from Wild Ganoderma Lucidum Suppressed Autophagy and Triggered Apoptosis in Colorectal Cancer Cells

OpenAIRE

Dan, Xiuli; Liu, Wenlong; Wong, Jack H.; Ng, Tzi B.

2016-01-01

The mushroom Ganoderma lucidum (G. lucidum) has been consumed in China as a medicine for promoting health and longevity for thousands of years. Due to its paramount and multiple pharmaceutical effects, G. lucidum has received considerable attention from researchers and its chemical constituents as well as their respective functions were gradually unveiled by using modern research methods. Herein, we reported the isolation of a protein (Ganoderma lucidum ribonuclease, GLR) with anti-colorectal...

Discovering a Reliable Heat-Shock Factor-1 Inhibitor to Treat Human Cancers: Potential Opportunity for Phytochemists

Directory of Open Access Journals (Sweden)

Murugesan Velayutham

2018-04-01

Full Text Available Heat-shock factor-1 (HSF-1 is an important transcription factor that regulates pathogenesis of many human diseases through its extensive transcriptional regulation. Especially, it shows pleiotropic effects in human cancer, and hence it has recently received increased attention of cancer researchers. After myriad investigations on HSF-1, the field has advanced to the phase where there is consensus that finding a potent and selective pharmacological inhibitor for this transcription factor will be a major break-through in the treatment of various human cancers. Presently, all reported inhibitors have their limitations, made evident at different stages of clinical trials. This brief account summarizes the advances with tested natural products as HSF-1 inhibitors and highlights the necessity of phytochemistry in this endeavor of discovering a potent pharmacological HSF-1 inhibitor.

Discovery and development of inhibitors selective for human constitutive proteasome and immunoproteasome active sites

NARCIS (Netherlands)

Xin, B.

2017-01-01

This thesis describes the design and development of subunit‐selective inhibitors of particular catalytically active subunits of human constitutive proteasomes and immunoproteasomes. Most existing proteasome inhibitors are oligopeptides composed of 2‐4 amino acid residues, N‐terminally

Primary structures of two ribonucleases from ginseng calluses - New members of the PR-10 family of intracellular pathogenesis-related plant proteins

NARCIS (Netherlands)

Moiseyev, GP; Fedoreyeva, LI; Zhuravlev, YN; Yasnetskaya, E; Jekel, PA; Beintema, JJ

1997-01-01

The amino acid sequences of two ribonucleases from a callus cell culture of Panax ginseng were determined, The two sequences differ at 26% of the amino acid positions, Homology was found with a large family of intracellular pathogenesis-related proteins, food allergens and tree pollen allergens from

Sensitization of multidrug-resistant human cancer cells to Hsp90 inhibitors by down-regulation of SIRT1

Science.gov (United States)

Kim, Hak-Bong; Lee, Su-Hoon; Um, Jee-Hyun; Oh, Won Keun; Kim, Dong-Wan; Kang, Chi-Dug; Kim, Sun-Hee

2015-01-01

The effectiveness of Hsp90 inhibitors as anticancer agents was limited in multidrug-resistant (MDR) human cancer cells due to induction of heat shock proteins (Hsps) such as Hsp70/Hsp27 and P-glycoprotein (P-gp)-mediated efflux. In the present study, we showed that resistance to Hsp90 inhibitors of MDR human cancer cells could be overcome with SIRT1 inhibition. SIRT1 knock-down or SIRT1 inhibitors (amurensin G and EX527) effectively suppressed the resistance to Hsp90 inhibitors (17-AAG and AUY922) in several MDR variants of human lymphoblastic leukemia and human breast cancer cell lines. SIRT1 inhibition down-regulated the expression of heat shock factor 1 (HSF1) and subsequently Hsps and facilitated Hsp90 multichaperone complex disruption via hyperacetylation of Hsp90/Hsp70. These findings were followed by acceleration of ubiquitin ligase CHIP-mediated mutant p53 (mut p53) degradation and subsequent down-regulation of P-gp in 17-AAG-treated MDR cancer cells expressing P-gp and mut p53 after inhibition of SIRT1. Therefore, combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be a more effective therapeutic approach for Hsp90 inhibitor-resistant MDR cells via down-regulation of HSF1/Hsps, mut p53 and P-gp. PMID:26416354

Molecular dynamics simulation of bovine pancreatic ribonuclease A-CpA and transition state-like complexes.

Science.gov (United States)

Formoso, Elena; Matxain, Jon M; Lopez, Xabier; York, Darrin M

2010-06-03

The mechanisms of enzymes are intimately connected with their overall structure and dynamics in solution. Experimentally, it is considerably challenging to provide detailed atomic level information about the conformational events that occur at different stages along the chemical reaction path. Here, theoretical tools may offer new potential insights that complement those obtained from experiments that may not yield an unambiguous mechanistic interpretation. In this study, we apply molecular dynamics simulations of bovine pancreatic ribonuclease A, an archetype ribonuclease, to study the conformational dynamics, structural relaxation, and differential solvation that occur at discrete stages of the transesterification and cleavage reaction. Simulations were performed with explicit solvation with rigorous electrostatics and utilize recently developed molecular mechanical force field parameters for transphosphorylation and hydrolysis transition state analogues. Herein, we present results for the enzyme complexed with the dinucleotide substrate cytidilyl-3',5'-adenosine (CpA) in the reactant, and transphosphorylation and hydrolysis transition states. A detailed analysis of active site structures and hydrogen-bond patterns is presented and compared. The integrity of the overall backbone structure is preserved in the simulations and supports a mechanism whereby His12 stabilizes accumulating negative charge at the transition states through hydrogen-bond donation to the nonbridge oxygens. Lys41 is shown to be highly versatile along the reaction coordinate and can aid in the stabilization of the dianionic transition state, while being poised to act as a general acid catalyst in the hydrolysis step.

ROCK inhibitor prevents the dedifferentiation of human articular chondrocytes

International Nuclear Information System (INIS)

Matsumoto, Emi; Furumatsu, Takayuki; Kanazawa, Tomoko; Tamura, Masanori; Ozaki, Toshifumi

2012-01-01

Highlights: ► ROCK inhibitor stimulates chondrogenic gene expression of articular chondrocytes. ► ROCK inhibitor prevents the dedifferentiation of monolayer-cultured chondrocytes. ► ROCK inhibitor enhances the redifferentiation of cultured chondrocytes. ► ROCK inhibitor is useful for preparation of un-dedifferentiated chondrocytes. ► ROCK inhibitor may be a useful reagent for chondrocyte-based regeneration therapy. -- Abstract: Chondrocytes lose their chondrocytic phenotypes in vitro. The Rho family GTPase ROCK, involved in organizing the actin cytoskeleton, modulates the differentiation status of chondrocytic cells. However, the optimum method to prepare a large number of un-dedifferentiated chondrocytes is still unclear. In this study, we investigated the effect of ROCK inhibitor (ROCKi) on the chondrogenic property of monolayer-cultured articular chondrocytes. Human articular chondrocytes were subcultured in the presence or absence of ROCKi (Y-27632). The expression of chondrocytic marker genes such as SOX9 and COL2A1 was assessed by quantitative real-time PCR analysis. Cellular morphology and viability were evaluated. Chondrogenic redifferentiation potential was examined by a pellet culture procedure. The expression level of SOX9 and COL2A1 was higher in ROCKi-treated chondrocytes than in untreated cells. Chondrocyte morphology varied from a spreading form to a round shape in a ROCKi-dependent manner. In addition, ROCKi treatment stimulated the proliferation of chondrocytes. The deposition of safranin O-stained proteoglycans and type II collagen was highly detected in chondrogenic pellets derived from ROCKi-pretreated chondrocytes. Our results suggest that ROCKi prevents the dedifferentiation of monolayer-cultured chondrocytes, and may be a useful reagent to maintain chondrocytic phenotypes in vitro for chondrocyte-based regeneration therapy.

Atomistic Details of the Associative Phosphodiester Cleavage in Human Ribonuclease H

International Nuclear Information System (INIS)

Elsasser, Brigitta M.; Fels, Gregor

2010-01-01

During translation of the genetic information of DNA into proteins, mRNA is synthesized by RNA polymerase and after the transcription process degraded by RNase H. The endoribonuclease RNase H is a member of the nucleotidyl-transferase (NT) superfamily and is known to hydrolyze the phosphodiester bonds of RNA which is hybridized to DNA. Retroviral RNase H is part of the viral reverse transcriptase enzyme that is indispensable for the proliferation of retroviruses, such as HIV. Inhibitors of this enzyme could therefore provide new drugs against diseases like AIDS. In our study we investigated the molecular mechanism of RNA cleavage by human RNase H using a comprehensive high level DFT/B3LYP QM/MM theoretical method for the calculation of the stationary points and nudged elastic band (NEB) and free energy calculations to identify the transition state structures, the rate limiting step and the reaction barrier. Our calculations reveal that the catalytic mechanism proceeds in two steps and that the nature of the nucleophile is a water molecule. In the first step, the water attack on the scissile phosphorous is followed by a proton transfer from the water to the O2P oxygen and a trigonal bipyramidal pentacoordinated phosphorane is formed. Subsequently, in the second step the proton is shuttled to the O30 oxygen to generate the product state. During the reaction mechanism two Mg2+ ions support the formation of a stable associated in-line SN2-type phosphorane intermediate. Our calculated energy barrier of 19.3 kcal mol*1 is in excellent agreement with experimental findings (20.5 kcal mol*1). These results may contribute to the clarification and understanding of the RNase H reaction mechanism and of further enzymes from the RNase family.

Synergistic efficacy in human ovarian cancer cells by histone deacetylase inhibitor TSA and proteasome inhibitor PS-341.

Science.gov (United States)

Fang, Yong; Hu, Yi; Wu, Peng; Wang, Beibei; Tian, Yuan; Xia, Xi; Zhang, Qinghua; Chen, Tong; Jiang, Xuefeng; Ma, Quanfu; Xu, Gang; Wang, Shixuan; Zhou, Jianfeng; Ma, Ding; Meng, Li

2011-05-01

Histone deacetylase inhibitors and proteasome inhibitor are all emerging as new classes of anticancer agents. We chose TSA and PS-341 to identify whether they have a synergistic efficacy on human ovarian cancer cells. After incubated with 500 nM TSA or/and 40 nM PS-341, we found that combined groups resulted in a striking increase of apoptosis and G2/M blocking rates, no matter in A2780, cisplatin-sensitive ovarian cancer cell line OV2008 or its resistant variant C13*. This demonstrated that TSA interacted synergistically with PS-341, which raised the possibility that combined the two drugs may represent a novel strategy in ovarian cancer.

Structure of a retro-binding peptide inhibitor complexed with human alpha-thrombin.

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Tabernero, L; Chang, C Y; Ohringer, S L; Lau, W F; Iwanowicz, E J; Han, W C; Wang, T C; Seiler, S M; Roberts, D G; Sack, J S

1995-02-10

The crystallographic structure of the ternary complex between human alpha-thrombin, hirugen and the peptidyl inhibitor Phe-alloThr-Phe-O-CH3, which is acylated at its N terminus with 4-guanidino butanoic acid (BMS-183507), has been determined at 2.6 A resolution. The structure reveals a unique "retro-binding" mode for this tripeptide active site inhibitor. The inhibitor binds with its alkyl-guanidine moiety in the primary specificity pocket and its two phenyl rings occupying the hydrophobic proximal and distal pockets of the thrombin active site. In this arrangement the backbone of the tripeptide forms a parallel beta-strand to the thrombin main-chain at the binding site. This is opposite to the orientation of the natural substrate, fibrinogen, and all the small active site-directed thrombin inhibitors whose bound structures have been previously reported. BMS-183507 is the first synthetic inhibitor proved to bind in a retro-binding fashion to thrombin, in a fashion similar to that of the N-terminal residues of the natural inhibitor hirudin. Furthermore, this new potent thrombin inhibitor (Ki = 17.2 nM) is selective for thrombin over other serine proteases tested and may be a template to be considered in designing hirudin-based thrombin inhibitors with interactions at the specificity pocket.

Human glioblastoma multiforme: p53 reactivation by a novel MDM2 inhibitor.

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Barbara Costa

Full Text Available Cancer development and chemo-resistance are often due to impaired functioning of the p53 tumor suppressor through genetic mutation or sequestration by other proteins. In glioblastoma multiforme (GBM, p53 availability is frequently reduced because it binds to the Murine Double Minute-2 (MDM2 oncoprotein, which accumulates at high concentrations in tumor cells. The use of MDM2 inhibitors that interfere with the binding of p53 and MDM2 has become a valid approach to inhibit cell growth in a number of cancers; however little is known about the efficacy of these inhibitors in GBM. We report that a new small-molecule inhibitor of MDM2 with a spirooxoindolepyrrolidine core structure, named ISA27, effectively reactivated p53 function and inhibited human GBM cell growth in vitro by inducing cell cycle arrest and apoptosis. In immunoincompetent BALB/c nude mice bearing a human GBM xenograft, the administration of ISA27 in vivo activated p53, inhibited cell proliferation and induced apoptosis in tumor tissue. Significantly, ISA27 was non-toxic in an in vitro normal human cell model and an in vivo mouse model. ISA27 administration in combination with temozolomide (TMZ produced a synergistic inhibitory effect on GBM cell viability in vitro, suggesting the possibility of lowering the dose of TMZ used in the treatment of GBM. In conclusion, our data show that ISA27 releases the powerful antitumor capacities of p53 in GBM cells. The use of this MDM2 inhibitor could become a novel therapy for the treatment of GBM patients.

ROCK inhibitor prevents the dedifferentiation of human articular chondrocytes

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Matsumoto, Emi [Department of Orthopaedic Surgery, Science of Functional Recovery and Reconstruction, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikatacho, Kitaku, Okayama 700-8558 (Japan); Furumatsu, Takayuki, E-mail: matino@md.okayama-u.ac.jp [Department of Orthopaedic Surgery, Science of Functional Recovery and Reconstruction, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikatacho, Kitaku, Okayama 700-8558 (Japan); Kanazawa, Tomoko; Tamura, Masanori; Ozaki, Toshifumi [Department of Orthopaedic Surgery, Science of Functional Recovery and Reconstruction, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikatacho, Kitaku, Okayama 700-8558 (Japan)

2012-03-30

Highlights: Black-Right-Pointing-Pointer ROCK inhibitor stimulates chondrogenic gene expression of articular chondrocytes. Black-Right-Pointing-Pointer ROCK inhibitor prevents the dedifferentiation of monolayer-cultured chondrocytes. Black-Right-Pointing-Pointer ROCK inhibitor enhances the redifferentiation of cultured chondrocytes. Black-Right-Pointing-Pointer ROCK inhibitor is useful for preparation of un-dedifferentiated chondrocytes. Black-Right-Pointing-Pointer ROCK inhibitor may be a useful reagent for chondrocyte-based regeneration therapy. -- Abstract: Chondrocytes lose their chondrocytic phenotypes in vitro. The Rho family GTPase ROCK, involved in organizing the actin cytoskeleton, modulates the differentiation status of chondrocytic cells. However, the optimum method to prepare a large number of un-dedifferentiated chondrocytes is still unclear. In this study, we investigated the effect of ROCK inhibitor (ROCKi) on the chondrogenic property of monolayer-cultured articular chondrocytes. Human articular chondrocytes were subcultured in the presence or absence of ROCKi (Y-27632). The expression of chondrocytic marker genes such as SOX9 and COL2A1 was assessed by quantitative real-time PCR analysis. Cellular morphology and viability were evaluated. Chondrogenic redifferentiation potential was examined by a pellet culture procedure. The expression level of SOX9 and COL2A1 was higher in ROCKi-treated chondrocytes than in untreated cells. Chondrocyte morphology varied from a spreading form to a round shape in a ROCKi-dependent manner. In addition, ROCKi treatment stimulated the proliferation of chondrocytes. The deposition of safranin O-stained proteoglycans and type II collagen was highly detected in chondrogenic pellets derived from ROCKi-pretreated chondrocytes. Our results suggest that ROCKi prevents the dedifferentiation of monolayer-cultured chondrocytes, and may be a useful reagent to maintain chondrocytic phenotypes in vitro for chondrocyte

Synergistic interactions between HDAC and sirtuin inhibitors in human leukemia cells.

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Michele Cea

Full Text Available Aberrant histone deacetylase (HDAC activity is frequent in human leukemias. However, while classical, NAD(+-independent HDACs are an established therapeutic target, the relevance of NAD(+-dependent HDACs (sirtuins in leukemia treatment remains unclear. Here, we assessed the antileukemic activity of sirtuin inhibitors and of the NAD(+-lowering drug FK866, alone and in combination with traditional HDAC inhibitors. Primary leukemia cells, leukemia cell lines, healthy leukocytes and hematopoietic progenitors were treated with sirtuin inhibitors (sirtinol, cambinol, EX527 and with FK866, with or without addition of the HDAC inhibitors valproic acid, sodium butyrate, and vorinostat. Cell death was quantified by propidium iodide cell staining and subsequent flow-cytometry. Apoptosis induction was monitored by cell staining with FITC-Annexin-V/propidium iodide or with TMRE followed by flow-cytometric analysis, and by measuring caspase3/7 activity. Intracellular Bax was detected by flow-cytometry and western blotting. Cellular NAD(+ levels were measured by enzymatic cycling assays. Bax was overexpressed by retroviral transduction. Bax and SIRT1 were silenced by RNA-interference. Sirtuin inhibitors and FK866 synergistically enhanced HDAC inhibitor activity in leukemia cells, but not in healthy leukocytes and hematopoietic progenitors. In leukemia cells, HDAC inhibitors were found to induce upregulation of Bax, a pro-apoptotic Bcl2 family-member whose translocation to mitochondria is normally prevented by SIRT1. As a result, leukemia cells become sensitized to sirtuin inhibitor-induced apoptosis. In conclusion, NAD(+-independent HDACs and sirtuins cooperate in leukemia cells to avoid apoptosis. Combining sirtuin with HDAC inhibitors results in synergistic antileukemic activity that could be therapeutically exploited.

Screening of protein kinase inhibitors identifies PKC inhibitors as inhibitors of osteoclastic acid secretion and bone resorption

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Boutin Jean A

2010-10-01

Full Text Available Abstract Background Bone resorption is initiated by osteoclastic acidification of the resorption lacunae. This process is mediated by secretion of protons through the V-ATPase and chloride through the chloride antiporter ClC-7. To shed light on the intracellular signalling controlling extracellular acidification, we screened a protein kinase inhibitor library in human osteoclasts. Methods Human osteoclasts were generated from CD14+ monocytes. The effect of different kinase inhibitors on lysosomal acidification in human osteoclasts was investigated using acridine orange for different incubation times (45 minutes, 4 and 24 hours. The inhibitors were tested in an acid influx assay using microsomes isolated from human osteoclasts. Bone resorption by human osteoclasts on bone slices was measured by calcium release. Cell viability was measured using AlamarBlue. Results Of the 51 compounds investigated only few inhibitors were positive in both acidification and resorption assays. Rottlerin, GF109203X, Hypericin and Ro31-8220 inhibited acid influx in microsomes and bone resorption, while Sphingosine and Palmitoyl-DL-carnitine-Cl showed low levels of inhibition. Rottlerin inhibited lysosomal acidification in human osteoclasts potently. Conclusions In conclusion, a group of inhibitors all indicated to inhibit PKC reduced acidification in human osteoclasts, and thereby bone resorption, indicating that acid secretion by osteoclasts may be specifically regulated by PKC in osteoclasts.

Inhibition of SRC-3 enhances sensitivity of human cancer cells to histone deacetylase inhibitors

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Zou, Zhengzhi, E-mail: zouzhengzhi@m.scnu.edu.cn [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510000 (China); Luo, Xiaoyong [Department of Oncology, The Affiliated Luoyang Central Hospital of Zhengzhou University, Luoyang 471000 (China); Nie, Peipei [KingMed Diagnostics and KingMed School of Laboratory Medicine, Guangzhou Medical University, Guangzhou 510000 (China); Wu, Baoyan; Zhang, Tao; Wei, Yanchun [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510000 (China); Wang, Wenyi [Xiamen Cancer Center, Department of Medical Oncology, The First Affiliated Hospital of Xiamen University, Xiamen 361000 (China); Geng, Guojun; Jiang, Jie [Xiamen Cancer Center, Department of Thoracic Surgery, The First Affiliated Hospital of Xiamen University, Xiamen 361000 (China); Mi, Yanjun, E-mail: myjgj_77@163.com [Xiamen Cancer Center, Department of Medical Oncology, The First Affiliated Hospital of Xiamen University, Xiamen 361000 (China)

2016-09-09

SRC-3 is widely expressed in multiple tumor types and involved in cancer cell proliferation and apoptosis. Histone deacetylase (HDAC) inhibitors are promising antitumor drugs. However, the poor efficacy of HDAC inhibitors in solid tumors has restricted its further clinical application. Here, we reported the novel finding that depletion of SRC-3 enhanced sensitivity of breast and lung cancer cells to HDAC inhibitors (SAHA and romidepsin). In contrast, overexpression of SRC-3 decreased SAHA-induced cancer cell apoptosis. Furthermore, we found that SRC-3 inhibitor bufalin increased cancer cell apoptosis induced by HDAC inhibitors. The combination of bufalin and SAHA was particular efficient in attenuating AKT activation and reducing Bcl-2 levels. Taken together, these accumulating data might guide development of new breast and lung cancer therapies. - Highlights: • Depletion of SRC-3 enhanced sensitivity of breast and lung cancer cells to HDAC inhibitors. • Overexpression of SRC-3 enhanced cancer cell resistance to HDAC inhibitors. • SRC-3 inhibitor bufalin increased cancer cell apoptosis induced by HDAC inhibitors. • Bufalin synergized with HDAC inhibitor attenuated AKT activation and reduced Bcl-2 levels in human cancer cell.

Prediction of the binding mode and resistance profile for a dual-target pyrrolyl diketo acid scaffold against HIV-1 integrase and reverse-transcriptase-associated ribonuclease H.

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Yang, Fengyuan; Zheng, Guoxun; Fu, Tingting; Li, Xiaofeng; Tu, Gao; Li, Ying Hong; Yao, Xiaojun; Xue, Weiwei; Zhu, Feng

2018-06-27

The rapid emergence of drug-resistant variants is one of the most common causes of highly active antiretroviral therapeutic (HAART) failure in patients infected with HIV-1. Compared with the existing HAART, the recently developed pyrrolyl diketo acid scaffold targeting both HIV-1 integrase (IN) and reverse transcriptase-associated ribonuclease H (RNase H) is an efficient approach to counteract the failure of anti-HIV treatment due to drug resistance. However, the binding mode and potential resistance profile of these inhibitors with important mechanistic principles remain poorly understood. To address this issue, an integrated computational method was employed to investigate the binding mode of inhibitor JMC6F with HIV-1 IN and RNase H. By using per-residue binding free energy decomposition analysis, the following residues: Asp64, Thr66, Leu68, Asp116, Tyr143, Gln148 and Glu152 in IN, Asp443, Glu478, Trp536, Lys541 and Asp549 in RNase H were identified as key residues for JMC6F binding. And then computational alanine scanning was carried to further verify the key residues. Moreover, the resistance profile of the currently known major mutations in HIV-1 IN and 2 mutations in RNase H against JMC6F was predicted by in silico mutagenesis studies. The results demonstrated that only three mutations in HIV-1 IN (Y143C, Q148R and N155H) and two mutations in HIV-1 RNase H (Y501R and Y501W) resulted in a reduction of JMC6F potency, thus indicating their potential role in providing resistance to JMC6F. These data provided important insights into the binding mode and resistance profile of the inhibitors with a pyrrolyl diketo acid scaffold in HIV-1 IN and RNase H, which would be helpful for the development of more effective dual HIV-1 IN and RNase H inhibitors.

Localization in the Nucleolus and Coiled Bodies of Protein Subunits of the Ribonucleoprotein Ribonuclease P

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Jarrous, Nayef; Wolenski, Joseph S.; Wesolowski, Donna; Lee, Christopher; Altman, Sidney

1999-01-01

The precise location of the tRNA processing ribonucleoprotein ribonuclease P (RNase P) and the mechanism of its intranuclear distribution have not been completely delineated. We show that three protein subunits of human RNase P (Rpp), Rpp14, Rpp29 and Rpp38, are found in the nucleolus and that each can localize a reporter protein to nucleoli of cells in tissue culture. In contrast to Rpp38, which is uniformly distributed in nucleoli, Rpp14 and Rpp29 are confined to the dense fibrillar component. Rpp29 and Rpp38 possess functional, yet distinct domains required for subnucleolar localization. The subunit Rpp14 lacks such a domain and appears to be dependent on a piggyback process to reach the nucleolus. Biochemical analysis suggests that catalytically active RNase P exists in the nucleolus. We also provide evidence that Rpp29 and Rpp38 reside in coiled bodies, organelles that are implicated in the biogenesis of several other small nuclear ribonucleoproteins required for processing of precursor mRNA. Because some protein subunits of RNase P are shared by the ribosomal RNA processing ribonucleoprotein RNase MRP, these two evolutionary related holoenzymes may share common intranuclear localization and assembly pathways to coordinate the processing of tRNA and rRNA precursors. PMID:10444065

Glucose dynamics and mechanistic implications of SGLT2 inhibitors in animals and humans.

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List, James F; Whaley, Jean M

2011-03-01

Glucose is freely filtered in the glomeruli before being almost entirely reabsorbed into circulation from the proximal renal tubules. The sodium-glucose cotransporter 2 (SGLT2), present in the S1 segment of the proximal tubule, is responsible for the majority of glucose reabsorption. SGLT2 inhibitors reduce glucose reabsorption and increase urinary glucose excretion. In animal models and humans with type 2 diabetes, this effect is associated with reduced fasting and postprandial blood glucose levels, and reduced hemoglobin A1c. Animal studies suggest that reduction of hyperglycemia with SGLT2 inhibitors may also improve insulin sensitivity and preserve β-cell function. Urinary excretion of excess calories with SGLT2 inhibitors is also associated with reduction in body weight. Modest reductions in blood pressure have been noted with SGLT2 inhibitors, consistent with a mild diuretic action. Some C-glucoside SGLT2 inhibitors, such as dapagliflozin, have pharmacokinetic properties that make them amenable to once-daily dosing.

Crystallization and preliminary crystallographic studies of a cysteine protease inhibitor from the human nematode parasite Ascaris lumbricoides

International Nuclear Information System (INIS)

Liu, Sanling; Dong, Jianmei; Mei, Guoqiang; Liu, Guiyun; Xu, Wei; Su, Zhong; Liu, Jinsong

2011-01-01

A recombinant cysteine protease inhibitor from the human nematode parasite A. lumbricoides has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.1 Å resolution. The cysteine protease inhibitor from Ascaris lumbricoides, a roundworm that lives in the human intestine, may be involved in the suppression of human immune responses. Here, the molecular cloning, protein expression and purification, preliminary crystallization and crystallographic characterization of the cysteine protease inhibitor from A. lumbricoides are reported. The rod-shaped crystal belonged to space group C2, with unit-cell parameters a = 99.40, b = 37.52, c = 62.92 Å, β = 118.26°. The crystal diffracted to 2.1 Å resolution and contained two molecules in the asymmetric unit

Discovery of a novel dual fungal CYP51/human 5-lipoxygenase inhibitor: implications for anti-fungal therapy.

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Eric K Hoobler

Full Text Available We report the discovery of a novel dual inhibitor targeting fungal sterol 14α-demethylase (CYP51 or Erg11 and human 5-lipoxygenase (5-LOX with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. A series of potent 5-LOX inhibitors containing a phenylenediamine core, were synthesized that exhibit nanomolar potency and >30-fold selectivity against the LOX paralogs, platelet-type 12-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity against ovine cyclooxygenase-1 and human cyclooxygnease-2. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual inhibitor against human 5-LOX (IC50 = 700 nM and CYP51 (IC50 = 43 nM in vitro. It was tested in whole blood and found to down-regulate LTB4 synthesis, displaying 45% inhibition at 10 µM. In addition, ketaminazole selectively inhibited yeast CYP51 relative to human CYP51 by 17-fold, which is greater selectivity than that of ketoconazole and could confer a therapeutic advantage. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have therapeutic uses against fungal infections that have an anti-inflammatory component.

Ticlopidine in Its Prodrug Form Is a Selective Inhibitor of Human NTPDase1

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Joanna Lecka

2014-01-01

Full Text Available Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, like other ectonucleotidases, controls extracellular nucleotide levels and consequently their (pathophysiological responses such as in thrombosis, inflammation, and cancer. Selective NTPDase1 inhibitors would therefore be very useful. We previously observed that ticlopidine in its prodrug form, which does not affect P2 receptor activity, inhibited the recombinant form of human NTPDase1 (Ki=14 μM. Here we tested whether ticlopidine can be used as a selective inhibitor of NTPDase1. We confirmed that ticlopidine inhibits NTPDase1 in different forms and in different assays. The ADPase activity of intact HUVEC as well as of COS-7 cells transfected with human NTPDase1 was strongly inhibited by 100 µM ticlopidine, 99 and 86%, respectively. Ticlopidine (100 µM completely inhibited the ATPase activity of NTPDase1 in situ as shown by enzyme histochemistry with human liver and pancreas sections. Ticlopidine also inhibited the activity of rat and mouse NTPDase1 and of potato apyrase. At 100 µM ticlopidine did not affect the activity of human NTPDase2, NTPDase3, and NTPDase8, nor of NPP1 and NPP3. Weak inhibition (10–20% of NTPDase3 and -8 was observed at 1 mM ticlopidine. These results show that ticlopidine is a specific inhibitor of NTPDase1 that can be used in enzymatic and histochemistry assays.

Influenza virus inactivated by artificial ribonucleases as a prospective killed virus vaccine.

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Fedorova, Antonina A; Goncharova, Elena P; Kovpak, Mikhail P; Vlassov, Valentin V; Zenkova, Marina A

2012-04-19

The inactivation of viral particles with agents causing minimal damage to the structure of surface epitopes is a well-established approach for the production of killed virus vaccines. Here, we describe new agents for the inactivation of influenza virus, artificial ribonucleases (aRNases), which are chemical compounds capable of cleaving RNA molecules. Several aRNases were identified, exhibiting significant virucidal activity against the influenza A virus and causing a minimal effect on the affinity of monoclonal antibodies for the inactivated virus. Using a murine model of the influenza virus infection, a high protective activity of the aRNase-inactivated virus as a vaccine was demonstrated. The results of the experiments demonstrate the efficacy of novel chemical agents in the preparation of vaccines against influenza and, perhaps, against other infections caused by RNA viruses. Copyright © 2012 Elsevier Ltd. All rights reserved.

A Ribonuclease Isolated from Wild Ganoderma Lucidum Suppressed Autophagy and Triggered Apoptosis in Colorectal Cancer Cells.

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Dan, Xiuli; Liu, Wenlong; Wong, Jack H; Ng, Tzi B

2016-01-01

The mushroom Ganoderma lucidum (G. lucidum) has been consumed in China as a medicine for promoting health and longevity for thousands of years. Due to its paramount and multiple pharmaceutical effects, G. lucidum has received considerable attention from researchers and its chemical constituents as well as their respective functions were gradually unveiled by using modern research methods. Herein, we reported the isolation of a protein (Ganoderma lucidum ribonuclease, GLR) with anti-colorectal cancer activities from G. lucidum. This protein is a 17.4-kDa RNA degrading enzyme (ribonuclease) and was purified by using liquid chromatography procedures. GLR manifested potent anti-proliferative and anti-colony formation activities on HT29 and HCT116 colorectal cancer cells by inducing cell cycle arrest in G1 phase through the regulation of cyclin D1 and P53 expression. GLR was demonstrated to induce cell apoptosis in HCT116 cells by activating unfolded protein response and caspase-9 regulated pathways. Besides, the ability to undergo autophagy which is a stress adaption mechanism to cope with metabolic crisis was significantly suppressed by GLR treatment in HCT116 cells. The activation of apoptosis in GLR-treated HT29 cells was, however, independent of caspase-9 and the suppression of autophagy was also relatively minor. Thus the apoptosis of HT29 cells triggered by GLR was much milder than that in HCT116 cells. Our findings show that the RNase from G. lucidum may be one of the bioactive components that contribute to the anti-colorectal cancer activity of G. lucidum.

Differential regulation of TNF-α and IL-1β production from endotoxin stimulated human monocytes by phosphodiesterase inhibitors

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K. L. Molnar-Kimber

1992-01-01

Full Text Available The effect of selective PDE-I (vinpocetine, PDE-III (milrinone, CI-930, PDE-IV (rolipram, nitroquazone, and PDE-V (zaprinast isozyme inhibitors on TNF-α and IL-1β production from LPS stimulated human monocytes was investigated. The PDE-IV inhibitors caused a concentration dependent inhibition of TNF-α production, but only partially inhibited IL-1β at high concentrations. High concentrations of the PDE-III inhibitors weakly inhibited TNF-α, but had no effect on IL-1β production. PDE-V inhibition was associated with an augmentation of cytokine secretion. Studies with combinations of PDE isozyme inhibitors indicated that PDE-III and PDE-V inhibitors modulate rolipram's suppression of TNF production in an additive manner. These data confirm that TNF-α and IL-1β production from LPS stimulated human monocytes are differentially regulated, and suggest that PDE-IV inhibitors have the potential to suppress TNF levels in man.

Glucocorticoids and Polyamine Inhibitors Synergize to Kill Human Leukemic CEM Cells

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Aaron L. Miller

2002-01-01

Full Text Available Glucocorticoids are well-known apoptotic agents in certain classes of lymphoid cell malignancies. Reduction of intracellular polyamine levels by use of inhibitors that block polyamine synthesis slows or inhibits growth of many cells in vitro. Several such inhibitors have shown efficacy in clinical trials, though the toxicity of some compounds has limited their usefulness. We have tested the effects of combinations of the glucocorticoid dexamethasone. (20Dex and two polyamine inhibitors, difluoromethylornithine. (20DFMO and methyl glyoxal bis guanylhydrazone. (20MGBG, on the clonal line of human acute lymphoblastic leukemia cells, CEM-C7-14. Dex alone kills these cells, though only after a delay of at least 24 hours. We also evaluated a partially glucocorticoid-resistant c-Myc-expressing CEM-C7-14 clone. We show that Dex downregulates ornithine decarboxylase. (20ODC, the rate-limiting enzyme in polyamine synthesis. Pretreatment with the ODC inhibitor DFMO, followed by addition of Dex, enhances steroid-evoked kill slightly. The combination of pretreatment with sublethal concentrations of both DFMO and the inhibitor of S-adenosylmethionine decarboxylase, MGBG, followed by addition of Dex, results in strong synergistic cell kill. Both the rapidity and extent of cell kill are enhanced compared to the effects of Dex alone. These results suggest that use of such combinations in vivo may result in apoptosis of malignant cells with lower overall toxicity.

Identification of a macromolecular crystal growth inhibitor in human urine as osteopontin

DEFF Research Database (Denmark)

Sørensen, Steen; Justesen, S J; Johnsen, A H

1995-01-01

, an unidentified protein rich in uronic acid, and uropontin have all been described as possessing such activity. We have recently isolated an unknown inhibitor of calcium oxalate crystal growth that co-eluted with trypsin inhibitor in several separation steps, which suggested its identity. The aim of the present......Macromolecules occurring in human urine inhibit the growth and/or aggregation of calcium oxalate crystals and may prevent the formation of kidney stones. Attention has focused particularly on proteins, as these seem to be most responsible for the inhibitory activity; three proteins, nephrocalcin...... study was to outline a simple procedure for isolating and identifying this inhibitor. Purification was done as follows: precipitation of the major proteins (albumin and uromucoid) with trichloroacetic acid, followed by anion exchange chromatography, hydroxyapatite chromatography, anion exchange...

Mechanism-based Inhibitors of the Human Sirtuin 5 Deacylase: Structure-Activity Relationship, Biostructural, and Kinetic Insight

DEFF Research Database (Denmark)

Rajabi, Nima; Auth, Marina; Troelsen, Kathrin Rentzius

2017-01-01

to date. We provide rationalization of the mode of binding by solving co-crystal structures of selected inhibitors in complex with both human and zebrafish SIRT5, which provide insight for future optimization of inhibitors with more "drug-like" properties. Importantly, enzyme kinetic evaluation revealed...

Discovery of natural mouse serum derived HIV-1 entry inhibitor(s).

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Wei, M; Chen, Y; Xi, J; Ru, S; Ji, M; Zhang, D; Fang, Q; Tang, B

Among rationally designed human immunodeficiency virus 1 (HIV-1) inhibitors, diverse natural factors have showed as potent anti-HIV activity in human blood. We have discovered that the boiled supernatant of healthy mouse serum could suppress HIV-1 entry, and exhibited reduced inhibitory activity after trypsin digestion. Further analysis demonstrated that only the fraction containing 10-25 K proteins could inhibit HIV-1 mediated cell-cell fusion. These results suggest that the 10-25 K protein(s) is novel natural HIV-1 entry inhibitor(s). Our findings provide important information about novel natural HIV entry inhibitors in mouse serum.

Pronounced radiosensitization of cultured human cancer cells by COX inhibitor under acidic microenvironment

International Nuclear Information System (INIS)

Shah, Tushar; Ryu, Samuel; Lee, Ho Jun; Brown, Stephen; Kim, Jae Ho

2002-01-01

Purpose: To demonstrate the influence of pH on the cytotoxicity and radiosensitization by COX (cyclooxygenase) -1 and -2 inhibitors using established human cancer cells in culture. Methods and Materials: Nonselective COX inhibitor, ibuprofen (IB), and selective COX-2 inhibitor, SC-236, were used to determine the cytotoxicity and radiosensitization at varying pH of culture media. Human colon carcinoma cell line (HT-29) was exposed to the drug alone and in combination with radiation at different pH of the cell culture media. The end point was clonogenic ability of the single-plated cells after the treatment. Results: Cytotoxicity and radiosensitization of IB increased with higher drug concentration and longer exposure time. The most significant radiosensitization was seen with IB (1.5 mM) for 2-h treatment at pH 6.7 before irradiation. The dose-modifying factor as defined by the ratio of radiation doses required to achieve the same effect on cell survival was 1.8 at 10% survival level. In contrast, SC-236 (50 μM for 2-8 h) showed no pH-dependent cytotoxicity. There was modest increase in the cell killing at lower doses of radiation. Conclusion: An acidic pH was an important factor affecting the increased cytotoxicity and radiosensitization by ibuprofen. Radiation response was enhanced at shoulder portion of the cell survival curve by selective COX-2 inhibitor

Flavonoids as Inhibitors of Human Butyrylcholinesterase Variants

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Maja Katalinić

2014-01-01

Full Text Available The inhibition of butyrylcholinesterase (BChE, EC 3.1.1.8 appears to be of interest in treating diseases with symptoms of reduced neurotransmitter levels, such as Alzheimer’s disease. However, BCHE gene polymorphism should not be neglected in research since it could have an effect on the expected outcome. Several well-known cholinergic drugs (e.g. galantamine, huperzine and rivastigmine originating from plants, or synthesised as derivatives of plant compounds, have shown that herbs could serve as a source of novel target-directed compounds. We focused our research on flavonoids, biologically active polyphenolic compounds found in many plants and plant-derived products, as BChE inhibitors. All of the tested flavonoids: galangin, quercetin, fisetin and luteolin reversibly inhibited usual, atypical, and fluoride-resistant variants of human BChE. The inhibition potency increased in the following order, identically for all three BChE variants: luteolininhibitor dissociation constants (Ki ranged from 10 to 170 mmol/L. We showed that no significant change in the inhibition potency of selected flavonoids exists in view of BChE polymorphism. Our results suggested that flavonoids could assist the further development of new BChE-targeted drugs for treating symptoms of neurodegenerative diseases and dementia.

Potent inhibition of mammalian ribonucleases by 3', 5'-pyrophosphate-linked nucleotides.

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Russo, N; Shapiro, R

1999-05-21

Molecular modeling based on the crystal structure of the complex of bovine pancreatic RNase A with the inhibitor 5'-diphosphoadenosine 3'-phosphate (ppAp) (Leonidas, D. D., Shapiro, R., Irons, L. I., Russo, N., and Acharya, K. R. (1997) Biochemistry 36, 5578-5588) was used to design new inhibitors that extend into unoccupied regions of the enzyme active site. These compounds are dinucleotides that contain an unusual 3',5'-pyrophosphate linkage and were synthesized in solution by a combined chemical and enzymatic procedure. The most potent of them, 5'-phospho-2'-deoxyuridine 3'-pyrophosphate, P' --> 5'-ester with adenosine 3'-phosphate (pdUppAp), binds to RNase A with Ki values of 27 and 220 nM at pH 5.9 and 7, respectively. These values are 6-9-fold lower than those for ppAp and 50-fold lower than that for the transition state analogue, uridine vanadate. pdUppAp has broad specificity; it is an effective inhibitor of at least two other members of the pancreatic RNase superfamily, human RNase-2 (eosinophil-derived neurotoxin) and RNase-4, which share only 36-44% sequence identity with the pancreatic enzyme. The potency of pdUppAp and the other inhibitors described here depends critically on the extended internucleotide linkage; the pyrophosphate group enhances dinucleotide binding to the three RNases by 2.1-2.9 orders of magnitude, as compared with a monophosphate. These data give further insight into the organization of the catalytic centers of the various RNases. Moreover, the new class of inhibitors provides a useful means by which to probe the biological actions of these and other related enzymes.

Blocking the proliferation of human tumor cell lines by peptidase inhibitors from Bauhinia seeds.

Science.gov (United States)

Nakahata, Adriana Miti; Mayer, Barbara; Neth, Peter; Hansen, Daiane; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela

2013-03-01

In cancer tumors, growth, invasion, and formation of metastasis at a secondary site play a pivotal role, participating in diverse processes in the development of the pathology, such as degradation of extracellular matrix. Bauhinia seeds contain relatively large quantities of peptidase inhibitors, and two Bauhinia inhibitors were obtained in a recombinant form from the Bauhinia bauhinioides species, B. bauhinoides cruzipain inhibitor, which is a cysteine and serine peptidase inhibitor, and B. bauhinioides kallikrein inhibitor, which is a serine peptidase inhibitor. While recombinant B. bauhinoides cruzipain inhibitor inhibits human neutrophil elastase cathepsin G and the cysteine proteinase cathepsin L, recombinant B. bauhinioides kallikrein inhibitor inhibits plasma kallikrein and plasmin. The effects of recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor on the viability of tumor cell lines with a distinct potential of growth from the same tissue were compared to those of the clinical cytotoxic drug 5-fluorouracil. At 12.5 µM concentration, recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor were more efficient than 5-fluorouracil in inhibiting MKN-28 and Hs746T (gastric), HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), and THP-1 and K562 (leukemia) cell lines. Additionally, recombinant B. bauhinoides cruzipain inhibitor inhibited 40 % of the migration of Hs746T, the most invasive gastric cell line, while recombinant B. bauhinioides kallikrein inhibitor did not affect cell migration. Recombinant B. bauhinioides kallikrein inhibitor and recombinant B. bauhinoides cruzipain inhibitor, even at high doses, did not affect hMSC proliferation while 5-fluorouracil greatly reduced the proliferation rates of hMSCs. Therefore, both recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor might be considered for further studies

Design, synthesis and biological evaluations of N-Hydroxy thienopyrimidine-2,4-diones as inhibitors of HIV reverse transcriptase-associated RNase H.

Science.gov (United States)

Kankanala, Jayakanth; Kirby, Karen A; Huber, Andrew D; Casey, Mary C; Wilson, Daniel J; Sarafianos, Stefan G; Wang, Zhengqiang

2017-12-01

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) is the only HIV enzymatic function not targeted by current antiviral drugs. Although various chemotypes have been reported to inhibit HIV RNase H, few have shown significant antiviral activities. We report herein the design, synthesis and biological evaluation of a novel N-hydroxy thienopyrimidine-2,3-dione chemotype (11) which potently and selectively inhibited RNase H with considerable potency against HIV-1 in cell culture. Current structure-activity-relationship (SAR) identified analogue 11d as a nanomolar inhibitor of RNase H (IC 50  = 0.04 μM) with decent antiviral potency (EC 50  = 7.4 μM) and no cytotoxicity (CC 50  > 100 μM). In extended biochemical assays compound 11d did not inhibit RT polymerase (pol) while inhibiting integrase strand transfer (INST) with 53 fold lower potency (IC 50  = 2.1 μM) than RNase H inhibition. Crystallographic and molecular modeling studies confirmed the RNase H active site binding mode. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Molecular docking and NMR binding studies to identify novel inhibitors of human phosphomevalonate kinase

Energy Technology Data Exchange (ETDEWEB)

Boonsri, Pornthip [Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI 53201 (United States); Department of Chemistry, NANOTEC Center of Nanotechnology, National Nanotechnology Center, Faculty of Science, Kasetsart University, Bangkok 10900 (Thailand); Neumann, Terrence S.; Olson, Andrew L.; Cai, Sheng [Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI 53201 (United States); Herdendorf, Timothy J.; Miziorko, Henry M. [Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110 (United States); Hannongbua, Supa [Department of Chemistry, NANOTEC Center of Nanotechnology, National Nanotechnology Center, Faculty of Science, Kasetsart University, Bangkok 10900 (Thailand); Sem, Daniel S., E-mail: daniel.sem@cuw.edu [Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI 53201 (United States)

2013-01-04

Highlights: Black-Right-Pointing-Pointer Natural and synthetic inhibitors of human phosphomevalonate kinase identified. Black-Right-Pointing-Pointer Virtual screening yielded a hit rate of 15%, with inhibitor K{sub d}'s of 10-60 {mu}M. Black-Right-Pointing-Pointer NMR studies indicate significant protein conformational changes upon binding. -- Abstract: Phosphomevalonate kinase (PMK) phosphorylates mevalonate-5-phosphate (M5P) in the mevalonate pathway, which is the sole source of isoprenoids and steroids in humans. We have identified new PMK inhibitors with virtual screening, using autodock. Promising hits were verified and their affinity measured using NMR-based {sup 1}H-{sup 15}N heteronuclear single quantum coherence (HSQC) chemical shift perturbation and fluorescence titrations. Chemical shift changes were monitored, plotted, and fitted to obtain dissociation constants (K{sub d}). Tight binding compounds with K{sub d}'s ranging from 6-60 {mu}M were identified. These compounds tended to have significant polarity and negative charge, similar to the natural substrates (M5P and ATP). HSQC cross peak changes suggest that binding induces a global conformational change, such as domain closure. Compounds identified in this study serve as chemical genetic probes of human PMK, to explore pharmacology of the mevalonate pathway, as well as starting points for further drug development.

Sodium valproate, a histone deacetylase inhibitor, modulates the vascular endothelial growth inhibitor-mediated cell death in human osteosarcoma and vascular endothelial cells.

Science.gov (United States)

Yamanegi, Koji; Kawabe, Mutsuki; Futani, Hiroyuki; Nishiura, Hiroshi; Yamada, Naoko; Kato-Kogoe, Nahoko; Kishimoto, Hiromitsu; Yoshiya, Shinichi; Nakasho, Keiji

2015-05-01

The level of vascular endothelial growth inhibitor (VEGI) has been reported to be negatively associated with neovascularization in malignant tumors. The soluble form of VEGI is a potent anti-angiogenic factor due to its effects in inhibiting endothelial cell proliferation. This inhibition is mediated by death receptor 3 (DR3), which contains a death domain in its cytoplasmic tail capable of inducing apoptosis that can be subsequently blocked by decoy receptor 3 (DcR3). We investigated the effects of sodium valproate (VPA) and trichostatin A (TSA), histone deacetylase inhibitors, on the expression of VEGI and its related receptors in human osteosarcoma (OS) cell lines and human microvascular endothelial (HMVE) cells. Consequently, treatment with VPA and TSA increased the VEGI and DR3 expression levels without inducing DcR3 production in the OS cell lines. In contrast, the effect on the HMVE cells was limited, with no evidence of growth inhibition or an increase in the DR3 and DcR3 expression. However, VPA-induced soluble VEGI in the OS cell culture medium markedly inhibited the vascular tube formation of HMVE cells, while VEGI overexpression resulted in enhanced OS cell death. Taken together, the HDAC inhibitor has anti-angiogenesis and antitumor activities that mediate soluble VEGI/DR3-induced apoptosis via both autocrine and paracrine pathways. This study indicates that the HDAC inhibitor may be exploited as a therapeutic strategy modulating the soluble VEGI/DR3 pathway in osteosarcoma patients.

Binding site of ribosomal proteins on prokaryotic 5S ribonucleic acids: a study with ribonucleases

DEFF Research Database (Denmark)

Douthwaite, S; Christensen, A; Garrett, R A

1982-01-01

The binding sites of ribosomal proteins L18 and L25 on 5S RNA from Escherichia coli were probed with ribonucleases A, T1, and T2 and a double helix specific cobra venom endonuclease. The results for the protein-RNA complexes, which were compared with those for the free RNA [Douthwaite, S...... stearothermophilus 5S RNA. Several protein-induced changes in the RNA structures were identified; some are possibly allosteric in nature. The two prokaryotic 5S RNAs were also incubated with total 50S subunit proteins from E. coli and B. stearothermophilus ribosomes. Homologous and heterologous reconstitution....... stearothermophilus 5S RNA, which may have been due to a third ribosomal protein L5....

Susceptibility of Human Endogenous Retrovirus Type K to Reverse Transcriptase Inhibitors.

Science.gov (United States)

Contreras-Galindo, Rafael; Dube, Derek; Fujinaga, Koh; Kaplan, Mark H; Markovitz, David M

2017-12-01

Human endogenous retroviruses (HERVs) make up 8% of the human genome. The HERV type K (HERV-K) HML-2 (HK2) family contains proviruses that are the most recent entrants into the human germ line and are transcriptionally active. In HIV-1 infection and cancer, HK2 genes produce retroviral particles that appear to be infectious, yet the replication capacity of these viruses and potential pathogenicity has been difficult to ascertain. In this report, we screened the efficacy of commercially available reverse transcriptase inhibitors (RTIs) at inhibiting the enzymatic activity of HK2 RT and HK2 genomic replication. Interestingly, only one provirus, K103, was found to encode a functional RT among those examined. Several nucleoside analogue RTIs (NRTIs) blocked K103 RT activity and consistently inhibited the replication of HK2 genomes. The NRTIs zidovudine (AZT), stavudine (d4T), didanosine (ddI), and lamivudine (3TC), and the nucleotide RTI inhibitor tenofovir (TDF), show efficacy in blocking K103 RT. HIV-1-specific nonnucleoside RTIs (NNRTIs), protease inhibitors (PIs), and integrase inhibitors (IIs) did not affect HK2, except for the NNRTI etravirine (ETV). The inhibition of HK2 infectivity by NRTIs appears to take place at either the reverse transcription step of the viral genome prior to HK2 viral particle formation and/or in the infected cells. Inhibition of HK2 by these drugs will be useful in suppressing HK2 infectivity if these viruses prove to be pathogenic in cancer, neurological disorders, or other diseases associated with HK2. The present studies also elucidate a key aspect of the life cycle of HK2, specifically addressing how they do, and/or did, replicate. IMPORTANCE Endogenous retroviruses are relics of ancestral virus infections in the human genome. The most recent of these infections was caused by HK2. While HK2 often remains silent in the genome, this group of viruses is activated in HIV-1-infected and cancer cells. Recent evidence suggests that these

Glucocorticoids and Polyamine Inhibitors Synergize to Kill Human Leukemic CEM Cells1

Science.gov (United States)

Miller, Aaron L; Johnson, Betty H; Medh, Rheem D; Townsend, Courtney M; Thompson, E Brad

2002-01-01

Abstract Glucocorticoids are well-known apoptotic agents in certain classes of lymphoid cell malignancies. Reduction of intracellular polyamine levels by use of inhibitors that block polyamine synthesis slows or inhibits growth of many cells in vitro. Several such inhibitors have shown efficacy in clinical trials, though the toxicity of some compounds has limited their usefulness. We have tested the effects of combinations of the glucocorticoid dexamethasone (Dex) and two polyamine inhibitors, difluoromethylornithine (DFMO) and methyl glyoxal bis guanylhydrazone (MGBG), on the clonal line of human acute lymphoblastic leukemia cells, CEM-C7-14. Dex alone kills these cells, though only after a delay of at least 24 hours. We also evaluated a partially glucocorticoid-resistant c-Myc-expressing CEM-C7-14 clone. We show that Dex downregulates ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis. Pretreatment with the ODC inhibitor DFMO, followed by addition of Dex, enhances steroid-evoked kill slightly. The combination of pretreatment with sublethal concentrations of both DFMO and the inhibitor of S-adenosylmethionine decarboxylase, MGBG, followed by addition of Dex, results in strong synergistic cell kill. Both the rapidity and extent of cell kill are enhanced compared to the effects of Dex alone. These results suggest that use of such combinations in vivo may result in apoptosis of malignant cells with lower overall toxicity. PMID:11922393

An in vitro reprogrammable antiviral RISC with size-preferential ribonuclease activity.

Science.gov (United States)

Omarov, Rustem T; Ciomperlik, Jessica; Scholthof, Herman B

2016-03-01

Infection of Nicotiana benthamiana plants with Tomato bushy stunt virus (TBSV) mutants compromised for silencing suppression induces formation of an antiviral RISC (vRISC) that can be isolated using chromatography procedures. The isolated vRISC sequence-specifically degrades TBSV RNA in vitro, its activity can be down-regulated by removing siRNAs, and re-stimulated by exogenous supply of siRNAs. vRISC is most effective at hydrolyzing the ~4.8kb genomic RNA, but less so for a ~2.2kb TBSV subgenomic mRNA (sgRNA1), while the 3' co-terminal sgRNA2 of ~0.9kb appears insensitive to vRISC cleavage. Moreover, experiments with in vitro generated 5' co-terminal viral transcripts show that RNAs of ~2.7kb are efficiently cleaved while those of ~1.1kb or shorter are unaffected. The isolated antiviral ribonuclease complex fails to degrade ~0.4kb defective interfering RNAs (DIs) in vitro, agreeing with findings that in plants DIs are not targeted by silencing. Copyright © 2016. Published by Elsevier Inc.

Recombinant yeast screen for new inhibitors of human acetyl-CoA carboxylase 2 identifies potential drugs to treat obesity

Science.gov (United States)

Marjanovic, Jasmina; Chalupska, Dominika; Patenode, Caroline; Coster, Adam; Arnold, Evan; Ye, Alice; Anesi, George; Lu, Ying; Okun, Ilya; Tkachenko, Sergey; Haselkorn, Robert; Gornicki, Piotr

2010-01-01

Acetyl-CoA carboxylase (ACC) is a key enzyme of fatty acid metabolism with multiple isozymes often expressed in different eukaryotic cellular compartments. ACC-made malonyl-CoA serves as a precursor for fatty acids; it also regulates fatty acid oxidation and feeding behavior in animals. ACC provides an important target for new drugs to treat human diseases. We have developed an inexpensive nonradioactive high-throughput screening system to identify new ACC inhibitors. The screen uses yeast gene-replacement strains depending for growth on cloned human ACC1 and ACC2. In “proof of concept” experiments, growth of such strains was inhibited by compounds known to target human ACCs. The screen is sensitive and robust. Medium-size chemical libraries yielded new specific inhibitors of human ACC2. The target of the best of these inhibitors was confirmed with in vitro enzymatic assays. This compound is a new drug chemotype inhibiting human ACC2 with 2.8 μM IC50 and having no effect on human ACC1 at 100 μM. PMID:20439761

Inhibitors for human glutaminyl cyclase by structure based design and bioisosteric replacement.

Science.gov (United States)

Buchholz, Mirko; Hamann, Antje; Aust, Susanne; Brandt, Wolfgang; Böhme, Livia; Hoffmann, Torsten; Schilling, Stephan; Demuth, Hans-Ulrich; Heiser, Ulrich

2009-11-26

The inhibition of human glutaminyl cyclase (hQC) has come into focus as a new potential approach for the treatment of Alzheimer's disease. The hallmark of this principle is the prevention of the formation of Abeta(3,11(pE)-40,42), as these Abeta-species were shown to be of elevated neurotoxicity and likely to act as a seeding core leading to an accelerated formation of Abeta-oligomers and fibrils. Starting from 1-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea, bioisosteric replacements led to the development of new classes of inhibitors. The optimization of the metal-binding group was achieved by homology modeling and afforded a first insight into the probable binding mode of the inhibitors in the hQC active site. The efficacy assessment of the hQC inhibitors was performed in cell culture, directly monitoring the inhibition of Abeta(3,11(pE)-40,42) formation.

Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors

DEFF Research Database (Denmark)

Ermert, David; Shaughnessy, Jutamas; Joeris, Thorsten

2015-01-01

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and c...... in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy.......Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement...... and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being...

Potent radiolabeled human renin inhibitor, [3H]SR42128: enzymatic, kinetic, and binding studies to renin and other aspartic proteases

International Nuclear Information System (INIS)

Cumin, F.; Nisato, D.; Gagnol, J.P.; Corvol, P.

1987-01-01

The in vitro binding of [ 3 H]SR42128 (Iva-Phe-Nle-Sta-Ala-Sta-Arg), a potent inhibitor of human renin activity, to purified human renin and a number of other aspartic proteases was examined. SR42128 was found to be a competitive inhibitor of human renin, with a K/sub i/ of 0.35 nM at pH 5.7 and 2.0 nM at pH 7.4; it was thus more effective at pH 5.7 than at pH 7.4. Scatchard analysis of the interaction binding of [ 3 H]SR42128 to human renin indicated that binding was reversible and saturable at both pH 5.7 and pH 7.4. There was a single class of binding sites, and the K/sub D/ was 0.9 nM at pH 5.7 and 1 nM at pH 7.4. The association rate was 10 times more rapid at pH 5.7 than at pH 7.4, but there was no difference between the rates of dissociation of the enzyme-inhibitor complex at the two pHs. The effect of pH on the binding of [ 3 H]SR42128 to human renin, cathepsin D, pepsin, and gastricsin was also examined over the pH range 3-8. All the aspartic proteases had a high affinity for the inhibitor at low pH. However, at pH 7.4, [ 3 H]SR42128 was bound only to human renin and to none of the other aspartic proteases. Competitive binding studies with [ 3 H]SR42128 and a number of other inhibitors on human renin or cathepsin D were used to examine the relationships between structure and activity in these systems. The study as a whole indicates that pH plays a major role in the binding of [ 3 H]SR42128 to aspartic proteases and that the nature of the inhibitor residue reacting with the renin S 2 subsites is of critical importance for the specificity of the renin-inhibitor interaction

Chromosomal localization of the human diazepam binding inhibitor gene

International Nuclear Information System (INIS)

DeBernardi, M.A.; Crowe, R.R.; Mocchetti, I.; Shows, T.B.; Eddy, R.L.; Costa, E.

1988-01-01

The authors have used in situ chromosome hybridization and human-mouse somatic cell hybrids to map the gene(s) for human diazepam binding inhibitor (DBI), an endogenous putative modulator of the γ-aminobutyric acid receptor acting at the allosteric regulatory center of this receptor that includes the benzodiazepine recognition site. In 784 chromosome spreads hybridized with human DBI cDNA, the distribution of 1,476 labeled sites revealed a significant clustering of autoradiographic grains (11.3% of total label) on the long arm of chromosome 2 (2q). Furthermore, 63.5% of the grains found on 2q were located on 2q12-21, suggesting regional mapping of DBI gene(s) to this segment. Secondary hybridization signals were frequently observed on other chromosomes and they were statistically significant mainly for chromosomes 5, 6, 11, and 14. In addition, DNA from 32 human-mouse cell hybrids was digested with BamHI and probed with human DBI cDNA. A 3.5-kilobase band, which probably represents the human DBI gene, was assigned to chromosome 2. Four higher molecular weight bands, also detected in BamHI digests, could not be unequivocally assigned. A chromosome 2 location was excluded for the 27-, 13-, and 10-kilobase bands. These results assign a human DBI gene to chromosome 2 (2q12-21) and indicate that three of the four homologous sequences detected by the human DBI probe are located on three other chromosomes

Highly Fluorescent Ribonuclease-A-Encapsulated Lead Sulfide Quantum Dots for Ultrasensitive Fluorescence in Vivo Imaging in the Second Near-Infrared Window

OpenAIRE

Kong, Yifei; Chen, Jun; Fang, Hongwei; Heath, George; Wo, Yan; Wang, Weili; Li, Yunxia; Guo, Yuan; Evans, Stephen D.; Chen, Shiyi; Zhou, Dejian

2016-01-01

Ribonuclease-A (RNase-A) encapsulated PbS quantum dots (RNase-A@PbS Qdots) which emit in the second near-infrared biological window (NIR-II, ca. 1000?1400 nm) are rapidly synthesized under microwave heating. Photoluminescence (PL) spectra of the Qdots can be tuned across the entire NIR-II range by simply controlling synthesis temperature. The size and morphology of the Qdots are examined by transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DL...

A potential human hepatocellular carcinoma inhibitor from Bauhinia purpurea L. seeds: from purification to mechanism exploration.

Science.gov (United States)

Fang, Evandro Fei; Bah, Clara Shui Fern; Wong, Jack Ho; Pan, Wen Liang; Chan, Yau Sang; Ye, Xiu Juan; Ng, Tzi Bun

2012-02-01

A 20-kDa Kunitz-type trypsin-chymotrypsin inhibitor, Bauhinia purpurea trypsin inhibitor (BPLTI), has been isolated from the seeds of B. purpurea L. by using liquid chromatography procedures that involved ion exchange chromatography on Sp-Sepharose and Mono S and gel filtration on Superdex 75. BPLTI demonstrated protease inhibitory activities of 7226 BAEE units/mg and 65 BTEE units/mg toward trypsin and α-chymotrypsin, respectively. BPLTI was relatively thermal (0-60°C) and pH (3-10) stable and its activity could be decreased by dithiothreitol treatment. BPLTI exhibited a wide spectrum of anti-proliferative and pro-apoptotic activities especially on human hepatocellular carcinoma Hep G2 cells. However, it was devoid of a significant antiproliferative effect on immortal human hepatic WRL 68 cells. We show here that BPLTI stimulates apoptosis in Hep G2 cells, including (1) evoking DNA damage including the production of chromatin condensation and apoptotic bodies; (2) induction of cell apoptosis/necrosis; (3) mitochondrial membrane depolarization; and (4) increasing the production of cytokines. Taken together, our findings show for the first time that purified protease inhibitor from B. purpurea L. seeds is a promising candidate for the treatment of human hepatocellular carcinoma.

Blockade of the ERK pathway enhances the therapeutic efficacy of the histone deacetylase inhibitor MS-275 in human tumor xenograft models

Energy Technology Data Exchange (ETDEWEB)

Sakamoto, Toshiaki; Ozaki, Kei-ichi; Fujio, Kohsuke; Kajikawa, Shu-hei [Laboratory of Cell Regulation, Department of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521 (Japan); Uesato, Shin-ichi [Department of Biotechnology, Faculty of Engineering, Kansai University, Osaka 564-8680 (Japan); Watanabe, Kazushi [Proubase Technology Inc., Kanagawa 211-0063 (Japan); Tanimura, Susumu [Laboratory of Cell Regulation, Department of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521 (Japan); Koji, Takehiko [Department of Histology and Cell Biology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8523 (Japan); Kohno, Michiaki, E-mail: kohnom@nagasaki-u.ac.jp [Laboratory of Cell Regulation, Department of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521 (Japan); Proubase Technology Inc., Kanagawa 211-0063 (Japan); Kyoto University Graduate School of Pharmaceutical Sciences, Kyoto 606-8501 (Japan)

2013-04-19

Highlights: •Blockade of the ERK pathway enhances the anticancer efficacy of HDAC inhibitors. •MEK inhibitors sensitize human tumor xenografts to HDAC inhibitor cytotoxicity. •Such the enhanced efficacy is achieved by a transient blockade of the ERK pathway. •This drug combination provides a promising therapeutic strategy for cancer patients. -- Abstract: The ERK pathway is up-regulated in various human cancers and represents a prime target for mechanism-based approaches to cancer treatment. Specific blockade of the ERK pathway alone induces mostly cytostatic rather than pro-apoptotic effects, however, resulting in a limited therapeutic efficacy of the ERK kinase (MEK) inhibitors. We previously showed that MEK inhibitors markedly enhance the ability of histone deacetylase (HDAC) inhibitors to induce apoptosis in tumor cells with constitutive ERK pathway activation in vitro. To evaluate the therapeutic efficacy of such drug combinations, we administered the MEK inhibitor PD184352 or AZD6244 together with the HDAC inhibitor MS-275 in nude mice harboring HT-29 or H1650 xenografts. Co-administration of the MEK inhibitor markedly sensitized the human xenografts to MS-275 cytotoxicity. A dose of MS-275 that alone showed only moderate cytotoxicity thus suppressed the growth of tumor xenografts almost completely as well as induced a marked reduction in tumor cellularity when administered with PD184352 or AZD6244. The combination of the two types of inhibitor also induced marked oxidative stress, which appeared to result in DNA damage and massive cell death, specifically in the tumor xenografts. The enhanced therapeutic efficacy of the drug combination was achieved by a relatively transient blockade of the ERK pathway. Administration of both MEK and HDAC inhibitors represents a promising chemotherapeutic strategy with improved safety for cancer patients.

Development of Pharmacophore Model for Indeno[1,2-b]indoles as Human Protein Kinase CK2 Inhibitors and Database Mining

Directory of Open Access Journals (Sweden)

Samer Haidar

2017-01-01

Full Text Available Protein kinase CK2, initially designated as casein kinase 2, is an ubiquitously expressed serine/threonine kinase. This enzyme, implicated in many cellular processes, is highly expressed and active in many tumor cells. A large number of compounds has been developed as inhibitors comprising different backbones. Beside others, structures with an indeno[1,2-b]indole scaffold turned out to be potent new leads. With the aim of developing new inhibitors of human protein kinase CK2, we report here on the generation of common feature pharmacophore model to further explain the binding requirements for human CK2 inhibitors. Nine common chemical features of indeno[1,2-b]indole-type CK2 inhibitors were determined using MOE software (Chemical Computing Group, Montreal, Canada. This pharmacophore model was used for database mining with the aim to identify novel scaffolds for developing new potent and selective CK2 inhibitors. Using this strategy several structures were selected by searching inside the ZINC compound database. One of the selected compounds was bikaverin (6,11-dihydroxy-3,8-dimethoxy-1-methylbenzo[b]xanthene-7,10,12-trione, a natural compound which is produced by several kinds of fungi. This compound was tested on human recombinant CK2 and turned out to be an active inhibitor with an IC50 value of 1.24 µM.

Nitrile bonds as infrared probes of electrostatics in ribonuclease S.

Science.gov (United States)

Fafarman, Aaron T; Boxer, Steven G

2010-10-28

Three different nitrile-containing amino acids, p-cyanophenylalanine, m-cyanophenylalanine, and S-cyanohomocysteine, have been introduced near the active site of the semisynthetic enzyme ribonuclease S (RNase S) to serve as probes of electrostatic fields. Vibrational Stark spectra, measured directly on the probe-modified proteins, confirm the predominance of the linear Stark tuning rate in describing the sensitivity of the nitrile stretch to external electric fields, a necessary property for interpreting observed frequency shifts as a quantitative measure of local electric fields that can be compared with simulations. The X-ray structures of these nitrile-modified RNase variants and enzymatic assays demonstrate minimal perturbation to the structure and function, respectively, by the probes and provide a context for understanding the influence of the environment on the nitrile stretching frequency. We examine the ability of simulation techniques to recapitulate the spectroscopic properties of these nitriles as a means to directly test a computational electrostatic model for proteins, specifically that in the ubiquitous Amber-99 force field. Although qualitative agreement between theory and experiment is observed for the largest shifts, substantial discrepancies are observed in some cases, highlighting the ongoing need for experimental metrics to inform the development of theoretical models of electrostatic fields in proteins.

A human fatty acid synthase inhibitor binds β-ketoacyl reductase in the keto-substrate site.

Science.gov (United States)

Hardwicke, Mary Ann; Rendina, Alan R; Williams, Shawn P; Moore, Michael L; Wang, Liping; Krueger, Julie A; Plant, Ramona N; Totoritis, Rachel D; Zhang, Guofeng; Briand, Jacques; Burkhart, William A; Brown, Kristin K; Parrish, Cynthia A

2014-09-01

Human fatty acid synthase (hFAS) is a complex, multifunctional enzyme that is solely responsible for the de novo synthesis of long chain fatty acids. hFAS is highly expressed in a number of cancers, with low expression observed in most normal tissues. Although normal tissues tend to obtain fatty acids from the diet, tumor tissues rely on de novo fatty acid synthesis, making hFAS an attractive metabolic target for the treatment of cancer. We describe here the identification of GSK2194069, a potent and specific inhibitor of the β-ketoacyl reductase (KR) activity of hFAS; the characterization of its enzymatic and cellular mechanism of action; and its inhibition of human tumor cell growth. We also present the design of a new protein construct suitable for crystallography, which resulted in what is to our knowledge the first co-crystal structure of the human KR domain and includes a bound inhibitor.

Conserved regions of ribonucleoprotein ribonuclease MRP are involved in interactions with its substrate.

Science.gov (United States)

Esakova, Olga; Perederina, Anna; Berezin, Igor; Krasilnikov, Andrey S

2013-08-01

Ribonuclease (RNase) MRP is a ubiquitous and essential site-specific eukaryotic endoribonuclease involved in the metabolism of a wide range of RNA molecules. RNase MRP is a ribonucleoprotein with a large catalytic RNA moiety that is closely related to the RNA component of RNase P, and multiple proteins, most of which are shared with RNase P. Here, we report the results of an ultraviolet-cross-linking analysis of interactions between a photoreactive RNase MRP substrate and the Saccharomyces cerevisiae RNase MRP holoenzyme. The results show that the substrate interacts with phylogenetically conserved RNA elements universally found in all enzymes of the RNase P/MRP family, as well as with a phylogenetically conserved RNA region that is unique to RNase MRP, and demonstrate that four RNase MRP protein components, all shared with RNase P, interact with the substrate. Implications for the structural organization of RNase MRP and the roles of its components are discussed.

Radiolytic and enzymatic dimerization of tyrosyl residues in insulin, ribonuclease, papain and collagen

Energy Technology Data Exchange (ETDEWEB)

Boguta, G; Dancewicz, A M [Institute of Nuclear Research, Warsaw (Poland)

1983-03-01

Insulin ribonuclease, papain and collagen solutions saturated with nitrogen, N/sub 2/O or air were irradiated with doses of 10 to 640 Gy of gamma rays. Protein solutions were also oxidized enzymatically in a system of horse-radish peroxidase: hydrogen peroxide. Column chromatography (Sephadex G-75 or Sephacryl S-200) of treated protein solutions revealed that they contain protein molecular aggregates. Nitrogen saturation of solution before irradiation was most favourable for radiation-induced aggregation of proteins. Fluorescence analysis of protein solutions resulted in detection of dityrosyl structures in irradiated as well as in enzymatically oxidized proteins. Concentration of dityrosine in proteins studied was determined fluorimetrically in their hydrolysates separated on BioGel P-2 column. In irradiated proteins, dityrosine was present almost exclusively in their aggregated forms. In proteins oxidized enzymatically, dityrosine was also present in fractions containing apparently unchanged protein. Mechanisms which could account for differences in the yield of dityrosine formation in radiolysis and in enzymatic oxidation of proteins are suggested.

Radiolytic and enzymatic dimerization of tyrosyl residues in insulin, ribonuclease, papain and collagen

International Nuclear Information System (INIS)

Boguta, G.; Dancewicz, A.M.

1983-01-01

Insulin ribonuclease, papain and collagen solutions saturated with nitrogen, N 2 O or air were irradiated with doses of 10 to 640 Gy of gamma rays. Protein solutions were also oxidized enzymatically in a system of horse-radish peroxidase: hydrogen peroxide. Column chromatography (Sephadex G-75 or Sephacryl S-200) of treated protein solutions revealed that they contain protein molecular aggregates. Nitrogen saturation of solution before irradiation was most favourable for radiation-induced aggregation of proteins. Fluorescence analysis of protein solutions resulted in detection of dityrosyl structures in irradiated as well as in enzymatically oxidized proteins. Concentration of dityrosine in proteins studied was determined fluorimetrically in their hydrolysates separated on BioGel P-2 column. In irradiated proteins, dityrosine was present almost exclusively in their aggregated forms. In proteins oxidized enzymatically, dityrosine was also present in fractions containing apparently unchanged protein. Mechanisms which could account for differences in the yield of dityrosine formation in radiolysis and in enzymatic oxidation of proteins are suggested. (author)

High throughput Screening to Identify Natural Human Monoamine Oxidase B Inhibitors

Science.gov (United States)

Mazzio, E; Deiab, S; Park, K; Soliman, KFA

2012-01-01

Age-related increase in monoamine oxidase B (MAO-B) may contribute to CNS neurodegenerative diseases. Moreover, MAO-B inhibitors are used in the treatment of idiopathic Parkinson disease as preliminary monotherapy or adjunct therapy with L-dopa. To date, meager natural sources of MAO-B inhibitors have been identified, and the relative strength, potency and rank of many plants relative to standard drugs such as Selegiline (L-deprenyl, Eldepryl) are not known. In this work, we developed and utilized a high throughput enzyme microarray format to screen and evaluate 905 natural product extracts (0.025–.7 mg/ml) to inhibit human MAO-B derived from BTI-TN-5B1-4 cells infected with recombinant baculovirus. The protein sequence of purified enzyme was confirmed using 1D gel electrophoresis-matrix assisted laser desorption ionization-time-of-flight-tandem mass spectroscopy, and enzyme activity was confirmed by [1] substrate conversion (3-mM benzylamine) to H202 and [2] benzaldehyde. Of the 905 natural extracts tested, the lowest IC50s [Comfrey, Bringraj, Skullcap, Kava-kava, Wild Indigo, Gentian and Green Tea. In conclusion, the data reflect relative potency information by rank of commonly used herbs and plants that contain human MAO-B inhibitory properties in their natural form. PMID:22887993

Inhibitors of serotonin reuptake and specific imipramine binding in human blood plasma

International Nuclear Information System (INIS)

Brusov, O.S.; Fomenko, A.M.; Katasonov, A.B.; Lidemann, R.R.

1985-01-01

This paper describes a method of extraction of endogenous inhibitors of specific IMI binding and of 5-HT reuptake, from human blood plasma and the heterogeneity of these compounds is demonstrated. Specific binding was determined as the difference between binding of 3 H-IMI in the absence and in the presence of 50 microM IMI. Under these conditions, specific binding amounted to 70-80% of total binding of 3 H-IMI. It is shown that extract obtained from human blood contains a material which inhibits dose-dependently both 5-HT reuptake and specific binding of 3 H-IMI. Gel-chromatography of extracts of human blood plasma on Biogel P-2 is also shown

VapD in Xylella fastidiosa Is a Thermostable Protein with Ribonuclease Activity.

Science.gov (United States)

Mendes, Juliano S; Santiago, André da S; Toledo, Marcelo A S; Rosselli-Murai, Luciana K; Favaro, Marianna T P; Santos, Clelton A; Horta, Maria Augusta C; Crucello, Aline; Beloti, Lilian L; Romero, Fabian; Tasic, Ljubica; de Souza, Alessandra A; de Souza, Anete P

2015-01-01

Xylella fastidiosa strain 9a5c is a gram-negative phytopathogen that is the causal agent of citrus variegated chlorosis (CVC), a disease that is responsible for economic losses in Brazilian agriculture. The most well-known mechanism of pathogenicity for this bacterial pathogen is xylem vessel occlusion, which results from bacterial movement and the formation of biofilms. The molecular mechanisms underlying the virulence caused by biofilm formation are unknown. Here, we provide evidence showing that virulence-associated protein D in X. fastidiosa (Xf-VapD) is a thermostable protein with ribonuclease activity. Moreover, protein expression analyses in two X. fastidiosa strains, including virulent (Xf9a5c) and nonpathogenic (XfJ1a12) strains, showed that Xf-VapD was expressed during all phases of development in both strains and that increased expression was observed in Xf9a5c during biofilm growth. This study is an important step toward characterizing and improving our understanding of the biological significance of Xf-VapD and its potential functions in the CVC pathosystem.

VapD in Xylella fastidiosa Is a Thermostable Protein with Ribonuclease Activity.

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Juliano S Mendes

Full Text Available Xylella fastidiosa strain 9a5c is a gram-negative phytopathogen that is the causal agent of citrus variegated chlorosis (CVC, a disease that is responsible for economic losses in Brazilian agriculture. The most well-known mechanism of pathogenicity for this bacterial pathogen is xylem vessel occlusion, which results from bacterial movement and the formation of biofilms. The molecular mechanisms underlying the virulence caused by biofilm formation are unknown. Here, we provide evidence showing that virulence-associated protein D in X. fastidiosa (Xf-VapD is a thermostable protein with ribonuclease activity. Moreover, protein expression analyses in two X. fastidiosa strains, including virulent (Xf9a5c and nonpathogenic (XfJ1a12 strains, showed that Xf-VapD was expressed during all phases of development in both strains and that increased expression was observed in Xf9a5c during biofilm growth. This study is an important step toward characterizing and improving our understanding of the biological significance of Xf-VapD and its potential functions in the CVC pathosystem.

A Specific Inhibitor of TGF-β Receptor Kinase, SB-431542, as a Potent Antitumor Agent for Human Cancers

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Sunil K. Halder

2005-05-01

Full Text Available Small molecule inhibitors of signaling pathways have proven to be extremely useful for the development of therapeutic strategies for human cancers. Blocking the tumor-promoting effects of transforming growth factor-β (TGF-β in advanced stage carcinogenesis provides a potentially interesting drug target for therapeutic intervention. Although very few TGF-β receptor kinase inhibitors (TRKI are now emerging in preclinical studies, nothing is known about how these inhibitors might regulate the tumor-suppressive or tumor-promoting effects of TGF-β, or when these inhibitors might be useful for treatment during cancer progression. We have investigated the potential of TRKI in new therapeutic approaches in preclinical models. Here, we demonstrate that the TRKI, SB-431542, inhibits TGF-β-induced transcription, gene expression, apoptosis, and growth suppression. We have observed that SB-431542 attenuates the tumor-promoting effects of TGF-β, including TGF-β-induced EMT, cell motility, migration and invasion, and vascular endothelial growth factor secretion in human cancer cell lines. Interestingly, SB-431542 induces anchorage independent growth of cells that are growth-inhibited by TGF-β, whereas it reduces colony formation by cells that are growth-promoted by TGF-β. However, SB-431542 has no effect on a cell line that failed to respond to TGF-β. This represents a novel potential application of these inhibitors as therapeutic agents for human cancers with the goal of blocking tumor invasion, angiogenesis, and metastasis, when tumors are refractory to TGF-β-induced tumor-suppressor functions but responsive to tumor-promoting effects of TGF-β.

Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells.

Science.gov (United States)

Kang, Minyong; Lee, Kyoung-Hwa; Lee, Hye Sun; Jeong, Chang Wook; Kwak, Cheol; Kim, Hyeon Hoe; Ku, Ja Hyeon

2017-02-04

Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR) inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1), chloroquine (CQ) and 3-methyladenine (3-MA) were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8) assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI) was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA) remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating a novel

Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells

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Minyong Kang

2017-02-01

Full Text Available Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1, chloroquine (CQ and 3-methyladenine (3-MA were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8 assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating

Covalent cross-linking of insulin-like growth factor-1 to a specific inhibitor from human serum

International Nuclear Information System (INIS)

Ooi, G.T.; Herington, A.C.

1986-01-01

Previous studies have shown that a specific inhibitor of insulin-like growth factor (IGF) action in vitro can be isolated from normal human serum and subsequently partially purified on an IGF-affinity column. The ability of the inhibitor to bind the IGFs has now been confirmed directly using covalent cross-linking techniques. When 125 I-IGF-1 was cross-linked to inhibitor using disuccinimidyl suberate, five specifically labelled bands were seen on SDS-PAGE and autoradiography. Two bands (MW 21.5 K and 25.5 K) were intensely labelled, while the remaining three (MW 37 K, 34 K and 18 K) appeared as minor bands only. Inhibitor bioactivity, following further analysis by hydrophobic interaction chromatography or Con A-Sepharose affinity chromatography, was always associated with the presence of the 21.5 K and/or 25.5 K bands

The human immunodeficiency virus protease inhibitor ritonavir is potentially active against urological malignancies

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Sato A

2015-04-01

Full Text Available Akinori Sato Department of Urology, National Defense Medical College, Tokorozawa, Japan Abstract: The human immunodeficiency virus protease inhibitor ritonavir has recently been shown to have antineoplastic activity, and its use in urological malignancies is under investigation with an eye toward drug repositioning. Ritonavir is thought to exert its antineoplastic activity by inhibiting multiple signaling pathways, including the Akt and nuclear factor-kappaB pathways. It can increase the amount of unfolded proteins in the cell by inhibiting both the proteasome and heat shock protein 90. Combinations of ritonavir with agents that increase the amount of unfolded proteins, such as proteasome inhibitors, histone deacetylase inhibitors, or heat shock protein 90 inhibitors, therefore, induce endoplasmic reticulum stress cooperatively and thereby kill cancer cells effectively. Ritonavir is also a potent cytochrome P450 3A4 and P-glycoprotein inhibitor, increasing the intracellular concentration of combined drugs by inhibiting their degradation and efflux from cancer cells and thereby enhancing their antineoplastic activity. Furthermore, riotnavir’s antineoplastic activity includes modulation of immune system activity. Therapies using ritonavir are thus an attractive new approach to cancer treatment and, due to their novel mechanisms of action, are expected to be effective against malignancies that are refractory to current treatment strategies. Further investigations using ritonavir are expected to find new uses for clinically available drugs in the treatment of urological malignancies as well as many other types of cancer. Keywords: drug repositioning, novel treatment

Inhibition of human carboxylesterases hCE1 and hiCE by cholinesterase inhibitors.

Science.gov (United States)

Tsurkan, Lyudmila G; Hatfield, M Jason; Edwards, Carol C; Hyatt, Janice L; Potter, Philip M

2013-03-25

Carboxylesterases (CEs) are ubiquitously expressed proteins that are responsible for the detoxification of xenobiotics. They tend to be expressed in tissues likely to be exposed to such agents (e.g., lung and gut epithelia, liver) and can hydrolyze numerous agents, including many clinically used drugs. Due to the considerable structural similarity between cholinesterases (ChE) and CEs, we have assessed the ability of a series of ChE inhibitors to modulate the activity of the human liver (hCE1) and the human intestinal CE (hiCE) isoforms. We observed inhibition of hCE1 and hiCE by carbamate-containing small molecules, including those used for the treatment of Alzheimer's disease. For example, rivastigmine resulted in greater than 95% inhibition of hiCE that was irreversible under the conditions used. Hence, the administration of esterified drugs, in combination with these carbamates, may inadvertently result in decreased hydrolysis of the former, thereby limiting their efficacy. Therefore drug:drug interactions should be carefully evaluated in individuals receiving ChE inhibitors. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Convergent evolution of ribonuclease h in LTR retrotransposons and retroviruses.

Science.gov (United States)

Ustyantsev, Kirill; Novikova, Olga; Blinov, Alexander; Smyshlyaev, Georgy

2015-05-01

Ty3/Gypsy long terminals repeat (LTR) retrotransposons are structurally and phylogenetically close to retroviruses. Two notable structural differences between these groups of genetic elements are 1) the presence in retroviruses of an additional envelope gene, env, which mediates infection, and 2) a specific dual ribonuclease H (RNH) domain encoded by the retroviral pol gene. However, similar to retroviruses, many Ty3/Gypsy LTR retrotransposons harbor additional env-like genes, promoting concepts of the infective mode of these retrotransposons. Here, we provide a further line of evidence of similarity between retroviruses and some Ty3/Gypsy LTR retrotransposons. We identify that, together with their additional genes, plant Ty3/Gypsy LTR retrotransposons of the Tat group have a second RNH, as do retroviruses. Most importantly, we show that the resulting dual RNHs of Tat LTR retrotransposons and retroviruses emerged independently, providing strong evidence for their convergent evolution. The convergent resemblance of Tat LTR retrotransposons and retroviruses may indicate similar selection pressures acting on these diverse groups of elements and reveal potential evolutionary constraints on their structure. We speculate that dual RNH is required to accelerate retrotransposon evolution through increased rates of strand transfer events and subsequent recombination events. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

VapC from the leptospiral VapBC toxin-antitoxin module displays ribonuclease activity on the initiator tRNA.

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Alexandre P Y Lopes

Full Text Available The prokaryotic ubiquitous Toxin-Antitoxin (TA operons encode a stable toxin and an unstable antitoxin. The most accepted hypothesis of the physiological function of the TA system is the reversible cessation of cellular growth under stress conditions. The major TA family, VapBC is present in the spirochaete Leptospira interrogans. VapBC modules are classified based on the presence of a predicted ribonucleasic PIN domain in the VapC toxin. The expression of the leptospiral VapC in E. coli promotes a strong bacterial growth arrestment, making it difficult to express the recombinant protein. Nevertheless, we showed that long term induction of expression in E. coli enabled the recovery of VapC in inclusion bodies. The recombinant protein was successfully refolded by high hydrostatic pressure, providing a new method to obtain the toxin in a soluble and active form. The structural integrity of the recombinant VapB and VapC proteins was assessed by circular dichroism spectroscopy. Physical interaction between the VapC toxin and the VapB antitoxin was demonstrated in vivo and in vitro by pull down and ligand affinity blotting assays, respectively, thereby indicating the ultimate mechanism by which the activity of the toxin is regulated in bacteria. The predicted model of the leptospiral VapC structure closely matches the Shigella's VapC X-ray structure. In agreement, the ribonuclease activity of the leptospiral VapC was similar to the activity described for Shigella's VapC, as demonstrated by the cleavage of tRNAfMet and by the absence of unspecific activity towards E. coli rRNA. This finding suggests that the cleavage of the initiator transfer RNA may represent a common mechanism to a larger group of bacteria and potentially configures a mechanism of post-transcriptional regulation leading to the inhibition of global translation.

A Dimeric Mutant of Human Pancreatic Ribonuclease with Selective Cytotoxicity toward Malignant Cells

Science.gov (United States)

Piccoli, Renata; di Gaetano, Sonia; de Lorenzo, Claudia; Grauso, Michela; Monaco, Carmen; Spalletti-Cernia, Daniela; Laccetti, Paolo; Cinatl, Jaroslav; Matousek, Josef; D'Alessio, Giuseppe

1999-07-01

Monomeric human pancreatic RNase, devoid of any biological activity other than its RNA degrading ability, was engineered into a dimeric protein with a cytotoxic action on mouse and human tumor cells, but lacking any appreciable toxicity on mouse and human normal cells. This dimeric variant of human pancreas RNase selectively sensitizes to apoptotic death cells derived from a human thyroid tumor. Because of its selectivity for tumor cells, and because of its human origin, this protein represents a potentially very attractive, novel tool for anticancer therapy.

Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation

DEFF Research Database (Denmark)

Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch

2015-01-01

) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced...... to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4...

Dysregulation of protease and protease inhibitors in a mouse model of human pelvic organ prolapse.

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Madhusudhan Budatha

Full Text Available Mice deficient for the fibulin-5 gene (Fbln5(-/- develop pelvic organ prolapse (POP due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5(-/- mice, herein named V1 (25 kDa. V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5(-/- mice. PRSS3 was (a localized in epithelial secretions, (b detected in media of vaginal organ culture from both Fbln5(-/- and wild type mice, and (c cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin and Elafin] was dysregulated in Fbln5(-/- epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice.

The Anti-Inflammatory Effects of Lipoxygenase and Cyclo-Oxygenase Inhibitors in Inflammation-Induced Human Fetal Glia Cells and the Aβ Degradation Capacity of Human Fetal Astrocytes in an Ex vivo Assay

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Rea Pihlaja

2017-05-01

Full Text Available Chronic inflammation is a common phenomenon present in the background of multiple neurodegenerative diseases, including Alzheimer's disease (AD. The arachidonic acid pathway overproduces proinflammatory eicosanoids during these states and glial cells in the brain gradually lose their vital functions of protecting and supporting neurons. In this study, the role of different key enzymes of the eicosanoid pathway mediating inflammatory responses was examined in vitro and ex vivo using human fetal glial cells. Astrocytes and microglia were exposed to proinflammatory agents i.e., cytokines interleukin 1-β (IL-1β and tumor necrosis factor (TNF-α. ELISA assays were used to examine the effects of inhibitors of key enzymes in the eicosanoid pathway. Inhibitors for 5-lipoxygenase (5-LOX and cyclo-oxygenase 2 (COX-2 in both cell types and 5-, 12-, and 15-LOX-inhibitor in astrocytes reduced significantly IL-6 secretion, compared to exposed glial cells without inhibitors. The cytokine antibody array showed that especially treatments with 5, -12, and -15 LOX inhibitor in astrocytes, 5-LOX inhibitor in microglia and COX-2 inhibitor in both glial cell types significantly reduced the expression of multiple proinflammatory cytokines. Furthermore, human fetal astrocytes and microglia were cultured on top of AD-affected and control human brain sections for 30 h. According to the immunochemical evaluation of the level of total Aβ, astrocytes were very efficient at degrading Aβ from AD-affected brain sections ex vivo; simultaneously added enzyme inhibitors did not increase their Aβ degradation capabilities. Microglia were not able to reduce the level of total Aβ during the 30 h incubation time.

Regulation of 2-5A Dependent RNase at the Level of its Phosphorylation

Science.gov (United States)

1991-06-26

extract as follows: 25 ul wheat germ extract 10 ul H2O 1 ul RNasin ribonuclease inhibitor (40 u/ml) 7 ul ImM amino acid mixture 1 ul IM...diacylglycerol (DAG) 2. TPA 3. Indolactam Figure 6. Chemical structure of: 1. H-7 (A kinase inhibitor) 2. okadaic acid (A phosphatase inhibitor) Figure 7...elevating agents: Forskolin and Cholera toxin Figure 17. Down-regulation of 2-5A-depRNase by Okadaic 77 acid : A phosphatase inhibitor Figure 18

Restricted fragmentation of poliovirus type 1, 2, and 3 RNAs by ribonuclease III

Energy Technology Data Exchange (ETDEWEB)

Nomoto, A. (State Univ. of New York, Stony Brook); Lee, Y.F.; Babich, A.; Jacobson, A.; Dunn, J.J.; Wimmer, E.

1979-01-01

Cleavage of the genome RNAs of poliovirus type 1, 2, and 3 with the ribonuclease III of Escherichia coli has been investigated with the following results: (1) at or above physiological salt concentration, the RNAs are completely resistant to the action of the enzyme, an observation suggesting that the RNAs lack primary cleavage sites; (2) lowering the salt concentration to 0.1 M or below allows RNase III to cleave the RNAs at secondary sites. Both large and small fragments can be obtained in a reproducible manner depending on salt conditions chosen for cleavage. Fingerprints of three large fragments of poliovirus type 2 RNA show that they originate from unique segments and represent most if not all sequences of the genome. Based upon binding to poly(U) filters of poly(A)-linked fragments, a physical map of the large fragments of poliovirus type 2 RNA was constructed. The data suggest that RNase III cleavage of single-stranded RNA provides a useful method to fragment the RNA for further studies.

Eukaryotic ribonucleases P/MRP: the crystal structure of the P3 domain.

Science.gov (United States)

Perederina, Anna; Esakova, Olga; Quan, Chao; Khanova, Elena; Krasilnikov, Andrey S

2010-02-17

Ribonuclease (RNase) P is a site-specific endoribonuclease found in all kingdoms of life. Typical RNase P consists of a catalytic RNA component and a protein moiety. In the eukaryotes, the RNase P lineage has split into two, giving rise to a closely related enzyme, RNase MRP, which has similar components but has evolved to have different specificities. The eukaryotic RNases P/MRP have acquired an essential helix-loop-helix protein-binding RNA domain P3 that has an important function in eukaryotic enzymes and distinguishes them from bacterial and archaeal RNases P. Here, we present a crystal structure of the P3 RNA domain from Saccharomyces cerevisiae RNase MRP in a complex with RNase P/MRP proteins Pop6 and Pop7 solved to 2.7 A. The structure suggests similar structural organization of the P3 RNA domains in RNases P/MRP and possible functions of the P3 domains and proteins bound to them in the stabilization of the holoenzymes' structures as well as in interactions with substrates. It provides the first insight into the structural organization of the eukaryotic enzymes of the RNase P/MRP family.

Why Do SGLT2 inhibitors inhibit only 30-50% of renal glucose reabsorption in humans?

Science.gov (United States)

Liu, Jiwen Jim; Lee, TaeWeon; DeFronzo, Ralph A

2012-09-01

Sodium glucose cotransporter 2 (SGLT2) inhibition is a novel and promising treatment for diabetes under late-stage clinical development. It generally is accepted that SGLT2 mediates 90% of renal glucose reabsorption. However, SGLT2 inhibitors in clinical development inhibit only 30-50% of the filtered glucose load. Why are they unable to inhibit 90% of glucose reabsorption in humans? We will try to provide an explanation to this puzzle in this perspective analysis of the unique pharmacokinetic and pharmacodynamic profiles of SGLT2 inhibitors in clinical trials and examine possible mechanisms and molecular properties that may be responsible.

A 96-well automated method to study inhibitors of human sodium-dependent D-glucose transport.

Science.gov (United States)

Castaneda, Francisco; Kinne, Rolf K-H

2005-12-01

The sodium-dependent D-glucose transporter (SGLT) family is involved in glucose uptake via intestinal absorption (SGLT1) or renal reabsorption (SGLT1 and SGLT2). Current methods for the screening of inhibitors of SGLT transporters are complex, expensive and very labor intensive, and have not been applied to human SGLT transporters. The purpose of the present study was to develop an alternative 96-well automated method to study the activity of human SGLT1 and SGLT2. Chinese hamster ovary (CHO) Flp-In cells were stably transfected with pcDNA5-SGLT1 or pcDNA5-SGLT2 plasmid and maintained in hygromycin-selection Ham's F12 culture medium until hygromycin-resistant clones were developed. SGLT1 and SGLT2 gene expression was evaluated by relative real-time reverse transcription-polymerase chain reaction (RT-PCR) quantification, Western blotting, and immunocytochemical analysis. The clones with higher expression of SGLT1 and SGLT2 were used for transport studies using [14C]-methyl-alpha-D-glucopyranoside ([14C]AMG). The advantage of using the 96-well format is the low amount of radioactive compounds and inhibitory substances required, and its ability to establish reproducibility because repetition into the assay. This method represents an initial approach in the development of transport-based high-throughput screening in the search for inhibitors of glucose transport. The proposed method can easily be performed to yield quantitative data regarding key aspects of glucose membrane transport and kinetic studies of potential inhibitors of human SGLT1 and SGLT2.

Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation*

Science.gov (United States)

Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch; Sinning, Steffen; Kristensen, Anders Skov; Strømgaard, Kristian; Andersen, Jacob

2015-01-01

The serotonin transporter (SERT) terminates serotonergic neurotransmission by performing reuptake of released serotonin, and SERT is the primary target for antidepressants. SERT mediates the reuptake of serotonin through an alternating access mechanism, implying that a central substrate site is connected to both sides of the membrane by permeation pathways, of which only one is accessible at a time. The coordinated conformational changes in SERT associated with substrate translocation are not fully understood. Here, we have identified a Leu to Glu mutation at position 406 (L406E) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced by L406E are not simply charge-related effects. Leu406 is located >10 Å from the central inhibitor binding site indicating that the mutation affects inhibitor binding in an indirect manner. We found that L406E decreased accessibility to a residue in the cytoplasmic pathway. The shift in equilibrium to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4 has an important role in controlling the conformational equilibrium of human SERT. PMID:25903124

Molecular Modulation of Inhibitors of Apoptosis as a Novel Approach for Radiosensitization of Human Prostate Cancer

Science.gov (United States)

2008-11-01

in multiple myeloma cells is associated with a cleavage of Mcl-1 and Bim and a decrease in the Mcl-1/Bim...improved tumor control by radiotherapy in vivo [66], advocating a distinct role for XIAP in radiation resistant phenotype of human cancers, and...about the role of endogenous Smac in cells treated with Smac- mimetic IAP-inhibitors and irradiation. In multiple human cancer models,

DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity

International Nuclear Information System (INIS)

Kim, Hak Jae; Kim, Jin Ho; Chie, Eui Kyu; Da Young, Park; Kim, In Ah; Kim, Il Han

2012-01-01

Histone modifications and DNA methylation are two major factors in epigenetic phenomenon. Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies thus far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers. The principal objective of this study was to evaluate the effects of DNMT inhibitors on the radiosensitivity of human cancer cell lines, and to elucidate the mechanisms relevant to that process. A549 (lung cancer) and U373MG (glioblastoma) cells were exposed to radiation with or without six DNMT inhibitors (5-azacytidine, 5-aza-2'-deoxycytidine, zebularine, hydralazine, epigallocatechin gallate, and psammaplin A) for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays. Cell cycle and apoptosis were analyzed via flow cytometry. Expressions of DNMT1, 3A/3B, and cleaved caspase-3 were detected via Western blotting. Expression of γH2AX, a marker of radiation-induced DNA double-strand break, was examined by immunocytochemistry. Pretreatment with psammaplin A, 5-aza-2'-deoxycytidine, and zebularine radiosensitized both A549 and U373MG cells. Pretreatment with psammaplin A increased the sub-G1 fraction of A549 cells, as compared to cells exposed to radiation alone. Prolongation of γH2AX expression was observed in the cells treated with DNMT inhibitors prior to radiation as compared with those treated by radiation alone. Psammaplin A, 5-aza-2'-deoxycytidine, and zebularine induce radiosensitivity in both A549 and U373MG cell lines, and suggest that this effect might be associated with the inhibition of DNA repair

Synthetic Routes to N-9 Alkylated 8-Oxoguanines; Weak Inhibitors of the Human DNA Glycosylase OGG1

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Tushar R. Mahajan

2015-09-01

Full Text Available The human 8-oxoguanine DNA glycosylase OGG1 is involved in base excision repair (BER, one of several DNA repair mechanisms that may counteract the effects of chemo- and radiation therapy for the treatment of cancer. We envisage that potent inhibitors of OGG1 may be found among the 9-alkyl-8-oxoguanines. Thus we explored synthetic routes to 8-oxoguanines and examined these as OGG1 inhibitors. The best reaction sequence started from 6-chloroguanine and involved N-9 alkylation, C-8 bromination, and finally simultaneous hydrolysis of both halides. Bromination before N-alkylation should only be considered when the N-substituent is not compatible with bromination conditions. The 8-oxoguanines were found to be weak inhibitors of OGG1. 6-Chloro-8-oxopurines, byproducts in the hydrolysis of 2,6-halopurines, turned out to be slightly better inhibitors than the corresponding 8-oxoguanines.

DPP4 inhibitors promote biological functions of human endothelial progenitor cells by targeting the SDF-1/CXCR4 signaling pathway

Directory of Open Access Journals (Sweden)

Liu Feng

2016-01-01

Full Text Available Dipeptidyl peptidase 4 (DPP4 inhibitors(oral hypoglycemic agentshave beneficial effects during the early stages of diabetes. In this study, we evaluated the role of DPP4inhibitorsonthe biological functions of cultured human endothelial progenitor cells (EPCs. After treating EPCs with the DPP4 inhibitors sitagliptin and vildagliptin, we examined the mRNA expression of DPP4, vascular endothelial growth factor (VEGF,VEGF receptor 2 (VEGFR-2,endothelial nitric oxide synthase (eNOS, caspase-3,stromal cell-derived factor-1 (SDF-1, chemokine (C-X-C motif receptor 4 (CXCR4 were measured by RT-PCR. The protein expression of SDF-1 and CXCR4 was determined by Western blot; cell proliferation was tested by the MTT method, and DPP4 activity was determined by a DPP4 assay. Our results revealed that DPP4 expression and activity were inhibited following the treatment with various doses of DPP4 inhibitors. Cell proliferation and the expression of VEGF, VEGFR-2andeNOS were up regulated, while cell apoptosis was inhibited by DPP4 inhibitors in a dose-dependent manner. DPP4 inhibitors activated the SDF-1/CXCR4 signaling pathway, shown by the elevated expression of SDF-1/CXCR4. This further proved that after the SDF-1/CXCR4 signaling pathway was blocked by its inhibitor ADM3100, the effects of DPP4 inhibitors on the proliferation and apoptosis, and the expression of VEGF, VEGFR-2and eNOS of EPCs were significantly reduced. These findings suggest that DPP4 inhibitors promote the biological functions of human EPCs by up regulating the SDF-1/CXCR4 signaling pathway.

Monoamine oxidase inhibitors l-deprenyl and clorgyline protect nonmalignant human cells from ionising radiation and chemotherapy toxicity.

LENUS (Irish Health Repository)

Seymour, C B

2003-11-17

l-Deprenyl (R-(-)-deprenyl, selegiline) is an inhibitor of monoamine oxidase-B (MAO-B) that is known to protect nerve cells from a variety of chemical and physical insults. As apoptosis is a common mechanism of radiation-induced cell death, the effect of l-deprenyl on the survival of cultured cells and tissue explants was studied following exposure to gamma radiation. The results obtained were compared with the effects of the less-selective MAO-B inhibitor pargyline and the MAO-A inhibitor clorgyline. l-Deprenyl at a concentration of 10(-9) M protected the nontumorigenic cell line (HaCaT) and normal human urothelial explants from the effects of cobalt-60 gamma radiation, but did not protect tumorigenic human cell lines HaCaT-ras, HPV-transfected human keratinocytes (HPV-G cells), or PC3. Human bladder carcinoma explants were not protected. Clorgyline showed a smaller protective effect of normal cells, whereas pargyline had no effect. Radiation-induced delayed effects (genomic instability measured as delayed cell death) were prevented in normal cells by l-deprenyl but, interestingly, deprenyl appeared to increase the amount of delayed death in the tumorigenic cell lines. Studies using l-deprenyl prior to the exposure of nonmalignant cells to cisplatin showed that cell death due to this agent was also reduced. Treatment of cultures of nontumorigenic cells with l-deprenyl or clorgyline significantly increased the levels of the protein Bcl-2 following irradiation, but there was no such effect on the already-elevated levels of this protein in the tumour samples. Since the Bcl-2 has been shown to be an inhibitor of apoptosis or programmed cell death, this would imply that the protective effects of l-deprenyl and clorgyline involve activation of antiapoptotic pathways within the normal cell. This hypothesis is supported by data showing reduced levels of apoptosis in HaCAT cells and in normal bladder explant cultures following treatment with l-deprenyl.

Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation.

Science.gov (United States)

Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch; Sinning, Steffen; Kristensen, Anders Skov; Strømgaard, Kristian; Andersen, Jacob

2015-06-05

The serotonin transporter (SERT) terminates serotonergic neurotransmission by performing reuptake of released serotonin, and SERT is the primary target for antidepressants. SERT mediates the reuptake of serotonin through an alternating access mechanism, implying that a central substrate site is connected to both sides of the membrane by permeation pathways, of which only one is accessible at a time. The coordinated conformational changes in SERT associated with substrate translocation are not fully understood. Here, we have identified a Leu to Glu mutation at position 406 (L406E) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced by L406E are not simply charge-related effects. Leu(406) is located >10 Å from the central inhibitor binding site indicating that the mutation affects inhibitor binding in an indirect manner. We found that L406E decreased accessibility to a residue in the cytoplasmic pathway. The shift in equilibrium to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4 has an important role in controlling the conformational equilibrium of human SERT. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1

Energy Technology Data Exchange (ETDEWEB)

Lewis-Ballester, Ariel; Pham, Khoa N.; Batabyal, Dipanwita; Karkashon, Shay; Bonanno, Jeffrey B.; Poulos, Thomas L.; Yeh, Syun-Ru (Einstein); (UCI)

2017-11-22

Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination of an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.

Myricetin is a novel inhibitor of human inosine 5′-monophosphate dehydrogenase with anti-leukemia activity

International Nuclear Information System (INIS)

Pan, Huiling; Hu, Qian; Wang, Jingyuan; Liu, Zehui; Wu, Dang; Lu, Weiqiang; Huang, Jin

2016-01-01

Human inosine 5′-monophosphate dehydrogenase (hIMPDH) is a rate-limiting enzyme in the de novo biosynthetic pathway of purine nucleotides, playing crucial roles in cellular proliferation, differentiation, and transformation. Dysregulation of hIMPDH expression and activity have been found in a variety of human cancers including leukemia. In this study, we found that myricetin, a naturally occurring phytochemical existed in berries, wine and tea, was a novel inhibitor of human type 1 and type 2 IMPDH (hIMPDH1/2) with IC_5_0 values of 6.98 ± 0.22 μM and 4.10 ± 0.14 μM, respectively. Enzyme kinetic analysis using Lineweaver-Burk plot revealed that myricetin is a mix-type inhibitor for hIMPDH1/2. Differential scanning fluorimetry and molecular docking simulation data demonstrate that myricetin is capable of binding with hIMPDH1/2. Myricetin treatment exerts potent anti-proliferative and pro-apoptotic effects on K562 human leukemia cells in a dose-dependent manner. Importantly, cytotoxicity of myricetin on K562 cells were markedly attenuated by exogenous addition of guanosine, a salvage pathway of maintaining intracellular pool of guanine nucleotides. Taking together, these results indicate that natural product myricetin exhibits potent anti-leukemia activity by interfering with purine nucleotides biosynthetic pathway through the suppression of hIMPDH1/2 catalytic activity. - Highlights: • Myricetin, a common dietary flavonoid, is a novel inhibitor of hIMPDH1/2. • Myricetin directly binds with hIMPDH1/2 and induces cell cycle arrest and apoptosis of leukemia cells. • The cytotoxicity of myricetin on K562 cells is markedly attenuated by exogenous addition of guanosine.

Myricetin is a novel inhibitor of human inosine 5′-monophosphate dehydrogenase with anti-leukemia activity

Energy Technology Data Exchange (ETDEWEB)

Pan, Huiling; Hu, Qian; Wang, Jingyuan; Liu, Zehui; Wu, Dang [Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237 (China); Lu, Weiqiang, E-mail: wqlu@bio.ecnu.edu.cn [Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241 (China); Huang, Jin, E-mail: huangjin@ecust.edu.cn [Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237 (China)

2016-09-02

Human inosine 5′-monophosphate dehydrogenase (hIMPDH) is a rate-limiting enzyme in the de novo biosynthetic pathway of purine nucleotides, playing crucial roles in cellular proliferation, differentiation, and transformation. Dysregulation of hIMPDH expression and activity have been found in a variety of human cancers including leukemia. In this study, we found that myricetin, a naturally occurring phytochemical existed in berries, wine and tea, was a novel inhibitor of human type 1 and type 2 IMPDH (hIMPDH1/2) with IC{sub 50} values of 6.98 ± 0.22 μM and 4.10 ± 0.14 μM, respectively. Enzyme kinetic analysis using Lineweaver-Burk plot revealed that myricetin is a mix-type inhibitor for hIMPDH1/2. Differential scanning fluorimetry and molecular docking simulation data demonstrate that myricetin is capable of binding with hIMPDH1/2. Myricetin treatment exerts potent anti-proliferative and pro-apoptotic effects on K562 human leukemia cells in a dose-dependent manner. Importantly, cytotoxicity of myricetin on K562 cells were markedly attenuated by exogenous addition of guanosine, a salvage pathway of maintaining intracellular pool of guanine nucleotides. Taking together, these results indicate that natural product myricetin exhibits potent anti-leukemia activity by interfering with purine nucleotides biosynthetic pathway through the suppression of hIMPDH1/2 catalytic activity. - Highlights: • Myricetin, a common dietary flavonoid, is a novel inhibitor of hIMPDH1/2. • Myricetin directly binds with hIMPDH1/2 and induces cell cycle arrest and apoptosis of leukemia cells. • The cytotoxicity of myricetin on K562 cells is markedly attenuated by exogenous addition of guanosine.

Protease inhibitors enhance extracellular collagen fibril deposition in human mesenchymal stem cells.

Science.gov (United States)

Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

2015-10-15

Collagen is a widely used naturally occurring biomaterial for scaffolding, whereas mesenchymal stem cells (MSCs) represent a promising cell source in tissue engineering and regenerative medicine. It is generally known that cells are able to remodel their environment by simultaneous degradation of the scaffolds and deposition of newly synthesized extracellular matrix. Nevertheless, the interactions between MSCs and collagen biomaterials are poorly known, and the strategies enhancing the extracellular matrix deposition are yet to be defined. In this study, we aim to investigate the fate of collagen when it is in contact with MSCs and hypothesize that protease inhibition will enhance their extracellular deposition of collagen fibrils. Specifically, human MSCs (hMSCs) were exposed to fluorescence-labeled collagen with and without intracellular or extracellular protease inhibitors (or both) before tracing the collagen at both intracellular and extracellular spaces. Collagen were internalized by hMSCs and degraded intracellularly in lysosomes. In the presence of protease inhibitors, both intracellular collagen fibril growth and extracellular deposition of collagen fibrils were enhanced. Moreover, protease inhibitors work synergistically with ascorbic acid, a well-known matrix deposition-enhancing reagent, in further enhancing collagen fibril deposition at the extracellular space. These findings provide a better understanding of the interactions between hMSCs and collagen biomaterials and suggest a method to manipulate matrix remodeling and deposition of hMSCs, contributing to better scaffolding for tissue engineering and regenerative medicine.

A new synthetic matrix metalloproteinase inhibitor reduces human mesenchymal stem cell adipogenesis.

Directory of Open Access Journals (Sweden)

Dale B Bosco

Full Text Available Development of adipose tissue requires the differentiation of less specialized cells, such as human mesenchymal stem cells (hMSCs, into adipocytes. Since matrix metalloproteinases (MMPs play critical roles in the cell differentiation process, we conducted investigations to determine if a novel mercaptosulfonamide-based MMP inhibitor (MMPI, YHJ-7-52, could affect hMSC adipogenic differentiation and lipid accumulation. Enzyme inhibition assays, adipogenic differentiation experiments, and quantitative PCR methods were employed to characterize this inhibitor and determine its effect upon adipogenesis. YHJ-7-52 reduced lipid accumulation in differentiated cells by comparable amounts as a potent hydroxamate MMPI, GM6001. However, YHJ-7-82, a non-inhibitory structural analog of YHJ-7-52, in which the zinc-binding thiol group is replaced by a hydroxyl group, had no effect on adipogenesis. The two MMPIs (YHJ-7-52 and GM6001 were also as effective in reducing lipid accumulation in differentiated cells as T0070907, an antagonist of peroxisome-proliferator activated receptor gamma (PPAR-gamma, at a similar concentration. PPAR-gamma is a typical adipogenic marker and a key regulatory protein for the transition of preadiopocyte to adipocyte. Moreover, MMP inhibition was able to suppress lipid accumulation in cells co-treated with Troglitazone, a PPAR-gamma agonist. Our results indicate that MMP inhibitors may be used as molecular tools for adipogenesis and obesity treatment research.

Why Do SGLT2 Inhibitors Inhibit Only 30–50% of Renal Glucose Reabsorption in Humans?

Science.gov (United States)

Liu, Jiwen (Jim); Lee, TaeWeon; DeFronzo, Ralph A.

2012-01-01

Sodium glucose cotransporter 2 (SGLT2) inhibition is a novel and promising treatment for diabetes under late-stage clinical development. It generally is accepted that SGLT2 mediates 90% of renal glucose reabsorption. However, SGLT2 inhibitors in clinical development inhibit only 30–50% of the filtered glucose load. Why are they unable to inhibit 90% of glucose reabsorption in humans? We will try to provide an explanation to this puzzle in this perspective analysis of the unique pharmacokinetic and pharmacodynamic profiles of SGLT2 inhibitors in clinical trials and examine possible mechanisms and molecular properties that may be responsible. PMID:22923645

Sensitization of human cells by inhibitors of DNA synthesis following the action of DNA-damaging agents

Energy Technology Data Exchange (ETDEWEB)

Filatov, M.V.; Noskin, L.A. (Leningrad Inst. of Nuclear Physics, Gatchina (USSR))

1983-08-01

Inhibitors of DNA synthesis 1-..beta..-arabinofuranosylcytosine (Ac) and hydroxyurea (Hu) taken together drastically sensitized human cells to the killing effect of DNA-damaging agents. For UV-irradiation this sensitization depended on the cells' ability for excision repair. By using viscoelastometric methods of measurement of double-strand breaks (DSB) in the genome, it was established that the first DSB were generated after incubation of the damaged cells in the mixture of inhibitors at about the same dose when sensitization appeared. A scheme is proposed to describe molecular events associated with the phenomenon studied. 35 refs.

A phenanthrene derived PARP inhibitor is an extra-centrosomes de-clustering agent exclusively eradicating human cancer cells

Directory of Open Access Journals (Sweden)

Izraeli Shai

2011-09-01

Full Text Available Abstract Background Cells of most human cancers have supernumerary centrosomes. To enable an accurate chromosome segregation and cell division, these cells developed a yet unresolved molecular mechanism, clustering their extra centrosomes at two poles, thereby mimicking mitosis in normal cells. Failure of this bipolar centrosome clustering causes multipolar spindle structures and aberrant chromosomes segregation that prevent normal cell division and lead to 'mitotic catastrophe cell death'. Methods We used cell biology and biochemical methods, including flow cytometry, immunocytochemistry and live confocal imaging. Results We identified a phenanthrene derived PARP inhibitor, known for its activity in neuroprotection under stress conditions, which exclusively eradicated multi-centrosomal human cancer cells (mammary, colon, lung, pancreas, ovarian while acting as extra-centrosomes de-clustering agent in mitosis. Normal human proliferating cells (endothelial, epithelial and mesenchymal cells were not impaired. Despite acting as PARP inhibitor, the cytotoxic activity of this molecule in cancer cells was not attributed to PARP inhibition alone. Conclusion We identified a water soluble phenanthridine that exclusively targets the unique dependence of most human cancer cells on their supernumerary centrosomes bi-polar clustering for their survival. This paves the way for a new selective cancer-targeting therapy, efficient in a wide range of human cancers.

Different effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors on sterol synthesis in various human cell types

NARCIS (Netherlands)

Vliet, A.K. van; Thiel, G.C.F. van; Huisman, R.H.; Moshage, H.; Yap, S.H.; Cohen, L.H.

1995-01-01

The three vastatins examined, lovastatin, simvastatin and pravastatin, are equally strong inhibitors of the sterol synthesis in human hepatocytes in culture with IC50-values of 4.1, 8.0 and 2.0 nM, respectively. However, in the human extrahepatic cells: umbilical vascular endothelial cells, retinal

[The primary structure of the alpha-amylase inhibitor Hoe 467A from Streptomyces tendae 4158. A new class of inhibitors].

Science.gov (United States)

Aschauer, H; Vértesy, L; Nesemann, G; Braunitzer, G

1983-10-01

The native or modified alpha-amylase inhibitor Hoe 467A - isolated from the culture medium of Streptomyces tendae 4158 - and overlapping peptides were degraded by the automatic Edman technique. The oxidized or aminoethylated or oxidized and maleoylated inhibitor was digested with trypsin and the native inhibitor with pepsin. Further digestion with Staphylococcus aureus proteinase was also carried out. After peptic digestion two cystin peptides were isolated, which allowed the establishment of the disulfide bonds. The alpha-amylase inhibitor is a polypeptid consisting of 74 amino-acid residues with a molecular mass of 7958 Da. The inhibitor is composed of all naturally occurring amino acids except methionine and phenylalanine and shows no sequence homology to known inhibitors. The clinical and pharmacological importance in respect to the inhibitors ability for inactivation of human salivary and pancreatic alpha-amylase is discussed. Especially the proteinase resistance of the inhibitor enables a clinical application in human (e.g. Diabetes mellitus) per os.

Effect of urea and glycerol on the adsorption of ribonuclease A at the air-water interface.

Science.gov (United States)

Hüsecken, Anne K; Evers, Florian; Czeslik, Claus; Tolan, Metin

2010-08-17

This study reports on the influence of nonionic cosolvents on the interfacial structure of ribonuclease A (RNase) adsorbed at the air-water interface. We applied X-ray reflectometry to obtain detailed volume fraction profiles of the adsorbed layers and to follow the effect of glycerol and urea on the adsorbate structure as a function of cosolvent concentration. Under all conditions studied, the adsorbed RNase layer maintains its compact shape, and the adsorbed RNase molecules adopt a flat-on orientation at the interface. Both kosmotropic glycerol and chaotropic urea exert profound effects on the adsorbate: The surface excess decreases linearly with glycerol content and is also reduced at low urea concentration. However, at high urea concentration, parts of the adsorbed layer are dehydrated and become exposed to air. The electron density and volume fraction profiles of the adsorbed protein provide clear evidence that these effects are ruled by different mechanisms.

Application of cultured human mast cells (CHMC) for the design and structure-activity relationship of IgE-mediated mast cell activation inhibitors.

Science.gov (United States)

Argade, Ankush; Bhamidipati, Somasekhar; Li, Hui; Carroll, David; Clough, Jeffrey; Keim, Holger; Sylvain, Catherine; Rossi, Alexander B; Coquilla, Christina; Issakani, Sarkiz D; Masuda, Esteban S; Payan, Donald G; Singh, Rajinder

2015-01-01

Here we report the optimization of small molecule inhibitors of human mast cell degranulation via anti-IgE-mediated tryptase release following cross-linking and activation of IgE-loaded FcεR1 receptors. The compounds are selective upstream inhibitors of FcεR1-dependent human mast cell degranulation and proved to be devoid of activity in downstream ionomycin mediated degranulation. Structure-activity relationship (SAR) leading to compound 26 is outlined. Copyright © 2015 Elsevier Ltd. All rights reserved.

Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia

Energy Technology Data Exchange (ETDEWEB)

Shimada, Hidenori [Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan); Hashimoto, Yoshiya [Department of Biomaterials, Osaka Dental University, 8-1, Hanazonocho, Kuzuha, Hirakatashi, Osaka 573-1121 (Japan); Nakada, Akira; Shigeno, Keiji [Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan); Nakamura, Tatsuo, E-mail: nakamura@frontier.kyoto-u.ac.jp [Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan)

2012-01-13

Highlights: Black-Right-Pointing-Pointer Very rapid generation of human iPS cells under optimized conditions. Black-Right-Pointing-Pointer Five chemical inhibitors under hypoxia boosted reprogramming. Black-Right-Pointing-Pointer We performed genome-wide DNA methylation analysis. -- Abstract: Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1 month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly

Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia

International Nuclear Information System (INIS)

Shimada, Hidenori; Hashimoto, Yoshiya; Nakada, Akira; Shigeno, Keiji; Nakamura, Tatsuo

2012-01-01

Highlights: ► Very rapid generation of human iPS cells under optimized conditions. ► Five chemical inhibitors under hypoxia boosted reprogramming. ► We performed genome-wide DNA methylation analysis. -- Abstract: Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1 month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly generating bona fide human iPS cells and facilitates the application of i

Human monoamine oxidase is inhibited by tobacco smoke: β-carboline alkaloids act as potent and reversible inhibitors

International Nuclear Information System (INIS)

Herraiz, Tomas; Chaparro, Carolina

2005-01-01

Monoamine oxidase (MAO) is a mitochondrial outer-membrane flavoenzyme involved in brain and peripheral oxidative catabolism of neurotransmitters and xenobiotic amines, including neurotoxic amines, and a well-known target for antidepressant and neuroprotective drugs. Recently, positron emission tomography imaging has shown that smokers have a much lower activity of peripheral and brain MAO-A (30%) and -B (40%) isozymes compared to non-smokers. This MAO inhibition results from a pharmacological effect of smoke, but little is known about its mechanism. Working with mainstream smoke collected from commercial cigarettes we confirmed that cigarette smoke is a potent inhibitor of human MAO-A and -B isozymes. MAO inhibition was partly reversible, competitive for MAO-A, and a mixed-type inhibition for MAO-B. Two β-carboline alkaloids, norharman (β-carboline) and harman (1-methyl-β-carboline), were identified by GC-MS, quantified, and isolated from the mainstream smoke by solid phase extraction and HPLC. Kinetics analysis revealed that β-carbolines from cigarette smoke were competitive, reversible, and potent inhibitors of MAO enzymes. Norharman was an inhibitor of MAO-A (K i = 1.2 ± 0.18 μM) and MAO-B (K i = 1.12 ± 0.19 μM), and harman of MAO-A (K i = 55.54 ± 5.3 nM). β-Carboline alkaloids are psychopharmacologically active compounds that may occur endogenously in human tissues, including the brain. These results suggest that β-carboline alkaloids from cigarette smoke acting as potent reversible inhibitors of MAO enzymes may contribute to the MAO-reduced activity produced by tobacco smoke in smokers. The presence of MAO inhibitors in smoke like β-carbolines and others may help us to understand some of the purported neuropharmacological effects associated with smoking

Synthesis and biological evaluation of lycorine derivatives as dual inhibitors of human acetylcholinesterase and butyrylcholinesterase

Directory of Open Access Journals (Sweden)

Wang Yue-Hu

2012-09-01

Full Text Available Abstract Background Alzheimer’s disease (AD is a neurologically degenerative disorder that affects more than 20 million people worldwide. The selective butyrylcholinesterase (BChE inhibitors and bivalent cholinesterase (ChE inhibitors represent new treatments for AD. Findings A series of lycorine derivatives (1–10 were synthesized and evaluated for anti-cholinesterase activity. Result showed that the novel compound 2-O-tert-butyldimethylsilyl-1-O-(methylthiomethyllycorine (7 was a dual inhibitor of human acetylcholinesterase (hAChE and butyrylcholinesterase (hBChE with IC50 values of 11.40 ± 0.66 μM and 4.17 ± 0.29 μM, respectively. The structure-activity relationships indicated that (i the 1-O-(methylthiomethyl substituent in lycorine was better than the 1-O-acetyl group for the inhibition of cholinesterase; (ii the acylated or etherified derivatives of lycorine and lycorin-2-one were more potent against hBChE than hAChE; and (iii the oxidation of lycorine at C-2 decreases the activity. Conclusion Acylated or etherified derivatives of lycorine are potential dual inhibitors of hBChE and hAChE. Hence, further study on the modification of lycorine for ChE inhibition is necessary.

Cytogenetic study of Ascaris trypsin inhibitor in cultured human ...

Indian Academy of Sciences (India)

2009-04-01

Apr 1, 2009 ... Although the physical and chemical properties of Ascaris trypsin inhibitors ... male of Ascaris suum according to the method of Pudles and. Rola (1967). ..... inhibitor isolated from Ascaris resulted in the appearance of dominant ...

Structure of Human G Protein-Coupled Receptor Kinase 2 in Complex with the Kinase Inhibitor Balanol

Energy Technology Data Exchange (ETDEWEB)

Tesmer, John J.G.; Tesmer, Valerie M.; Lodowski, David T.; Steinhagen, Henning; Huber, Jochen (Sanofi); (Michigan); (Texas)

2010-07-19

G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with G{beta}{gamma} in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.

Design, synthesis and antiviral evaluation of novel heteroarylcarbothioamide derivatives as dual inhibitors of HIV-1 reverse transcriptase-associated RNase H and RDDP functions.

Science.gov (United States)

Corona, Angela; Onnis, Valentina; Deplano, Alessandro; Bianco, Giulia; Demurtas, Monica; Distinto, Simona; Cheng, Yung-Chi; Alcaro, Stefano; Esposito, Francesca; Tramontano, Enzo

2017-08-31

In the continuous effort to identify new HIV-1 inhibitors endowed with innovative mechanisms, the dual inhibition of different viral functions would provide a significant advantage against drug-resistant variants. The HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) is the only viral-encoded enzymatic activity that still lacks an efficient inhibitor. We synthesized a library of 3,5-diamino-N-aryl-1H-pyrazole-4-carbothioamide and 4-amino-5-benzoyl-N-phenyl-2-(substituted-amino)-1H-pyrrole-3-carbothioamide derivatives and tested them against RNase H activity. We identified the pyrazolecarbothioamide derivative A15, able to inhibit viral replication and both RNase H and RNA-dependent DNA polymerase (RDDP) RT-associated activities in the low micromolar range. Docking simulations hypothesized its binding to two RT pockets. Site-directed mutagenesis experiments showed that, with respect to wt RT, V108A substitution strongly reduced A15 IC50 values (12.6-fold for RNase H inhibition and 4.7-fold for RDDP), while substitution A502F caused a 9.0-fold increase in its IC50 value for RNase H, not affecting the RDDP inhibition, reinforcing the hypothesis of a dual-site inhibition. Moreover, A15 retained good inhibition potency against three non-nucleoside RT inhibitor (NNRTI)-resistant enzymes, confirming a mode of action unrelated to NNRTIs and suggesting its potential as a lead compound for development of new HIV-1 RT dual inhibitors active against drug-resistant viruses. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Recombinant human tissue factor pathway inhibitor exerts anticoagulant, anti-inflammatory and antimicrobial effects in murine pneumococcal pneumonia

NARCIS (Netherlands)

van den Boogaard, F. E.; Brands, X.; Schultz, M. J.; Levi, M. [=Marcel M.; Roelofs, J. J. T. H.; van 't Veer, C.; van der Poll, T.

2011-01-01

Background: Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia and a major cause of sepsis. Recombinant human tissue factor pathway inhibitor (rh-TFPI) attenuates sepsis-induced coagulation and has been evaluated in clinical trials involving patients

Structure-Activity Relationships of Pentacyclic Triterpenoids as Potent and Selective Inhibitors against Human Carboxylesterase 1

Directory of Open Access Journals (Sweden)

Li-Wei Zou

2017-06-01

Full Text Available Human carboxylesterase 1 (hCE1, one of the most important serine hydrolases distributed in liver and adipocytes, plays key roles in endobiotic homeostasis and xenobiotic metabolism. This study aimed to find potent and selective inhibitors against hCE1 from phytochemicals and their derivatives. To this end, a series of natural triterpenoids were collected and their inhibitory effects against human carboxylesterases (hCEs were assayed using D-Luciferin methyl ester (DME and 6,8-dichloro-9,9-dimethyl-7-oxo-7,9-dihydroacridin-2-yl benzoate (DDAB as specific optical substrate for hCE1, and hCE2, respectively. Following screening of a series of natural triterpenoids, oleanolic acid (OA, and ursolic acid (UA were found with strong inhibitory effects on hCE1 and relative high selectivity over hCE2. In order to get the highly selective and potent inhibitors of hCE1, a series of OA and UA derivatives were synthesized from OA and UA by chemical modifications including oxidation, reduction, esterification, and amidation. The inhibitory effects of these derivatives on hCEs were assayed and the structure-activity relationships of tested triterpenoids as hCE1 inhibitors were carefully investigated. The results demonstrated that the carbonyl group at the C-28 site is essential for hCE1 inhibition, the modifications of OA or UA at this site including esters, amides and alcohols are unbeneficial for hCE1 inhibition. In contrast, the structural modifications on OA and UA at other sites, such as converting the C-3 hydroxy group to 3-O-β-carboxypropionyl (compounds 20 and 22, led to a dramatically increase of the inhibitory effects against hCE1 and very high selectivity over hCE2. 3D-QSAR analysis of all tested triterpenoids including OA and UA derivatives provide new insights into the fine relationships linking between the inhibitory effects on hCE1 and the steric-electrostatic properties of triterpenoids. Furthermore, both inhibition kinetic analyses and docking

A376S in the connection subdomain of HIV-1 reverse transcriptase confers increased risk of virological failure to nevirapine therapy

NARCIS (Netherlands)

Paredes, Roger; Puertas, Maria Carmen; Bannister, Wendy; Kisic, Mónica; Cozzi-Lepri, Alessandro; Pou, Christian; Bellido, Rocío; Betancor, Gilberto; Bogner, Johannes; Gargalianos, Panagiotis; Bánhegyi, Dénes; Clotet, Bonaventura; Lundgren, Jens; Menéndez-Arias, Luis; Martinez-Picado, Javier; Losso, M.; Elias, C.; Vetter, N.; Zangerle, R.; Karpov, I.; Vassilenko, A.; Mitsura, V. M.; Suetnov, O.; Clumeck, N.; de Wit, S.; Poll, B.; Colebunders, R.; Vandekerckhove, L.; Hadziosmanovic, V.; Kostov, K.; Begovac, J.; Machala, L.; Rozsypal, H.; Sedlacek, D.; Nielsen, J.; Kronborg, G.; Benfield, T.; Larsen, M.; Gerstoft, J.; Katzenstein, T.; Hansen, A.-B. E.; Skinhøj, P.; Pedersen, C.; Oestergaard, L.; Zilmer, K.; Ristola, M.; Katlama, C.; Viard, J.-P.; Girard, P.-M.; Livrozet, J. M.; Vanhems, P.; Pradier, C.; Dabis, F.; Neau, D.; Rockstroh, J.; Schmidt, R.; van Lunzen, J.; Degen, O.; Stellbrink, H. J.; Staszewski, S.; Fätkenheuer, G.; Kosmidis, J.; Gargalianos, P.; Xylomenos, G.; Perdios, J.; Panos, G.; Filandras, A.; Karabatsaki, E.; Sambatakou, H.; Banhegyi, D.; Mulcahy, F.; Yust, I.; Turner, D.; Burke, M.; Pollack, S.; Hassoun, G.; Maayan, S.; Vella, S.; Esposito, R.; Mazeu, I.; Mussini, C.; Arici, C.; Pristera, R.; Mazzotta, F.; Gabbuti, A.; Vullo, V.; Lichtner, M.; Chirianni, A.; Montesarchio, E.; Gargiulo, M.; Antonucci, G.; Iacomi, F.; Narciso, P.; Vlassi, C.; Zaccarelli, M.; Lazzarin, A.; Finazzi, R.; Galli, M.; Ridolfo, A.; d'Arminio, A.; Rozentale, B.; Aldins, P.; Chaplinskas, S.; Hemmer, R.; Staub, T.; Reiss, P.; Ormaasen, V.; Maeland, A.; Brunn, J.; Knysz, B.; Gasiorowski, J.; Horban, A.; Bakowska, E.; Prokopowicz, D.; Flisiak, R.; Boron-Kaczmarska, A.; Pynka, M.; Beniowski, M.; Mularska, E.; Trocha, H.; Jablonowska, E.; Malolepsza, E.; Wojcik, K.; Antunes, F.; Valadas, E.; Mansinho, K.; Maltez, F.; Duiculescu, D.; Rakhmanova, A.; Vinogradova, E.; Buzunova, S.; Jevtovic, D.; Mokrás, M.; Staneková, D.; Tomazic, J.; González-Lahoz, J.; Soriano, V.; Martin-Carbonero, L.; Labarga, P.; Moreno, S.; Clotet, B.; Jou, A.; Paredes, R.; Tural, C.; Puig, J.; Bravo, I.; Gatell, J. M.; Miró, J. M.; Domingo, P.; Gutierrez, M.; Mateo, G.; Sambeat, M. A.; Karlsson, A.; Persson, P. O.; Ledergerber, B.; Weber, R.; Francioli, P.; Cavassini, M.; Hirschel, B.; Boffi, E.; Furrer, H.; Battegay, M.; Elzi, L.; Kravchenko, E.; Chentsova, N.; Kutsyna, G.; Servitskiy, S.; Krasnov, M.; Barton, S.; Johnson, A. M.; Mercey, D.; Phillips, A.; Johnson, M. A.; Murphy, M.; Weber, J.; Scullard, G.; Fisher, M.; Leen, C.; Gatell, J.; Gazzard, B.; Lundgren, J.; d'Arminio Monforte, A.; Kirk, O.; Mocroft, A.; Cozzi-Lepri, A.; Grint, D.; Ellefson, M.; Podlekareva, D.; Kjaer, J.; Peters, L.; Reekie, J.; Kowalska, J.; Tverland, J.; Fischer, A. H.

2011-01-01

The clinical relevance of mutations in the connection subdomain and the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT) is uncertain. The risk of virological failure to nonnucleoside RT inhibitor (NNRTI)-based antiretroviral therapy (ART) was evaluated in NNRTI-naive patients who

The HSP90 Inhibitor Ganetespib Radiosensitizes Human Lung Adenocarcinoma Cells

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Roberto Gomez-Casal

2015-05-01

Full Text Available The molecular chaperone HSP90 is involved in stabilization and function of multiple client proteins, many of which represent important oncogenic drivers in NSCLC. Utilization of HSP90 inhibitors as radiosensitizing agents is a promising approach. The antitumor activity of ganetespib, HSP90 inhibitor, was evaluated in human lung adenocarcinoma (AC cells for its ability to potentiate the effects of IR treatment in both in vitro and in vivo. The cytotoxic effects of ganetespib included; G2/M cell cycle arrest, inhibition of DNA repair, apoptosis induction, and promotion of senescence. All of these antitumor effects were both concentration- and time-dependent. Both pretreatment and post-radiation treatment with ganetespib at low nanomolar concentrations induced radiosensitization in lung AC cells in vitro. Ganetespib may impart radiosensitization through multiple mechanisms: such as down regulation of the PI3K/Akt pathway; diminished DNA repair capacity and promotion of cellular senescence. In vivo, ganetespib reduced growth of T2821 tumor xenografts in mice and sensitized tumors to IR. Tumor irradiation led to dramatic upregulation of β-catenin expression in tumor tissues, an effect that was mitigated in T2821 xenografts when ganetespib was combined with IR treatments. These data highlight the promise of combining ganetespib with IR therapies in the treatment of AC lung tumors.

Synthesis and in vitro evaluation of PNA-peptide-DETA conjugates as potential cell penetrating artificial ribonucleases.

Science.gov (United States)

Petersen, Lene; de Koning, Martijn C; van Kuik-Romeijn, Petra; Weterings, Jimmy; Pol, Christine J; Platenburg, Gerard; Overhand, Mark; van der Marel, Gijsbert A; van Boom, Jacques H

2004-01-01

We report the synthesis of novel artificial ribonucleases with potentially improved cellular uptake. The design of trifunctional conjugates 1a and 1b is based on the specific RNA-recognizing properties of PNA, the RNA-cleaving abilities of diethylenetriamine (DETA), and the peptide (KFF)(3)K for potential uptake into E. coli. The conjugates were assembled in a convergent synthetic route involving native chemical ligation of a PNA, containing an N-terminal cysteine, with the C-terminal thioester of the cell-penetrating (KFF)(3)K peptide to give 12a and 12b. These hybrids contained a free cysteine side-chain, which was further functionalized with an RNA-hydrolyzing diethylenetriamine (DETA) moiety. The trifunctional conjugates (1a, 1b) were evaluated for RNA-cleaving properties in vitro and showed efficient degradation of the target RNA at two major cleavage sites. It was also established that the cleavage efficiency strongly depended on the type of spacer connecting the PNA and the peptide.

Crystal structure of the pestivirus envelope glycoprotein E(rns) and mechanistic analysis of its ribonuclease activity.

Science.gov (United States)

Krey, Thomas; Bontems, Francois; Vonrhein, Clemens; Vaney, Marie-Christine; Bricogne, Gerard; Rümenapf, Till; Rey, Félix A

2012-05-09

Pestiviruses, which belong to the Flaviviridae family of RNA viruses, are important agents of veterinary diseases causing substantial economical losses in animal farming worldwide. Pestivirus particles display three envelope glycoproteins at their surface: E(rns), E1, and E2. We report here the crystal structure of the catalytic domain of E(rns), the ribonucleolytic activity of which is believed to counteract the innate immunity of the host. The structure reveals a three-dimensional fold corresponding to T2 ribonucleases from plants and fungi. Cocrystallization experiments with mono- and oligonucleotides revealed the structural basis for substrate recognition at two binding sites previously identified for T2 RNases. A detailed analysis of poly-U cleavage products using (31)P-NMR and size exclusion chromatography, together with molecular docking studies, provides a comprehensive mechanistic picture of E(rns) activity on its substrates and reveals the presence of at least one additional nucleotide binding site. Copyright © 2012 Elsevier Ltd. All rights reserved.

Computational drug design strategies applied to the modelling of human immunodeficiency virus-1 reverse transcriptase inhibitors

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Lucianna Helene Santos

2015-11-01

Full Text Available Reverse transcriptase (RT is a multifunctional enzyme in the human immunodeficiency virus (HIV-1 life cycle and represents a primary target for drug discovery efforts against HIV-1 infection. Two classes of RT inhibitors, the nucleoside RT inhibitors (NRTIs and the nonnucleoside transcriptase inhibitors are prominently used in the highly active antiretroviral therapy in combination with other anti-HIV drugs. However, the rapid emergence of drug-resistant viral strains has limited the successful rate of the anti-HIV agents. Computational methods are a significant part of the drug design process and indispensable to study drug resistance. In this review, recent advances in computer-aided drug design for the rational design of new compounds against HIV-1 RT using methods such as molecular docking, molecular dynamics, free energy calculations, quantitative structure-activity relationships, pharmacophore modelling and absorption, distribution, metabolism, excretion and toxicity prediction are discussed. Successful applications of these methodologies are also highlighted.

Tumor necrosis factor increases the production of plasminogen activator inhibitor in human endothelial cells in vitro and in rats in vivo

NARCIS (Netherlands)

Hinsbergh, V.W.M. van; Kooistra, T.; Berg, E.A. van den; Princen, H.M.G.; Fiers, W.; Emeis, J.J.

1988-01-01

The vascular endothelium plays an important role in fibrinolysis by producing tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI). The monokine tumor necrosis factor (human recombinant TNF) increased the production of PAI by cultured human endothelial cells from

TAS-116, a novel Hsp90 inhibitor, selectively enhances radio-sensitivity of human cancer cells to X-rays and carbon ion radiation

Science.gov (United States)

Lee, Younghyun; Sunada, Shigeaki; Hirakawa, Hirokazu; Fujimori, Akira; Nickoloff, Jac A.; Okayasu, Ryuichi

2016-01-01

Hsp90 inhibitors have been investigated as cancer therapeutics in mono-therapy and to augment radiotherapy, however serious adverse effects of early generation Hsp90 inhibitors limited their development. TAS-116 is a novel Hsp90 inhibitor with lower adverse effects than other Hsp90 inhibitors, and here we investigated the radio-sensitizing effects of TAS-116 in low LET X-ray, and high LET carbon ion irradiated human cancer cells and mouse tumor xenografts. TAS-116 decreased cell survival of both X-ray and carbon ion-irradiated human cancer cell lines (HeLa and H1299 cells), and similar to other Hsp90 inhibitors, it did not affect radiosensitivity of non-cancerous human fibroblasts. TAS-116 increased the number of radiation-induced γ-H2AX foci, and delayed the repair of DNA double-strand breaks (DSBs). TAS-116 reduced the expression of proteins that mediate repair of DSBs by homologous recombination (RAD51) and non-homologous end joining (Ku, DNA-PKcs), and suppressed formation of RAD51 foci and phosphorylation/activation of DNA-PKcs. TAS-116 also decreased expression of the cdc25 cell cycle progression marker, markedly increasing G2/M arrest. Combined treatment of mouse tumor xenografts with carbon ions and TAS-116 showed promising delay in tumor growth compared to either individual treatment. These results demonstrate that TAS-116 radio-sensitizes human cancer cells to both X rays and carbon ions by inhibiting the two major DSB repair pathways, and these effects were accompanied by marked cell cycle arrest. The promising results of combination TAS-116 + carbon ion radiation therapy of tumor xenografts justify further exploration of TAS-116 as an adjunct to radiotherapy using low or high LET radiation. PMID:28062703

Single-molecule supercoil-relaxation assay as a screening tool to determine the mechanism and efficacy of human topoisomerase IB inhibitors

Science.gov (United States)

Seol, Yeonee; Zhang, Hongliang; Agama, Keli; Lorence, Nicholas; Pommier, Yves; Neuman, Keir C.

2015-01-01

Human nuclear type IB topoisomerase (Top1) inhibitors are widely used and powerful anti-cancer agents. In this study, we introduce and validate a single-molecule supercoil relaxation assay as a molecular pharmacology tool for characterizing therapeutically relevant Top1 inhibitors. Using this assay, we determined the effects on Top1 supercoil relaxation activity of four Top1 inhibitors; three clinically relevant: camptothecin, LMP-400, LMP-776 (both indenoisoquinoline derivatives), and one natural product in preclinical development, lamellarin-D. Our results demonstrate that Top1 inhibitors have two distinct effects on Top1 activity: a decrease in supercoil relaxation rate and an increase in religation inhibition. The type and magnitude of the inhibition mode depend both on the specific inhibitor and on the topology of the DNA substrate. In general, the efficacy of inhibition is significantly higher with supercoiled than with relaxed DNA substrates. Comparing single-molecule inhibition with cell growth inhibition (IC50) measurements showed a correlation between the binding time of the Top1 inhibitors and their cytotoxic efficacy, independent of the mode of inhibition. This study demonstrates that the single-molecule supercoil relaxation assay is a sensitive method to elucidate the detailed mechanisms of Top1 inhibitors and is relevant for the cellular efficacy of Top1 inhibitors. PMID:26351326

Determination of human serum alpha1-acid glycoprotein and albumin binding of various marketed and preclinical kinase inhibitors.

Science.gov (United States)

Zsila, Ferenc; Fitos, Ilona; Bencze, Gyula; Kéri, György; Orfi, László

2009-01-01

There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.

Stabilization versus inhibition of TAFIa by competitive inhibitors in vitro

NARCIS (Netherlands)

Walker, J.B.; Hughes, B.; James, I.; Haddock, P.; Kluft, C.; Bajzar, L.

2003-01-01

Two competitive inhibitors of TAFIa (activated thrombin-activable fibrinolysis inhibitor), 2-guanidinoethyl-mercaptosuccinic acid and potato tuber carboxypeptidase inhibitor, variably affect fibrinolysis of clotted human plasma. Depending on their concentration, the inhibitors shortened, prolonged,

Curcumin, a Natural Antioxidant, Acts as a Noncompetitive Inhibitor of Human RNase L in Presence of Its Cofactor 2-5A In Vitro

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Ankush Gupta

2014-01-01

Full Text Available Ribonuclease L (RNase L is an antiviral endoribonuclease of the innate immune system, which is induced and activated by viral infections, interferons, and double stranded RNA (dsRNA in mammalian cells. Although, RNase L is generally protective against viral infections, abnormal RNase L expression and activity have been associated with a number of diseases. Here, we show that curcumin, a natural plant-derived anti-inflammatory active principle, inhibits RNase L activity; hence, it may be exploited for therapeutic interventions in case of pathological situations associated with excess activation of RNase L.

Curcumin, a natural antioxidant, acts as a noncompetitive inhibitor of human RNase L in presence of its cofactor 2-5A in vitro.

Science.gov (United States)

Gupta, Ankush; Rath, Pramod C

2014-01-01

Ribonuclease L (RNase L) is an antiviral endoribonuclease of the innate immune system, which is induced and activated by viral infections, interferons, and double stranded RNA (dsRNA) in mammalian cells. Although, RNase L is generally protective against viral infections, abnormal RNase L expression and activity have been associated with a number of diseases. Here, we show that curcumin, a natural plant-derived anti-inflammatory active principle, inhibits RNase L activity; hence, it may be exploited for therapeutic interventions in case of pathological situations associated with excess activation of RNase L.

Inhibitors of cysteine cathepsin and calpain do not prevent ultraviolet-B-induced apoptosis in human keratinocytes and HeLa cells

DEFF Research Database (Denmark)

Bang, Bo; Baadsgaard, Ole; Skov, Lone

2004-01-01

been demonstrated to play a role in the execution of programmed cell death induced by other stimuli, e.g. TNF-alpha. The purpose of the present study was therefore to investigate whether inhibitors of cysteine cathepsins and calpains could prevent UVB-induced apoptosis in HeLa cells and keratinocytes....... This was done by investigating the effect of the irreversible cysteine protease inhibitor zFA-fmk, the cathepsin B inhibitor CA-074-Me and the calpain inhibitor ALLN on the viability of UVB-irradiated human keratinocytes and HeLa cells. At concentrations of 10 microM and above zVAD-fmk conferred partial dose......-dependent protection against UVB-induced apoptosis in HeLa cells and keratinocytes. Moreover, caspase-3 activity was completely blocked at zVAD-fmk concentrations of 1 microM in HeLa cells. This indicates that caspase-independent mechanisms could be involved in UVB-induced apoptosis. However, the protease inhibitors z...

Structure-activity relationships of amide-phosphonate derivatives as inhibitors of the human soluble epoxide hydrolase.

Science.gov (United States)

Kim, In-Hae; Park, Yong-Kyu; Nishiwaki, Hisashi; Hammock, Bruce D; Nishi, Kosuke

2015-11-15

Structure-activity relationships of amide-phosphonate derivatives as inhibitors of the human soluble epoxide hydrolase (sEH) were investigated. First, a series of alkyl or aryl groups were substituted on the carbon alpha to the phosphonate function in amide compounds to see whether substituted phosphonates can act as a secondary pharmacophore. A tert-butyl group (16) on the alpha carbon was found to yield most potent inhibition on the target enzyme. A 4-50-fold drop in inhibition was induced by other substituents such as aryls, substituted aryls, cycloalkyls, and alkyls. Then, the modification of the O-substituents on the phosphonate function revealed that diethyl groups (16 and 23) were preferable for inhibition to other longer alkyls or substituted alkyls. In amide compounds with the optimized diethylphosphonate moiety and an alkyl substitution such as adamantane (16), tetrahydronaphthalene (31), or adamantanemethane (36), highly potent inhibitions were gained. In addition, the resulting potent amide-phosphonate compounds had reasonable water solubility, suggesting that substituted phosphonates in amide inhibitors are effective for both inhibition potency on the human sEH and water solubility as a secondary pharmacophore. Copyright © 2015 Elsevier Ltd. All rights reserved.

PDE5 inhibitors blunt inflammation in human BPH: a potential mechanism of action for PDE5 inhibitors in LUTS.

Science.gov (United States)

Vignozzi, Linda; Gacci, Mauro; Cellai, Ilaria; Morelli, Annamaria; Maneschi, Elena; Comeglio, Paolo; Santi, Raffaella; Filippi, Sandra; Sebastianelli, Arcangelo; Nesi, Gabriella; Serni, Sergio; Carini, Marco; Maggi, Mario

2013-09-01

Metabolic syndrome (MetS) and benign prostate hyperplasia (BPH)/low urinary tract symptoms (LUTS) are often comorbid. Chronic inflammation is one of the putative links between these diseases. Phosphodiesterase type 5 inhibitors (PDE5i) are recognized as an effective treatment of BPH-related LUTS. One proposed mechanism of action of PDE5 is the inhibition of intraprostatic inflammation. In this study we investigate whether PDE5i could blunt inflammation in the human prostate. Evaluation of the effect of tadalafil and vardenafil on secretion of interleukin 8 (IL-8, a surrogate marker of prostate inflammation) by human myofibroblast prostatic cells (hBPH) exposed to different inflammatory stimuli. We preliminary evaluate histological features of prostatic inflammatory infiltrates in BPH patients enrolled in a randomized, double bind, placebo controlled study aimed at investigating the efficacy of vardenafil (10 mg/day, for 12 weeks) on BPH/LUTS. In vitro treatment with tadalafil or vardenafil on hBPH reduced IL-8 secretion induced by either TNFα or metabolic factors, including oxidized low-density lipoprotein, oxLDL, to the same extent as a PDE5-insensitive PKG agonist Sp-8-Br-PET-cGMP. These effects were reverted by the PKG inhibitor KT5823, suggesting a cGMP/PKG-dependency. Treatment with tadalafil or vardenafil significantly suppressed oxLDL receptor (LOX-1) expression. Histological evaluation of anti-CD45 staining (CD45 score) in prostatectomy specimens of BPH patients showed a positive association with MetS severity. Reduced HDL-cholesterol and elevated triglycerides were the only MetS factors significantly associated with CD45 score. In the MetS cohort there was a significant lower CD45 score in the vardenafil-arm versus the placebo-one. © 2013 Wiley Periodicals, Inc.

Discovery and characterization of inhibitors of human palmitoyl acyltransferases.

Science.gov (United States)

Ducker, Charles E; Griffel, Lindsay K; Smith, Ryan A; Keller, Staci N; Zhuang, Yan; Xia, Zuping; Diller, John D; Smith, Charles D

2006-07-01

The covalent attachment of palmitate to specific proteins by the action of palmitoyl acyltransferases (PAT) plays critical roles in the biological activities of several oncoproteins. Two PAT activities are expressed by human cells: type 1 PATs that modify the farnesyl-dependent palmitoylation motif found in H- and N-Ras, and type 2 PATs that modify the myristoyl-dependent palmitoylation motif found in the Src family of tyrosine kinases. We have previously shown that the type 1 PAT HIP14 causes cellular transformation. In the current study, we show that mRNA encoding HIP14 is up-regulated in a number of types of human tumors. To assess the potential of HIP14 and other PATs as targets for new anticancer drugs, we developed three cell-based assays suitable for high-throughput screening to identify inhibitors of these enzymes. Using these screens, five chemotypes, with activity toward either type 1 or type 2 PAT activity, were identified. The activity of the hits were confirmed using assays that quantify the in vitro inhibition of PAT activity, as well as a cell-based assay that determines the abilities of the compounds to prevent the localization of palmitoylated green fluorescent proteins to the plasma membrane. Representative compounds from each chemotype showed broad antiproliferative activity toward a panel of human tumor cell lines and inhibited the growth of tumors in vivo. Together, these data show that PATs, and HIP14 in particular, are interesting new targets for anticancer compounds, and that small molecules with such activity can be identified by high-throughput screening.

Retro-binding thrombin active site inhibitors: identification of an orally active inhibitor of thrombin catalytic activity.

Science.gov (United States)

Iwanowicz, Edwin J; Kimball, S David; Lin, James; Lau, Wan; Han, W-C; Wang, Tammy C; Roberts, Daniel G M; Schumacher, W A; Ogletree, Martin L; Seiler, Steven M

2002-11-04

A series of retro-binding inhibitors of human alpha-thrombin was prepared to elucidate structure-activity relationships (SAR) and optimize in vivo performance. Compounds 9 and 11, orally active inhibitors of thrombin catalytic activity, were identified to be efficacious in a thrombin-induced lethality model in mice.

A synthetic cryptochrome inhibitor induces anti-proliferative effects and increases chemosensitivity in human breast cancer cells

Energy Technology Data Exchange (ETDEWEB)

Chun, Sung Kook [Department of Brain & Cognitive Sciences, Daegu-Gyeongbuk Institute of Science & Technology, Daegu, 711-873 (Korea, Republic of); Department of Biological Sciences, Seoul National University, Seoul, 151-747 (Korea, Republic of); Department of Brain & Cognitive Sciences, Seoul National University, Seoul, 151-747 (Korea, Republic of); Chung, Sooyoung [Department of Biological Sciences, Seoul National University, Seoul, 151-747 (Korea, Republic of); Department of Biomedical Sciences, College of Medicine, Korea University, Seoul, 136-705 (Korea, Republic of); Kim, Hee-Dae [Department of Biological Sciences, Seoul National University, Seoul, 151-747 (Korea, Republic of); Lee, Ju Hyung [Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749 (Korea, Republic of); Jang, Jaebong [College of Pharmacy, Seoul National University, Seoul, 151-742 (Korea, Republic of); Kim, Jeongah; Kim, Doyeon [Department of Brain & Cognitive Sciences, Daegu-Gyeongbuk Institute of Science & Technology, Daegu, 711-873 (Korea, Republic of); Department of Biological Sciences, Seoul National University, Seoul, 151-747 (Korea, Republic of); Department of Brain & Cognitive Sciences, Seoul National University, Seoul, 151-747 (Korea, Republic of); Son, Gi Hoon [Department of Biomedical Sciences, College of Medicine, Korea University, Seoul, 136-705 (Korea, Republic of); Oh, Young J. [Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749 (Korea, Republic of); Suh, Young-Ger [College of Pharmacy, Seoul National University, Seoul, 151-742 (Korea, Republic of); Lee, Cheol Soon [Gachon Clinical Trials Center, Gachon University, Incheon, 417-842 (Korea, Republic of); and others

2015-11-13

Disruption of circadian rhythm is a major cause of breast cancer in humans. Cryptochrome (CRY), a circadian transcription factor, is a risk factor for initiation of breast cancer, and it is differentially expressed between normal and breast cancer tissues. Here, we evaluated the anti-proliferative and pro-apoptotic activity of KS15, a recently discovered small-molecule inhibitor of CRY, in human breast cancer cells. First, we investigated whether KS15 treatment could promote E-box-mediated transcription by inhibiting the activity of CRY in MCF-7 human breast cancer cells. Protein and mRNA levels of regulators of cell cycle and apoptosis, as well as core clock genes, were differentially modulated in response to KS15. Next, we investigated whether KS15 could inhibit proliferation and increase sensitivity to anti-tumor drugs in MCF-7 cells. We found that KS15 decreased the speed of cell growth and increased the chemosensitivity of MCF-7 cells to doxorubicin and tamoxifen, but had no effect on MCF-10A cells. These findings suggested that pharmacological inhibition of CRY by KS15 exerts an anti-proliferative effect and increases sensitivity to anti-tumor drugs in a specific type of breast cancer. - Highlights: • Cryptochrome inhibitor (KS15) has anti-tumor activity to human breast cancer cells. • KS15 induces differential changes in cell cycle regulators and pro-apoptotic genes. • KS15 inhibits MCF-7 cell growth and enhances susceptibility to anti-tumor drugs.

A synthetic cryptochrome inhibitor induces anti-proliferative effects and increases chemosensitivity in human breast cancer cells

International Nuclear Information System (INIS)

Chun, Sung Kook; Chung, Sooyoung; Kim, Hee-Dae; Lee, Ju Hyung; Jang, Jaebong; Kim, Jeongah; Kim, Doyeon; Son, Gi Hoon; Oh, Young J.; Suh, Young-Ger; Lee, Cheol Soon

2015-01-01

Disruption of circadian rhythm is a major cause of breast cancer in humans. Cryptochrome (CRY), a circadian transcription factor, is a risk factor for initiation of breast cancer, and it is differentially expressed between normal and breast cancer tissues. Here, we evaluated the anti-proliferative and pro-apoptotic activity of KS15, a recently discovered small-molecule inhibitor of CRY, in human breast cancer cells. First, we investigated whether KS15 treatment could promote E-box-mediated transcription by inhibiting the activity of CRY in MCF-7 human breast cancer cells. Protein and mRNA levels of regulators of cell cycle and apoptosis, as well as core clock genes, were differentially modulated in response to KS15. Next, we investigated whether KS15 could inhibit proliferation and increase sensitivity to anti-tumor drugs in MCF-7 cells. We found that KS15 decreased the speed of cell growth and increased the chemosensitivity of MCF-7 cells to doxorubicin and tamoxifen, but had no effect on MCF-10A cells. These findings suggested that pharmacological inhibition of CRY by KS15 exerts an anti-proliferative effect and increases sensitivity to anti-tumor drugs in a specific type of breast cancer. - Highlights: • Cryptochrome inhibitor (KS15) has anti-tumor activity to human breast cancer cells. • KS15 induces differential changes in cell cycle regulators and pro-apoptotic genes. • KS15 inhibits MCF-7 cell growth and enhances susceptibility to anti-tumor drugs.

Apoptosis inhibitor 5 (API-5; AAC-11; FIF) is upregulated in human carcinomas in vivo

Czech Academy of Sciences Publication Activity Database

Kočí, Lenka; Chlebová, K.; Hýžďalová, Martina; Hofmanová, Jiřina; Jíra, M.; Kysela, P.; Kozubík, Alois; Kala, Z.; Krejčí, Pavel

2012-01-01

Roč. 3, č. 4 (2012), s. 913-916 ISSN 1792-1074 R&D Projects: GA ČR(CZ) GA305/09/1526; GA ČR(CZ) GD303/09/H048; GA ČR(CZ) GAP301/11/1730 Institutional research plan: CEZ:AV0Z50040702 Keywords : apoptosis inhibitor 5 * apoptosis * human carcinoma Subject RIV: BO - Biophysics Impact factor: 0.237, year: 2012

[Computational prediction of human immunodeficiency resistance to reverse transcriptase inhibitors].

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Tarasova, O A; Filimonov, D A; Poroikov, V V

2017-10-01

Human immunodeficiency virus (HIV) causes acquired immunodeficiency syndrome (AIDS) and leads to over one million of deaths annually. Highly active antiretroviral treatment (HAART) is a gold standard in the HIV/AIDS therapy. Nucleoside and non-nucleoside inhibitors of HIV reverse transcriptase (RT) are important component of HAART, but their effect depends on the HIV susceptibility/resistance. HIV resistance mainly occurs due to mutations leading to conformational changes in the three-dimensional structure of HIV RT. The aim of our work was to develop and test a computational method for prediction of HIV resistance associated with the mutations in HIV RT. Earlier we have developed a method for prediction of HIV type 1 (HIV-1) resistance; it is based on the usage of position-specific descriptors. These descriptors are generated using the particular amino acid residue and its position; the position of certain residue is determined in a multiple alignment. The training set consisted of more than 1900 sequences of HIV RT from the Stanford HIV Drug Resistance database; for these HIV RT variants experimental data on their resistance to ten inhibitors are presented. Balanced accuracy of prediction varies from 80% to 99% depending on the method of classification (support vector machine, Naive Bayes, random forest, convolutional neural networks) and the drug, resistance to which is obtained. Maximal balanced accuracy was obtained for prediction of resistance to zidovudine, stavudine, didanosine and efavirenz by the random forest classifier. Average accuracy of prediction is 89%.

Intrinsic thermodynamics of inhibitor binding to human carbonic anhydrase IX.

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Linkuvienė, Vaida; Matulienė, Jurgita; Juozapaitienė, Vaida; Michailovienė, Vilma; Jachno, Jelena; Matulis, Daumantas

2016-04-01

Human carbonic anhydrase 9th isoform (CA IX) is an important marker of numerous cancers and is increasingly interesting as a potential anticancer drug target. Various synthetic aromatic sulfonamide-bearing compounds are being designed as potent inhibitors of CA IX. However, sulfonamide compound binding to CA IX is linked to several reactions, the deprotonation of the sulfonamide amino group and the protonation of the CA active site Zn(II)-bound hydroxide. These linked reactions significantly affect the affinities and other thermodynamic parameters such as enthalpies and entropies of binding. The observed and intrinsic affinities of compound binding to CA IX were determined by the fluorescent thermal shift assay. The enthalpies and entropies of binding were determined by the isothermal titration calorimetry. The pKa of CA IX was determined to be 6.8 and the enthalpy of CA IX-Zn(II)-bound hydroxide protonation was -24 kJ/mol. These values enabled the analysis of intrinsic thermodynamics of a library of compounds binding to CA IX. The most strongly binding compounds exhibited the intrinsic affinity of 0.01 nM and the observed affinity of 2 nM. The intrinsic thermodynamic parameters of compound binding to CA IX helped to draw the compound structure to thermodynamics relationship. It is important to distinguish the intrinsic from observed parameters of any disease target protein interaction with its inhibitors as drug candidates when drawing detailed compound structure to thermodynamics correlations. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel imidazopyridine analogue as a phosphatidylinositol 3-kinase inhibitor against human breast cancer.

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Lee, Hyunseung; Li, Guang-Yong; Jeong, Yujeong; Jung, Kyung Hee; Lee, Ju-Hee; Ham, Kyungrok; Hong, Sungwoo; Hong, Soon-Sun

2012-05-01

Potentiation of anti-breast cancer activity of an imidazopyridine-based PI3Kα inhibitor, HS-104, was investigated in human breast cancer cells. HS-104 shows strong inhibitory activity against recombinant PI3Kα isoform and the PI3K signaling pathway, resulting in anti-proliferative activity in breast cancer cells. It also induced cell cycle arrest at the G(2)/M phase as well as apoptosis. Furthermore, oral administration of HS-104 significantly inhibited the growth of tumor in SkBr3 mouse xenograft models. Therefore, HS-104 could be considered as a potential candidate for the treatment of human breast cancer. Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.

Positron emitter labeled enzyme inhibitors

International Nuclear Information System (INIS)

Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

1990-01-01

This invention involves a new strategy for imagining and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography

HSP90 Inhibitors, Geldanamycin and Radicicol, Enhance Fisetin-Induced Cytotoxicity via Induction of Apoptosis in Human Colonic Cancer Cells

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Ming-Shun Wu

2013-01-01

Full Text Available We revealed the cytotoxic effect of the flavonoid, fisetin (FIS, on human COLO205 colon cancer cells in the presence and absence of the HSP90 inhibitors, geldanamycin (GA and radicicol (RAD. Compared to FIS treatment alone of COLO205 cells, GA and RAD significantly enhanced FIS-induced cytotoxicity, increased expression of cleaved caspase-3 and the PAPR protein, and produced a greater density of DNA ladder formation. GA and RAD also reduced the MMPs with induction of caspase-9 protein cleavage in FIS-treated COLO205 cells. Increased caspase-3 and -9 activities were detected in COLO205 cells treated with FIS+GA or FIS+RAD, and the intensity of DNA ladder formation induced by FIS+GA was reduced by adding the caspase-3 inhibitor, DEVD-FMK. A decrease in Bcl-2 but not Bcl-XL or Bax protein by FIS+GA or FIS+RAD was identified in COLO205 cells by Western blotting. A reduction in p53 protein with increased ubiquitin-tagged proteins was observed in COLO205 cells treated with FIS+GA or FIS+RAD. Furthermore, GA and RAD reduced the stability of the p53 protein in COLO205 cells under FIS stimulation. The evidence supports HSP90 inhibitors possibly sensitizing human colon cancer cells to FIS-induced apoptosis, and treating colon cancer by combining HSP90 inhibitors with FIS deserves further in vivo study.

Structural characterization of human heme oxygenase-1 in complex with azole-based inhibitors.

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Rahman, Mona N; Vlahakis, Jason Z; Roman, Gheorghe; Vukomanovic, Dragic; Szarek, Walter A; Nakatsu, Kanji; Jia, Zongchao

2010-03-01

The development of inhibitors specific for heme oxygenases (HO) aims to provide powerful tools in understanding the HO system. Based on the lead structure (2S, 4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[((4-aminophenyl)thio)methyl]-1,3-dioxolane (azalanstat, QC-1) we have synthesized structural modifications to develop novel and selective HO inhibitors. The structural study of human HO-1 (hHO-1) in complex with a select group of the inhibitors was initiated using X-ray crystallographic techniques. Comparison of the structures of four such compounds each in complex with hHO-1 revealed a common binding mode, despite having different structural fragments. The compounds bind to the distal side of heme through an azole "anchor" which coordinates with the heme iron. An expansion of the distal pocket, mainly due to distal helix flexibility, allows accommodation of the compounds without displacing heme or the critical Asp140 residue. Rather, binding displaces a catalytically critical water molecule and disrupts an ordered hydrogen-bond network involving Asp140. The presence of a triazole "anchor" may provide further stability via a hydrogen bond with the protein. A hydrophobic pocket acts to stabilize the region occupied by the phenyl or adamantanyl moieties of these compounds. Further, a secondary hydrophobic pocket is formed via "induced fit" to accommodate bulky substituents at the 4-position of the dioxolane ring. Copyright 2009 Elsevier Inc. All rights reserved.

Pharmacological characterization of a novel phosphodiesterase type 5 (PDE5) inhibitor lodenafil carbonate on human and rabbit corpus cavernosum.

Science.gov (United States)

Toque, Haroldo A; Teixeira, Cleber E; Lorenzetti, Raquel; Okuyama, Cristina E; Antunes, Edson; De Nucci, Gilberto

2008-09-04

Nitrergic nerves and endothelial cells release nitric oxide (NO) in the corpus cavernosum, a key mediator that stimulates soluble guanylyl cyclase to increase cGMP levels causing penile erection. Phosphodiesterase 5 (PDE5) inhibitors, such as sildenafil, prolong the NO effects by inhibiting cGMP breakdown. Here, we report a novel PDE5 inhibitor, lodenafil carbonate, (Bis-(2-{4-[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-benzenesulfonyl]piperazin-1-yl}-ethyl)carbonate) that is a dimer of lodenafil. We therefore aimed to compare the effects of sildenafil, lodenafil and lodenafil carbonate on in vitro human and rabbit cavernosal relaxations, activity of crude PDE extracts from human platelets, as well as stability and metabolic studies in rat, dog and human plasma. Pharmacokinetic evaluations after intravenous and oral administration were performed in male beagles. Functional experiments were conducted using organ bath techniques. Pharmacokinetics was studied in beagles by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), following oral or intravascular administration. All PDE5 inhibitors tested concentration-dependently relaxed (0.001-100 microM) phenylephrine-precontracted rabbit and human corpus cavernosum. The cavernosal relaxations evoked by either acetylcholine (0.01-100 microM) or electrical field stimulation (EFS, 1-20 Hz) were markedly potentiated by sildenafil, lodenafil and lodenafil carbonate. Lodenafil carbonate was more potent to inhibit the cGMP hydrolysis in PDE extracts compared with lodenafil and sildenafil. Following intravascular and single oral administration of lodenafil carbonate, only lodenafil and norlodenafil were detected in vivo. These results indicate that lodenafil carbonate works as a prodrug, being lodenafil the active moiety of lodenafil carbonate.

Potent Inhibitors against Newcastle Disease Virus Hemagglutinin-Neuraminidase.

Science.gov (United States)

Rota, Paola; La Rocca, Paolo; Piccoli, Marco; Montefiori, Marco; Cirillo, Federica; Olsen, Lars; Orioli, Marica; Allevi, Pietro; Anastasia, Luigi

2018-02-06

Neuraminidase activity is essential for the infection and propagation of paramyxoviruses, including human parainfluenza viruses (hPIVs) and the Newcastle disease virus (NDV). Thus, many inhibitors have been developed based on the 2-deoxy-2,3-didehydro-d-N-acetylneuraminic acid inhibitor (DANA) backbone. Along this line, herein we report a series of neuraminidase inhibitors, having C4 (p-toluenesulfonamido and azido substituents) and C5 (N-perfluorinated chains) modifications to the DANA backbone, resulting in compounds with 5- to 15-fold greater potency than the currently most active compound, the N-trifluoroacetyl derivative of DANA (FANA), toward the NDV hemagglutinin-neuraminidase (NDV-HN). Remarkably, these inhibitors were found to be essentially inactive against the human sialidase NEU3, which is present on the outer layer of the cell membrane and is highly affected by the current NDV inhibitor FANA. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A study of ribonuclease activity in venom of vietnam cobra

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Thiet Van Nguyen

2017-09-01

Full Text Available Abstract Background Ribonuclease (RNase is one of the few toxic proteins that are present constantly in snake venoms of all types. However, to date this RNase is still poorly studied in comparison not only with other toxic proteins of snake venom, but also with the enzymes of RNase group. The objective of this paper was to investigate some properties of RNase from venom of Vietnam cobra Naja atra. Methods Kinetic methods and gel filtration chromatography were used to investigate RNase from venom of Vietnam cobra. Results RNase from venom of Vietnam cobra Naja atra has some characteristic properties. This RNase is a thermostable enzyme and has high conformational stability. This is the only acidic enzyme of the RNase A superfamily exhibiting a high catalytic activity in the pH range of 1–4, with pHopt = 2.58 ± 0.35. Its activity is considerably reduced with increasing ionic strength of reaction mixture. Venom proteins are separated by gel filtration into four peaks with ribonucleolytic activity, which is abnormally distributed among the isoforms: only a small part of the RNase activity is present in fractions of proteins with molecular weights of 12–15 kDa and more than 30 kDa, but most of the enzyme activity is detected in fractions of polypeptides, having molecular weights of less than 9 kDa, that is unexpected. Conclusions RNase from the venom of Vietnam cobra is a unique member of RNase A superfamily according to its acidic optimum pH (pHopt = 2.58 ± 0.35 and extremely low molecular weights of its major isoforms (approximately 8.95 kDa for RNase III and 5.93 kDa for RNase IV.

Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors

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Gozzo A.J.

2003-01-01

Full Text Available Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 µg/ml on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates reduced (1.2 to 3.0 times the catalytic efficiency of kallikrein (in a nanomolar range on the hydrolysis of plasminogen (0.3 to 1.8 µM and increased (1.9 to 7.7 times the enzyme efficiency in factor XII (0.1 to 10 µM activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times kallikrein inhibition by antithrombin (1.4 µM, while chondroitin 4- and 6-sulfates reduced it (1.3 times. Heparin and heparan sulfate increased (1.4 times the enzyme inhibition by the C1-inhibitor (150 nM.

Comparative studies on the human serum albumin binding of the clinically approved EGFR inhibitors gefitinib, erlotinib, afatinib, osimertinib and the investigational inhibitor KP2187.

Science.gov (United States)

Dömötör, Orsolya; Pelivan, Karla; Borics, Attila; Keppler, Bernhard K; Kowol, Christian R; Enyedy, Éva A

2018-05-30

Binding interactions between human serum albumin (HSA) and four approved epidermal growth factor receptor (EGFR) inhibitors gefitinib (GEF), erlotinib (ERL), afatinib (AFA), osimertinib (OSI), as well as the experimental drug KP2187, were investigated by means of spectrofluorometric and molecular modelling methods. Steady-state and time resolved spectrofluorometric techniques were carried out, including direct quenching of protein fluorescence and site marker displacement measurements. Proton dissociation processes and solvent dependent fluorescence properties were investigated as well. The EGFR inhibitors were predominantly presented in their single protonated form (HL + ) at physiological pH except ERL, which is charge-neutral. Significant solvent dependent fluorescence properties were found for GEF, ERL and KP2187, namely their emission spectra show strong dependence on the polarity and the hydrogen bonding ability of the solvents. The inhibitors proved to be bound at site I of HSA (in subdomain IIA) in a weak-to-moderate fashion (logK' 3.9-4.9) using spectrofluorometry. OSI (logK' 4.3) and KP2187 can additionally bind in site II (in subdomain IIIA), while GEF, ERL and AFA clearly show no interaction here. Docking methods qualitatively confirmed binding site preferences of compounds GEF and KP2187, and indicated that they probably bind to HSA in their neutral forms. Binding constants calculated on the basis of the various experimental data indicate a weak-to-moderate binding on HSA, only OSI exhibits somewhat higher affinity towards this protein. However, model calculations performed at physiological blood concentrations of HSA resulted in high (ca. 90%) bound fractions for the inhibitors, highlighting the importance of plasma protein binding. Copyright © 2018 Elsevier B.V. All rights reserved.

Pharmacology of a selective cyclooxygenase-2 inhibitor, HN-56249: a novel compound exhibiting a marked preference for the human enzyme in intact cells.

Science.gov (United States)

Berg, J; Fellier, H; Christoph, T; Kremminger, P; Hartmann, M; Blaschke, H; Rovensky, F; Towart, R; Stimmeder, D

2000-04-01

HN-56249 (3-(2,4-dichlorothiophenoxy)-4-methylsulfonylamino-benzenesu lfonamide), a highly selective cyclooxygenase (COX)-2 inhibitor, is the prototype of a novel series of COX inhibitors comprising bicyclic arylethersulfonamides; of this series HN-56249 is the most potent and selective human COX-2 inhibitor. HN-56249 inhibited platelet aggregation as a measure of COX-1 activity only moderately (IC50 26.5+/-1.7 microM). In LPS-stimulated monocytic cells the release of prostaglandin (PG) F1alpha as a measure of COX-2 was markedly inhibited (IC50 0.027+/-0.001 microM). Thus, HN-56249 showed an approximately 1000-fold selectivity for COX-2 in intact cells. In whole blood assays HN-56249 showed a potent inhibitory activity for COX-2 (IC50 0.78+/-0.37 microM) only. COX-1 was only weakly inhibited (IC50 867+/-181 microM). Hence, HN-56249 exhibited a greater than 1000-fold selectivity for whole blood COX-2. HN-56249 surpassed the COX-2 selectivities of the COX-2 selective inhibitors 3-cyclohexyloxy-4-methylsulfonylamino-nitrobenzene (NS-398) and 6-(2,4-difluorophenoxy)-5-methyl-sulfonylamino-1-indanone (flosulide) in the intact cell assays by eight- and threefold, respectively, and in the whole blood assays by approximately 40-fold. Following i.v. administration HN-56249 inhibited carrageenan-induced rat paw oedema only moderately (ID50 26.2+/-5.7 mg/kg, mean +/- SEM), approximately tenfold less potent than indomethacin (ID50 2.1+/-0.2 mg/kg, mean +/- SEM). After oral administration HN-56249 reversed thermal hyperalgesia in the carrageenan-induced rat paw oedema test, however, some 30-fold less potently than diclofenac. Comparing the inhibitory potency of HN-56249 against human COX-2 with that against murine COX-2 in intact cells revealed a 300-fold selectivity for the human enzyme. Similar effects were observed with other COX-2-selective arylethersulfonamides. In contrast, non-COX-2-selective arylethersulfonamides, including a highly selective COX-1 inhibitor, inhibited

In silico screening of potent natural inhibitor compounds against Human DOPA Decarboxylase for management of Parkinson’s Disease

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Surya Narayan Rath

2017-12-01

Full Text Available Loss of dopaminergic neurons of the substantia nigra of the mid brain is a well studied pathophysiology of Parkinson’s disease (PD, is the second most common neurodegenerative disorder. To compensate dopamine levels at the Central Nervous System (CNS exogenous L-Dopa is generally administered. But the major part of the L-Dopa is metabolized by Dopa decarboxylase (DDC, E.C. 4.1.1.28, a pyridoxal 5’ –phosphate (PLP enzyme, which is abundant in CNS and hence, only 1-5% of L-Dopa reaches to dopaminergic neurons. In this context, co-administration of peripheral DDC inhibitors (carbidopa or benserazide has been successfully used for the symptomatic treatment of PD patients. But, due to use of synthetic drugs many adverse effects have been reported during treatment. Therefore, the current study is planned to discover some plant based potent natural inhibitors against human DDC as an alternative way for the management of PD. This study was conducted through virtual screening and molecular docking of DDC enzyme with phytochemicals like withania somnifera (ashwagandha, glycine max (soybean, vicia faba (broad bean, and marsilea quadrifolia (sunsunia etc to evaluate their inhibition properties. In silico study results shown a good binding affinity and predicted some of the phytochemicals as potent natural inhibitors against human DDC. This work could be validated further through experimental procedures.

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity.

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Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L

2015-06-05

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity*

Science.gov (United States)

Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L.

2015-01-01

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. PMID:25903123

BstXI RFLP in the human inter-alpha-trypsin inhibitor light chain gene

Energy Technology Data Exchange (ETDEWEB)

Leveillard, T; Bourguignon, J; Sesbouee, R; Hanauer, A; Salier, J P; Diarra-Mehrpour, M; Martin, J P

1988-03-25

The 1.2 kb EcoRI/SmaI fragment of lambdaHuLITI2 was used as probe. lambdaHuLITI2 is a full length cDNA clone coding for human inter-alpha-trypsin inhibitor light chain isolated from immunochemical screening of a lambdagt11 library. Its sequence coding for HI-30 and alpha-1-microglobulin is in agreement. BstXI identifies five invariant bands at 5.0 kb, 2.3 kb, 1.5 kb, 1.1 kb, and 0.7 kb and a diallelic polymorphism with DNA fragments at 2.0 kb or 1.7 kb.

The effects of metal ion PCR inhibitors on results obtained with the Quantifiler(®) Human DNA Quantification Kit.

Science.gov (United States)

Combs, Laura Gaydosh; Warren, Joseph E; Huynh, Vivian; Castaneda, Joanna; Golden, Teresa D; Roby, Rhonda K

2015-11-01

Forensic DNA samples may include the presence of PCR inhibitors, even after extraction and purification. Studies have demonstrated that metal ions, co-purified at specific concentrations, inhibit DNA amplifications. Metal ions are endogenous to sample types, such as bone, and can be introduced from environmental sources. In order to examine the effect of metal ions as PCR inhibitors during quantitative real-time PCR, 2800 M DNA was treated with 0.0025-18.750 mM concentrations of aluminum, calcium, copper, iron, nickel, and lead. DNA samples, both untreated and metal-treated, were quantified using the Quantifiler(®) Human DNA Quantification Kit. Quantification cycle (Cq) values for the Quantifiler(®) Human DNA and internal PCR control (IPC) assays were measured and the estimated concentrations of human DNA were obtained. Comparisons were conducted between metal-treated and control DNA samples to determine the accuracy of the quantification estimates and to test the efficacy of the IPC inhibition detection. This kit is most resistant to the presence of calcium as compared to all metals tested; the maximum concentration tested does not affect the amplification of the IPC or quantification of the sample. This kit is most sensitive to the presence of aluminum; concentrations greater than 0.0750 mM negatively affected the quantification, although the IPC assay accurately assessed the presence of PCR inhibition. The Quantifiler(®) Human DNA Quantification Kit accurately quantifies human DNA in the presence of 0.5000 mM copper, iron, nickel, and lead; however, the IPC does not indicate the presence of PCR inhibition at this concentration of these metals. Unexpectedly, estimates of DNA quantity in samples treated with 18.750 mM copper yielded values in excess of the actual concentration of DNA in the samples; fluorescence spectroscopy experiments indicated this increase was not a direct interaction between the copper metal and 6-FAM dye used to label the probe that

Further insight into the roles of the glycans attached to human blood protein C inhibitor

DEFF Research Database (Denmark)

Sun, Wei; Parry, Simon; Ubhayasekera, Wimal

2010-01-01

Protein C inhibitor (PCI) is a 57-kDa glycoprotein that exists in many tissues and secretions in human. As a member of the serpin superfamily of proteins it displays unusually broad protease specificity. PCI is implicated in the regulation of a wide range of processes, including blood coagulation......, fertilization, prevention of tumors and pathogen defence. It has been reported that PCI isolated from human blood plasma is highly heterogeneous, and that this heterogeneity is caused by differences in N-glycan structures, N-glycosylation occupancy, and the presence of two forms that differ by the presence...... or absence of 6 amino acids at the amino-terminus. In this study we have verified that such heterogeneity exists in PCI purified from single individuals, and that individuals of two different ethnicities possess a similar PCI pattern, verifying that the micro-heterogeneity is conserved among humans...

Validation of Simultaneous Quantitative Method of HIV Protease Inhibitors Atazanavir, Darunavir and Ritonavir in Human Plasma by UPLC-MS/MS

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Tulsidas Mishra

2014-01-01

Full Text Available Objectives. HIV protease inhibitors are used in the treatment of patients suffering from AIDS and they act at the final stage of viral replication by interfering with the HIV protease enzyme. The paper describes a selective, sensitive, and robust method for simultaneous determination of three protease inhibitors atazanavir, darunavir and ritonavir in human plasma by ultra performance liquid chromatography-tandem mass spectrometry. Materials and Methods. The sample pretreatment consisted of solid phase extraction of analytes and their deuterated analogs as internal standards from 50 μL human plasma. Chromatographic separation of analytes was performed on Waters Acquity UPLC C18 (50 × 2.1 mm, 1.7 μm column under gradient conditions using 10 mM ammonium formate, pH 4.0, and acetonitrile as the mobile phase. Results. The method was established over a concentration range of 5.0–6000 ng/mL for atazanavir, 5.0–5000 ng/mL for darunavir and 1.0–500 ng/mL for ritonavir. Accuracy, precision, matrix effect, recovery, and stability of the analytes were evaluated as per US FDA guidelines. Conclusions. The efficiency of sample preparation, short analysis time, and high selectivity permit simultaneous estimation of these inhibitors. The validated method can be useful in determining plasma concentration of these protease inhibitors for therapeutic drug monitoring and in high throughput clinical studies.

Carbohydrate as covalent crosslink in human inter-alpha-trypsin inhibitor

DEFF Research Database (Denmark)

Jessen, T E; Faarvang, K L; Ploug, M

1988-01-01

The primary structure of inter-alpha-trypsin inhibitor is partially elucidated, but controversy about the construction of the polypeptide backbone still exists. We present evidence suggesting that inter-alpha-trypsin inhibitor represents a novel plasma protein structure with two separate polypept...... polypeptide chains covalently crosslinked only by carbohydrate (chondroitin sulphate)....

Calcineurin inhibitors acutely improve insulin sensitivity without affecting insulin secretion in healthy human volunteers

DEFF Research Database (Denmark)

Øzbay, Aygen; Møller, Niels; Juhl, Claus

2012-01-01

and tacrolimus has been attributed to both beta cell dysfunction and impaired insulin sensitivity. WHAT THIS STUDY ADDS: This is the first trial to investigate beta cell function and insulin sensitivity using gold standard methodology in healthy human volunteers treated with clinically relevant doses...... of ciclosporin and tacrolimus. We document that both drugs acutely increase insulin sensitivity, while first phase and pulsatile insulin secretion remain unaffected. This study demonstrates that ciclosporin and tacrolimus have similar acute effects on glucose metabolism in healthy humans. AIM The introduction...... of calcineurin inhibitors (CNIs) ciclosporin (CsA) and tacrolimus (Tac) has improved the outcome of organ transplants, but complications such as new onset diabetes mellitus after transplantation (NODAT) cause impairment of survival rates. The relative contribution of each CNI to the pathogenesis and development...

Structures of human Golgi-resident glutaminyl cyclase and its complexes with inhibitors reveal a large loop movement upon inhibitor binding.

Science.gov (United States)

Huang, Kai-Fa; Liaw, Su-Sen; Huang, Wei-Lin; Chia, Cho-Yun; Lo, Yan-Chung; Chen, Yi-Ling; Wang, Andrew H-J

2011-04-08

Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD150, was shown to be able to reduce the deposition of pyroglutamate-modified amyloid-β peptides in brain of transgenic mouse models of Alzheimer disease, leading to a significant improvement of learning and memory in those transgenic animals. Here, we report the 1.05-1.40 Å resolution structures, solved by the sulfur single-wavelength anomalous dispersion phasing method, of the Golgi-luminal catalytic domain of the recently identified Golgi-resident QC (gQC) and its complex with PBD150. We also describe the high-resolution structures of secretory QC (sQC)-PBD150 complex and two other gQC-inhibitor complexes. gQC structure has a scaffold similar to that of sQC but with a relatively wider and negatively charged active site, suggesting a distinct substrate specificity from sQC. Upon binding to PBD150, a large loop movement in gQC allows the inhibitor to be tightly held in its active site primarily by hydrophobic interactions. Further comparisons of the inhibitor-bound structures revealed distinct interactions of the inhibitors with gQC and sQC, which are consistent with the results from our inhibitor assays reported here. Because gQC and sQC may play different biological roles in vivo, the different inhibitor binding modes allow the design of specific inhibitors toward gQC and sQC.

Kinetic and Thermodynamic Rationale for SAHA Being a Preferential Human HDAC8 Inhibitor as Compared to the Structurally Similar Ligand, TSA

Science.gov (United States)

Singh, Raushan K.; Lall, Naveena; Leedahl, Travis S.; McGillivray, Abigail; Mandal, Tanmay; Haldar, Manas; Mallik, Sanku; Cook, Gregory; Srivastava, D.K.

2013-01-01

Of the different hydroxamate-based histone deacetylase (HDAC) inhibitors, Suberoylanilide hydroxamic acid (SAHA) has been approved by the FDA for treatment of T-cell lymphoma. Interestingly, a structurally similar inhibitor, Trichostatin A (TSA), which has a higher in vitro inhibitory-potency against HDAC8, reportedly shows a poor efficacy in clinical settings. In order to gain the molecular insight into the above discriminatory feature, we performed transient kinetic and isothermal titration calorimetric studies for the interaction of SAHA and TSA to the recombinant form of human HDAC8. The transient kinetic data revealed that the binding of both the inhibitors to the enzyme showed the biphasic profiles, which represented an initial encounter of enzyme with the inhibitor followed by the isomerization of the transient enzyme-inhibitor complexes. The temperature-dependent transient kinetic studies with the above inhibitors revealed that the bimolecular process is primarily dominated by favorable enthalpic changes, as opposed to the isomerization step; which is solely contributed by entropic changes. The standard binding-enthalpy (ΔH0) of SAHA, deduced from the transient kinetic as well as the isothermal titration calorimetric experiments, was 2–3 kcal/mol higher as compared to TSA. The experimental data presented herein suggests that SAHA serves as a preferential (target-specific/selective) HDAC8 inhibitor as compared to TSA. Arguments are presented that the detailed kinetic and thermodynamic studies may guide in the rational design of HDAC inhibitors as therapeutic agents. PMID:24079912

ELISA analysis of soybean trypsin inhibitors in processed foods.

Science.gov (United States)

Brandon, D L; Bates, A H; Friedman, M

1991-01-01

Soybean proteins are widely used in human foods in a variety of forms, including infant formulas, flour, protein concentrates, protein isolates, soy sauces, textured soy fibers, and tofu. The presence of inhibitors of digestive enzymes in soy proteins impairs the nutritional quality and possibly the safety of soybeans and other legumes. Processing, based on the use of heat or fractionation of protein isolates, does not completely inactivate or remove these inhibitors, so that residual amounts of inhibitors are consumed by animals and humans. New monoclonal antibody-based immunoassays can measure low levels of the soybean Kunitz trypsin inhibitor (KTI) and the Bowman-Birk trypsin and chymotrypsin inhibitor (BBI) and the Bowman-Birk foods. The enzyme-linked immunosorbent assay (ELISA) was used to measure the inhibitor content of soy concentrates, isolates, and flours, both heated and unheated; a commercial soy infant formula; KTI and BBI with rearranged disulfide bonds; browning products derived from heat-treatment of KTI with glucose and starch; and KTI exposed to high pH. The results indicate that even low inhibitor isolates contain significant amounts of specific inhibitors. Thus, infants on soy formula consume about 10 mg of KTI plus BBI per day. The immunoassays complement the established enzymatic assays of trypsin and chymotrypsin inhibitors, and have advantages in (a) measuring low levels of inhibitors in processed foods; and (b) differentiating between the Kunitz and Bowman-Birk inhibitors. The significance of our findings for food safety are discussed.

Human Kunitz-type protease inhibitor engineered for enhanced matrix retention extends longevity of fibrin biomaterials.

Science.gov (United States)

Briquez, Priscilla S; Lorentz, Kristen M; Larsson, Hans M; Frey, Peter; Hubbell, Jeffrey A

2017-08-01

Aprotinin is a broad-spectrum serine protease inhibitor used in the clinic as an anti-fibrinolytic agent in fibrin-based tissue sealants. However, upon re-exposure, some patients suffer from hypersensitivity immune reactions likely related to the bovine origin of aprotinin. Here, we aimed to develop a human-derived substitute to aprotinin. Based on sequence homology analyses, we identified the Kunitz-type protease inhibitor (KPI) domain of human amyloid-β A4 precursor protein as being a potential candidate. While KPI has a lower intrinsic anti-fibrinolytic activity than aprotinin, we reasoned that its efficacy is additionally limited by its fast release from fibrin material, just as aprotinin's is. Thus, we engineered KPI variants for controlled retention in fibrin biomaterials, using either covalent binding through incorporation of a substrate for the coagulation transglutaminase Factor XIIIa or through engineering of extracellular matrix protein super-affinity domains for sequestration into fibrin. We showed that both engineered KPI variants significantly slowed plasmin-mediated fibrinolysis in vitro, outperforming aprotinin. In vivo, our best engineered KPI variant (incorporating the transglutaminase substrate) extended fibrin matrix longevity by 50%, at a dose at which aprotinin did not show efficacy, thus qualifying it as a competitive substitute of aprotinin in fibrin sealants. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Liquid chromatography-tandem mass spectrometric assay for the T790M mutant EGFR inhibitor osimertinib (AZD9291) in human plasma

NARCIS (Netherlands)

Rood, Johannes J M; van Bussel, Mark T J; Schellens, Jan H M; Beijnen, Jos H; Sparidans, Rolf W

2016-01-01

A method for the quantitative analysis by ultra-performance liquid chromatography-tandem mass spectrometry of the highly selective irreversible covalent inhibitor of EGFR-TK, osimertinib in human plasma was developed and validated, using pazopanib as an internal standard. The validation was

Discovery and preclinical pharmacology of a selective ATP-competitive Akt inhibitor (GDC-0068) for the treatment of human tumors.

Science.gov (United States)

Blake, James F; Xu, Rui; Bencsik, Josef R; Xiao, Dengming; Kallan, Nicholas C; Schlachter, Stephen; Mitchell, Ian S; Spencer, Keith L; Banka, Anna L; Wallace, Eli M; Gloor, Susan L; Martinson, Matthew; Woessner, Richard D; Vigers, Guy P A; Brandhuber, Barbara J; Liang, Jun; Safina, Brian S; Li, Jun; Zhang, Birong; Chabot, Christine; Do, Steven; Lee, Leslie; Oeh, Jason; Sampath, Deepak; Lee, Brian B; Lin, Kui; Liederer, Bianca M; Skelton, Nicholas J

2012-09-27

The discovery and optimization of a series of 6,7-dihydro-5H-cyclopenta[d]pyrimidine compounds that are ATP-competitive, selective inhibitors of protein kinase B/Akt is reported. The initial design and optimization was guided by the use of X-ray structures of inhibitors in complex with Akt1 and the closely related protein kinase A. The resulting compounds demonstrate potent inhibition of all three Akt isoforms in biochemical assays and poor inhibition of other members of the cAMP-dependent protein kinase/protein kinase G/protein kinase C extended family and block the phosphorylation of multiple downstream targets of Akt in human cancer cell lines. Biological studies with one such compound, 28 (GDC-0068), demonstrate good oral exposure resulting in dose-dependent pharmacodynamic effects on downstream biomarkers and a robust antitumor response in xenograft models in which the phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin pathway is activated. 28 is currently being evaluated in human clinical trials for the treatment of cancer.

Repurposing a Library of Human Cathepsin L Ligands: Identification of Macrocyclic Lactams as Potent Rhodesain and Trypanosoma brucei Inhibitors.

Science.gov (United States)

Giroud, Maude; Dietzel, Uwe; Anselm, Lilli; Banner, David; Kuglstatter, Andreas; Benz, Jörg; Blanc, Jean-Baptiste; Gaufreteau, Delphine; Liu, Haixia; Lin, Xianfeng; Stich, August; Kuhn, Bernd; Schuler, Franz; Kaiser, Marcel; Brun, Reto; Schirmeister, Tanja; Kisker, Caroline; Diederich, François; Haap, Wolfgang

2018-04-26

Rhodesain (RD) is a parasitic, human cathepsin L (hCatL) like cysteine protease produced by Trypanosoma brucei ( T. b.) species and a potential drug target for the treatment of human African trypanosomiasis (HAT). A library of hCatL inhibitors was screened, and macrocyclic lactams were identified as potent RD inhibitors ( K i < 10 nM), preventing the cell-growth of Trypanosoma brucei rhodesiense (IC 50 < 400 nM). SARs addressing the S2 and S3 pockets of RD were established. Three cocrystal structures with RD revealed a noncovalent binding mode of this ligand class due to oxidation of the catalytic Cys25 to a sulfenic acid (Cys-SOH) during crystallization. The P-glycoprotein efflux ratio was measured and the in vivo brain penetration in rats determined. When tested in vivo in acute HAT model, the compounds permitted up to 16.25 (vs 13.0 for untreated controls) mean days of survival.

SNX-25a, a novel Hsp90 inhibitor, inhibited human cancer growth more potently than 17-AAG.

Science.gov (United States)

Wang, Shaoxiang; Wang, Xiao; Du, Zhan; Liu, Yuting; Huang, Dane; Zheng, Kai; Liu, Kaisheng; Zhang, Yi; Zhong, Xueyun; Wang, Yifei

2014-07-18

17-Allylamino-17-demethoxygeldanamycin (17-AAG), a typical Hsp90 inhibitor derived from geldanamycin (GA), has entered Phase III clinical trials for cancer therapy. However, it has several significant limitations such as poor solubility, limited bioavailability and unacceptable hepatotoxicity. In this study, the anticancer activity and mechanism of SNX-25a, a novel Hsp90 inhibitor, was investigated comparing with that of 17-AAG. We showed that SNX-25a triggered growth inhibition more sensitively than 17-AAG against many human cancer cells, including K562, SW-620, A375, Hep-2, MCF-7, HepG2, HeLa, and A549 cell lines, especially at low concentrations (AAG, SNX-25a was more potent in arresting the cell cycle at G2 phase, and displayed more potent effects on human cancer cell apoptosis and Hsp90 client proteins. It also exhibited a stronger binding affinity to Hsp90 than 17-AAG using molecular docking. Considering the superiority effects on Hsp90 affinity, cell growth, cell cycle, apoptosis, and Hsp90 client proteins, SNX-25a is supposed as a potential anticancer agent that needs to be explored in detail. Copyright © 2014 Elsevier Inc. All rights reserved.

Safety, pharmacokinetics, and antiviral activity of A77003, a C2 symmetry-based human immunodeficiency virus protease inhibitor

NARCIS (Netherlands)

Reedijk, M.; Boucher, C. A.; van Bommel, T.; Ho, D. D.; Tzeng, T. B.; Sereni, D.; Veyssier, P.; Jurriaans, S.; Granneman, R.; Hsu, A.

1995-01-01

A77003, an inhibitor of the human immunodeficiency virus type 1 (HIV-1) protease, was administered to asymptomatic HIV-1-infected patients in a phase I trial. The drug was given by continuous intravenous infusion at dosages of 0.035, 0.07, 0.14, and 0.28 mg/kg of body weight per h. The drug was

A direct thrombin inhibitor suppresses protein C activation and factor Va degradation in human plasma: Possible mechanisms of paradoxical enhancement of thrombin generation.

Science.gov (United States)

Kamisato, Chikako; Furugohri, Taketoshi; Morishima, Yoshiyuki

2016-05-01

We have demonstrated that antithrombin (AT)-independent thrombin inhibitors paradoxically increase thrombin generation (TG) in human plasma in a thrombomodulin (TM)- and protein C (PC)-dependent manner. We determined the effects of AT-independent thrombin inhibitors on the negative-feedback system, activation of PC and production and degradation of factor Va (FVa), as possible mechanisms underlying the paradoxical enhancement of TG. TG in human plasma containing 10nM TM was assayed by means of the calibrated automated thrombography. As an index of PC activation, plasma concentration of activated PC-PC inhibitor complex (aPC-PCI) was measured. The amounts of FVa heavy chain and its degradation product (FVa(307-506)) were examined by western blotting. AT-independent thrombin inhibitors, melagatran and dabigatran (both at 25-600nM) and 3-30μg/ml active site-blocked thrombin (IIai), increased peak levels of TG. Melagatran, dabigatran and IIai significantly decreased plasma concentration of aPC-PCI complex at 25nM or more, 75nM or more, and 10 and 30μg/ml, respectively. Melagatran (300nM) significantly increased FVa and decreased FVa(307-506). In contrast, a direct factor Xa inhibitor edoxaban preferentially inhibited thrombin generation (≥25nM), and higher concentrations were required to inhibit PC activation (≥150nM) and FVa degradation (300nM). The present study suggests that the inhibitions of protein C activation and subsequent degradation of FVa and increase in FVa by antithrombin-independent thrombin inhibitors may contribute to the paradoxical TG enhancement, and edoxaban may inhibit PC activation and FVa degradation as a result of TG suppression. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fem1b, a proapoptotic protein, mediates proteasome inhibitor-induced apoptosis of human colon cancer cells.

Science.gov (United States)

Subauste, M Cecilia; Sansom, Owen J; Porecha, Nehal; Raich, Natacha; Du, Liqin; Maher, Joseph F

2010-02-01

In the treatment of colon cancer, the development of resistance to apoptosis is a major factor in resistance to therapy. New molecular approaches to overcome apoptosis resistance, such as selectively upregulating proapoptotic proteins, are needed in colon cancer therapy. In a mouse model with inactivation of the adenomatous polyposis coli (Apc) tumor suppressor gene, reflecting the pathogenesis of most human colon cancers, the gene encoding feminization-1 homolog b (Fem1b) is upregulated in intestinal epithelium following Apc inactivation. Fem1b is a proapoptotic protein that interacts with apoptosis-inducing proteins Fas, tumor necrosis factor receptor-1 (TNFR1), and apoptotic protease activating factor-1 (Apaf-1). Increasing Fem1b expression induces apoptosis of cancer cells, but effects on colon cancer cells have not been reported. Fem1b is a homolog of feminization-1 (FEM-1), a protein in Caenorhabditis elegans that is regulated by proteasomal degradation, but whether Fem1b is likewise regulated by proteasomal degradation is unknown. Herein, we found that Fem1b protein is expressed in primary human colon cancer specimens, and in malignant SW620, HCT-116, and DLD-1 colon cancer cells. Increasing Fem1b expression, by transfection of a Fem1b expression construct, induced apoptosis of these cells. We found that proteasome inhibitor treatment of SW620, HCT-116, and DLD-1 cells caused upregulation of Fem1b protein levels, associated with induction of apoptosis. Blockade of Fem1b upregulation with morpholino antisense oligonucleotide suppressed the proteasome inhibitor-induced apoptosis of these cells. In conclusion, the proapoptotic protein Fem1b is downregulated by the proteasome in malignant colon cancer cells and mediates proteasome inhibitor-induced apoptosis of these cells. Therefore, Fem1b could represent a novel molecular target to overcome apoptosis resistance in therapy of colon cancer.

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity

Energy Technology Data Exchange (ETDEWEB)

Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L. (UW-MED); (UCB)

2015-04-22

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.

Biochemical analysis of leishmanial and human GDP-Mannose Pyrophosphorylases and selection of inhibitors as new leads.

Science.gov (United States)

Mao, Wei; Daligaux, Pierre; Lazar, Noureddine; Ha-Duong, Tâp; Cavé, Christian; van Tilbeurgh, Herman; Loiseau, Philippe M; Pomel, Sébastien

2017-04-07

Leishmaniases are an ensemble of diseases caused by the protozoan parasite of the genus Leishmania. Current antileishmanial treatments are limited and present main issues of toxicity and drug resistance emergence. Therefore, the generation of new inhibitors specifically directed against a leishmanial target is an attractive strategy to expand the chemotherapeutic arsenal. GDP-Mannose Pyrophosphorylase (GDP-MP) is a prominent therapeutic target involved in host-parasite recognition which has been described to be essential for parasite survival. In this work, we produced and purified GDP-MPs from L. mexicana (LmGDP-MP), L. donovani (LdGDP-MP), and human (hGDP-MP), and compared their enzymatic properties. From a rationale design of 100 potential inhibitors, four compounds were identified having a promising and specific inhibitory effect on parasite GDP-MP and antileishmanial activities, one of them exhibits a competitive inhibition on LdGDP-MP and belongs to the 2-substituted quinoline series.

Immunological changes in human immunodeficiency virus (HIV)-infected individuals during HIV-specific protease inhibitor treatment

DEFF Research Database (Denmark)

Ullum, H; Katzenstein, T; Aladdin, H

1999-01-01

The present study examines the influence of effective anti-retroviral treatment on immune function, evaluated by a broad array of immunological tests. We followed 12 individuals infected with human immunodeficiency virus (HIV) for 6 months after initiation of combination anti-retroviral treatment...... including a protease inhibitor. Unstimulated and pokeweed mitogen (PWM)-, interleukin (IL)-2- and phytohaemagglutinin (PHA)-stimulated lymphocyte proliferative responses increased during follow-up reaching average levels from 1.3-fold (PHA) to 3.7-fold (PWM) above baseline values. The total CD4+ lymphocyte...

Cloning, expression and location of RNase9 in human epididymis

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Lin YQ

2008-11-01

Full Text Available Abstract Background Mammalian spermatozoa become fully motile and fertile during transit through the luminal fluid of the epididymis. At least 200 proteins are present in the epididymal lumen, but the potential roles of these luminal proteins in male fertility are unknown. Investigation of the function of these proteins will elucidate the mechanism of sperm maturation, and also provide new drug targets for male contraception. We cloned RNase9 from a human epididymis cDNA library for characterization and analysis of its functions. Findings It was predicted that human RNase9 gene was located on chromosome 14q11.2 and encoded a 205 amino acids protein with a signal peptide of 26 amino acids at the N-terminus. The protein had eight conserved cysteine residues characteristic of the RNase A family members and several potential post-translational modification sites. At the transcriptional level, RNase9 was expressed in a wide variety of tissues, and the expression was higher in men than in boys. RNase9 was localized to the post-equatorial region of the sperms' head. Immunofluorescence staining showed that RNase9 protein was present mostly in the epithelium of the epididymal tubule. Recombinant RNase9 had no ribonuclease activity. In addition, RNase9 had no detectable effect on sperm motility and fertilization as demonstrated by blocking spermatozoa with anti-RNase9 polyclonal serum. Conclusion RNase9 is expressed in a wide variety of tissues. It is located on the post-equatorial region of the sperm head and the epithelium of epididymal tubule. Although RNase9 belongs to the RNase A family, it has no ribonuclease activity.

Lysosomal degradation of receptor-bound urokinase-type plasminogen activator is enhanced by its inhibitors in human trophoblastic choriocarcinoma cells

DEFF Research Database (Denmark)

Jensen, Poul Henning; Christensen, Erik Ilsø; Ebbesen, P.

1990-01-01

We have studied the effect of plasminogen activator inhibitors PAI-1 and PAI-2 on the binding of urokinase-type plasminogen activator (u-PA) to its receptor in the human choriocarcinoma cell line JAR. With 125I-labeled ligands in whole-cell binding assays, both uncomplexed u-PA and u......, with the highest density of grains over the membrane at cell-cell interphases, but, after incubation at 37 degrees C, 17 and 27% of the grains for u-PA and u-PA-PAI-1 complexes, respectively, appeared over lysosomal-like bodies. These findings suggest that the u-PA receptor possesses a clearance function......-PA-inhibitor complexes bound to the receptor with a Kd of approximately 100 pM at 4 degrees C. Transferring the cells to 37 degrees C led to degradation to amino acids of up to 50% of the cell-bound u-PA-inhibitor complexes, whereas the degradation of uncomplexed u-PA was 15%; the remaining ligand was recovered...

An Updated Review of Tyrosinase Inhibitors

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Te-Sheng Chang

2009-05-01

Full Text Available Tyrosinase is a multifunctional, glycosylated, and copper-containing oxidase, which catalyzes the first two steps in mammalian melanogenesis and is responsible for enzymatic browning reactions in damaged fruits during post-harvest handling and processing. Neither hyperpigmentation in human skin nor enzymatic browning in fruits are desirable. These phenomena have encouraged researchers to seek new potent tyrosinase inhibitors for use in foods and cosmetics. This article surveys tyrosinase inhibitors newly discovered from natural and synthetic sources. The inhibitory strength is compared with that of a standard inhibitor, kojic acid, and their inhibitory mechanisms are discussed.

Identification of human flap endonuclease 1 (FEN1) inhibitors using a machine learning based consensus virtual screening.

Science.gov (United States)

Deshmukh, Amit Laxmikant; Chandra, Sharat; Singh, Deependra Kumar; Siddiqi, Mohammad Imran; Banerjee, Dibyendu

2017-07-25

Human Flap endonuclease1 (FEN1) is an enzyme that is indispensable for DNA replication and repair processes and inhibition of its Flap cleavage activity results in increased cellular sensitivity to DNA damaging agents (cisplatin, temozolomide, MMS, etc.), with the potential to improve cancer prognosis. Reports of the high expression levels of FEN1 in several cancer cells support the idea that FEN1 inhibitors may target cancer cells with minimum side effects to normal cells. In this study, we used large publicly available, high-throughput screening data of small molecule compounds targeted against FEN1. Two machine learning algorithms, Support Vector Machine (SVM) and Random Forest (RF), were utilized to generate four classification models from huge PubChem bioassay data containing probable FEN1 inhibitors and non-inhibitors. We also investigated the influence of randomly selected Zinc-database compounds as negative data on the outcome of classification modelling. The results show that the SVM model with inactive compounds was superior to RF with Matthews's correlation coefficient (MCC) of 0.67 for the test set. A Maybridge database containing approximately 53 000 compounds was screened and top ranking 5 compounds were selected for enzyme and cell-based in vitro screening. The compound JFD00950 was identified as a novel FEN1 inhibitor with in vitro inhibition of flap cleavage activity as well as cytotoxic activity against a colon cancer cell line, DLD-1.

Thermostabilisation of human serum butyrylcholinesterase for detection of its inhibitors in water and biological fluids

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Lakshmanan Jaganathan

1999-01-01

Full Text Available The ability of gelatine-trehalose to convert the normally fragile, dry human serum BChE into a thermostable enzyme and its use in the detection of cholinesterase inhibitors in water and biological fluids is described. Gelatine or trehalose alone is unable to protect the dry enzyme against exposure to high temperature, while a combination of gelatine and trehalose were able to protect the enzyme activity against prolonged exposure to temperature as high as +50°C. A method for rapid, simple and inexpensive means of screening for cholinesterase inhibitors such as carbamates and organophosphates in water, vegetables and human blood has been developed.A capacidade da gelatina-trehalose em converter a frágil BChE do soro humano em uma enzima termoestável e seu uso na descoberta de inibidores de colinesterase em água e fluidos biológicos é apresentado. A Gelatina ou trehalose são incapazes de proteger a enzima seca BchE do soro humano contra exposição a elevadas temperaturas, enquanto que uma combinação de gelatina e trehalose são capazes de proteger a atividade de enzima contra exposição prolongada a temperaturas elevadas e da ordem de 50° C. Um método barato, simples e rápido de screening para inibidores de colinesterase tal como carbamatos e organofosfatos em água, verduras e sangue humano foi desenvolvido.

Discovering Bisdemethoxycurcumin from Curcuma longa rhizome as a potent small molecule inhibitor of human pancreatic α-amylase, a target for type-2 diabetes.

Science.gov (United States)

Ponnusamy, Sudha; Zinjarde, Smita; Bhargava, Shobha; Rajamohanan, P R; Ravikumar, Ameeta

2012-12-15

Curcuma longa rhizome is used extensively in culinary preparations in Far East and South-East Asia. Health benefits of curcuminoids from C. longa as antioxidants, anti-cancer and anti-inflammatory molecules have been well documented. We report here for the first time that Bisdemethoxycurcumin (BDMC) from C. longa, acts as an inhibitor to inactivate human pancreatic α-amylase, a therapeutic target for oral hypoglycemic agents in type-2 diabetes. Bioactivity guided isolation of rhizome isopropanol extract led to the identification by HPLC and NMR of BDMC as a lead small molecule inhibitor of porcine and human pancreatic α-amylase with an IC(50) value of 0.026 and 0.025 mM, respectively. Kinetic analysis revealed that using starch as the substrate, HPA exhibited an uncompetitive mode of inhibition with an apparent K(i) of 3.0 μM. The study gains importance as BDMC could be a good drug candidate in development of new inhibitors of HPA and of functional foods for controlling starch digestion in order to reduce post-prandial hyperglycemia. Copyright © 2012 Elsevier Ltd. All rights reserved.

pH-dependent antitumor activity of proton pump inhibitors against human melanoma is mediated by inhibition of tumor acidity.

Science.gov (United States)

De Milito, Angelo; Canese, Rossella; Marino, Maria Lucia; Borghi, Martina; Iero, Manuela; Villa, Antonello; Venturi, Giulietta; Lozupone, Francesco; Iessi, Elisabetta; Logozzi, Mariantonia; Della Mina, Pamela; Santinami, Mario; Rodolfo, Monica; Podo, Franca; Rivoltini, Licia; Fais, Stefano

2010-07-01

Metastatic melanoma is associated with poor prognosis and still limited therapeutic options. An innovative treatment approach for this disease is represented by targeting acidosis, a feature characterizing tumor microenvironment and playing an important role in cancer malignancy. Proton pump inhibitors (PPI), such as esomeprazole (ESOM) are prodrugs functionally activated by acidic environment, fostering pH neutralization by inhibiting proton extrusion. We used human melanoma cell lines and xeno-transplated SCID mice to provide preclinical evidence of ESOM antineoplastic activity. Human melanoma cell lines, characterized by different mutation and signaling profiles, were treated with ESOM in different pH conditions and evaluated for proliferation, viability and cell death. SCID mice engrafted with human melanoma were used to study ESOM administration effects on tumor growth and tumor pH by magnetic resonance spectroscopy (MRS). ESOM inhibited proliferation of melanoma cells in vitro and induced a cytotoxicity strongly boosted by low pH culture conditions. ESOM-induced tumor cell death occurred via rapid intracellular acidification and activation of several caspases. Inhibition of caspases activity by pan-caspase inhibitor z-vad-fmk completely abrogated the ESOM-induced cell death. ESOM administration (2.5 mg kg(-1)) to SCID mice engrafted with human melanoma reduced tumor growth, consistent with decrease of proliferating cells and clear reduction of pH gradients in tumor tissue. Moreover, systemic ESOM administration dramatically increased survival of human melanoma-bearing animals, in absence of any relevant toxicity. These data show preclinical evidence supporting the use of PPI as novel therapeutic strategy for melanoma, providing the proof of concept that PPI target human melanoma modifying tumor pH gradients.

Identification and characterization of metabolites of ASP015K, a novel oral Janus kinase inhibitor, in rats, chimeric mice with humanized liver, and humans.

Science.gov (United States)

Nakada, Naoyuki; Oda, Kazuo

2015-01-01

1. Here, we elucidated the structure of metabolites of novel oral Janus kinase inhibitor ASP015K in rats and humans and evaluated the predictability of human metabolites using chimeric mice with humanized liver (PXB mice). 2. Rat biological samples collected after oral dosing of (14)C-labelled ASP015K were examined using a liquid chromatography-radiometric detector and mass spectrometer (LC-RAD/MS). The molecular weight of metabolites in human and the liver chimeric mouse biological samples collected after oral dosing of non-labelled ASP015K was also investigated via LC-MS. Metabolites were also isolated from rat bile samples and analyzed using nuclear magnetic resonance. 3. Metabolic pathways of ASP015K in rats and humans were found to be glucuronide conjugation, methyl conjugation, sulfate conjugation, glutathione conjugation, hydroxylation of the adamantane ring and N-oxidation of the 1H-pyrrolo[2,3-b]pyridine ring. The main metabolite of ASP015K in rats was the glucuronide conjugate, while the main metabolite in humans was the sulfate conjugate. Given that human metabolites were produced by human hepatocytes in chimeric mice with humanized liver, this human model mouse was believed to be useful in predicting the human metabolic profile of various drug candidates.

New approaches of PARP-1 inhibitors in human lung cancer cells and cancer stem-like cells by some selected anthraquinone-derived small molecules.

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Yu-Ru Lee

Full Text Available Poly (ADP-ribose polymerase-1 (PARP-1 and telomerase, as well as DNA damage response pathways are targets for anticancer drug development, and specific inhibitors are currently under clinical investigation. The purpose of this work is to evaluate anticancer activities of anthraquinone-derived tricyclic and tetracyclic small molecules and their structure-activity relationships with PARP-1 inhibition in non-small cell lung cancer (NSCLC and NSCLC-overexpressing Oct4 and Nanog clone, which show high-expression of PARP-1 and more resistance to anticancer drug. We applied our library selected compounds to NCI's 60 human cancer cell-lines (NCI-60 in order to generate systematic profiling data. Based on our analysis, it is hypothesized that these drugs might be, directly and indirectly, target components to induce mitochondrial permeability transition and the release of pro-apoptotic factors as potential anti-NSCLC or PARP inhibitor candidates. Altogether, the most active NSC747854 showed its cytotoxicity and dose-dependent PARP inhibitory manner, thus it emerges as a promising structure for anti-cancer therapy with no significant negative influence on normal cells. Our studies present evidence that telomere maintenance should be taken into consideration in efforts not only to overcome drug resistance, but also to optimize the use of telomere-based therapeutics. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and telomerase inhibitors, in particular. Together, the data presented here expand our insight into the PARP inhibitors and support the resource-demanding lead optimization of structurally related small molecules for human cancer therapy.

New Approaches of PARP-1 Inhibitors in Human Lung Cancer Cells and Cancer Stem-Like Cells by Some Selected Anthraquinone-Derived Small Molecules

Science.gov (United States)

Yu, Dah-Shyong; Huang, Kuo-Feng; Chou, Shih-Jie; Chen, Tsung-Chih; Lee, Chia-Chung; Chen, Chun-Liang; Chiou, Shih-Hwa; Huang, Hsu-Shan

2013-01-01

Poly (ADP-ribose) polymerase-1 (PARP-1) and telomerase, as well as DNA damage response pathways are targets for anticancer drug development, and specific inhibitors are currently under clinical investigation. The purpose of this work is to evaluate anticancer activities of anthraquinone-derived tricyclic and tetracyclic small molecules and their structure-activity relationships with PARP-1 inhibition in non-small cell lung cancer (NSCLC) and NSCLC-overexpressing Oct4 and Nanog clone, which show high-expression of PARP-1 and more resistance to anticancer drug. We applied our library selected compounds to NCI's 60 human cancer cell-lines (NCI-60) in order to generate systematic profiling data. Based on our analysis, it is hypothesized that these drugs might be, directly and indirectly, target components to induce mitochondrial permeability transition and the release of pro-apoptotic factors as potential anti-NSCLC or PARP inhibitor candidates. Altogether, the most active NSC747854 showed its cytotoxicity and dose-dependent PARP inhibitory manner, thus it emerges as a promising structure for anti-cancer therapy with no significant negative influence on normal cells. Our studies present evidence that telomere maintenance should be taken into consideration in efforts not only to overcome drug resistance, but also to optimize the use of telomere-based therapeutics. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and telomerase inhibitors, in particular. Together, the data presented here expand our insight into the PARP inhibitors and support the resource-demanding lead optimization of structurally related small molecules for human cancer therapy. PMID:23451039

Effects of GSK3 inhibitors on in vitro expansion and differentiation of human adipose-derived stem cells into adipocytes

Directory of Open Access Journals (Sweden)

Peraldi Pascal

2008-02-01

Full Text Available Abstract Background Multipotent stem cells exist within adipose tissue throughout life. An abnormal recruitment of these adipose precursor cells could participate to hyperplasia of adipose tissue observed in severe obesity or to hypoplasia of adipose tissue observed in lipodystrophy. Therefore, pharmacological molecules that control the pool of stem cells in adipose tissue are of great interest. Glycogen Synthase Kinase (GSK 3 has been previously described as involved in differentiation of preadipose cells and might be a potential therapeutic target to modulate proliferation and differentiation of adipocyte precursors. However, the impact of GSK3 inhibition on human adipose-derived stem cells remained to be investigated. The aim of this study was to investigate GSK3 as a possible target for pharmacological inhibition of stem cell adipogenesis. To reach this goal, we studied the effects of pharmacological inhibitors of GSK3, i.e. lithium chloride (LiCl and BIO on proliferation and adipocyte differentiation of multipotent stem cells derived from human adipose tissue. Results Our results showed that GSK3 inhibitors inhibited proliferation and clonogenicity of human stem cells, strongly suggesting that GSK3 inhibitors could be potent regulators of the pool of adipocyte precursors in adipose tissue. The impact of GSK3 inhibition on differentiation of hMADS cells was also investigated. Adipogenic and osteogenic differentiations were inhibited upon hMADS treatment with BIO. Whereas a chronic treatment was required to inhibit osteogenesis, a treatment that was strictly restricted to the early step of differentiation was sufficient to inhibit adipogenesis. Conclusion These results demonstrated the feasibility of a pharmacological approach to regulate adipose-derived stem cell function and that GSK3 could represent a potential target for controlling adipocyte precursor pool under conditions where fat tissue formation is impaired.

Particle-bound phytochrome: differential pigment release by surfactants, ribonuclease and phospholipase C

International Nuclear Information System (INIS)

Gressel, J.; Quail, P.H.

1976-01-01

Surfactants and hydrolytic enzymes were used to probe the nature of the constituent(s) to which phytochrome binds in particulate fractions from red-irradiated Cucurbita, [ 14 C]-choline and [ 3 H]-uridine pre-labelled tissue was used to monitor the release of phospholipids and RNA by these agents. Ribonuclease (RNase) digestion of 20,000 x g pellets eliminates both the phytochrome and ribonucleprotein (RNP) which cosediment at 31S. Little [ 14 C]-choline occurs in the 31S fraction and the amount is not changed by RNase digestion. This is further evidence that phytochrome binds directly to the RNP in the 31S fraction rather than to any membranous material present. The distribution profile of the RNA in a second (='heavy') phytochrome fraction does not correlate with that of the pigment. This suggests that the phytochrome in this fraction is not bound to RNP. The RNA is of ribosomal origin but much less degraded than that of the 31S RNP and is resistant to RNase digestion. Phospholipase C releases 80% of the [ 14 C]-choline from the 'heavy' fraction without freeing phytochrome. This indicates that the pigment does not bind to the polar head groups of the membrane phospholipids present. Low concentrations of deoxycholate dissociate phytochrome from this fraction without releasing substantial quantities of integral membrane proteins or phospholipids. Some RNP is dislodged by the surfactant but the phytochrome and RNP are not released as a complex. The data suggest that the pigment in the 'heavy' fraction may be loosely bound to a protein constituent rather than to RNP or polar phospholipids. (auth.)

The ribonuclease Dis3 is an essential regulator of the developmental transcriptome

Directory of Open Access Journals (Sweden)

Hou Dezhi

2012-08-01

Full Text Available Abstract Background Dis3 is ribonuclease that acts directly in the processing, turnover, and surveillance of a large number of distinct RNA species. Evolutionarily conserved from eubacteria to eukaryotes and a crucial component of the RNA processing exosome, Dis3 has been shown to be essential in yeast and fly S2 cells. However, it is not known whether Dis3 has essential functions in a metazoan. This study inquires whether Dis3 is required for Drosophila development and viability and how Dis3 regulates the transcriptome in the developing fly. Results Using transgenic flies, we show that Dis3 knock down (Dis3KD retards growth, induces melanotic tumor formation, and ultimately results in 2nd instar larval lethality. In order to determine whether Dis3KD fly phenotypes were a consequence of disrupting developmentally regulated RNA turnover, we performed RNA deep sequencing analysis on total RNA isolated from developmentally staged animals. Bioinformatic analysis of transcripts from Dis3KD flies reveals substantial transcriptomic changes, most notably down-regulation in early expressed RNAs. Finally, gene ontology analysis of this early stage shows that Dis3 regulates transcripts related to extracellular structure and remodelling, neurogenesis, and nucleotide metabolism. Conclusions We conclude that Dis3 is essential for early Drosophila melanogaster development and has specific and important stage-specific roles in regulating RNA metabolism. In showing for the first time that Dis3 is required for the development of a multicellular organism, our work provides mechanistic insight into how Dis3—either independent of or associated with the RNA processing exosome—participates in cell type-specific RNA turnover in metazoan development.

RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

International Nuclear Information System (INIS)

Rumi, Mohammad; Ishihara, Shunji; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

2006-01-01

RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor α-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use

Activities of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor nelfinavir mesylate in combination with reverse transcriptase and protease inhibitors against acute HIV-1 infection in vitro.

OpenAIRE

Patick, A K; Boritzki, T J; Bloom, L A

1997-01-01

Nelfinavir mesylate (formerly AG1343) is a potent and selective, nonpeptidic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease that was discovered by protein structure-based design methodologies. We evaluated the antiviral and cytotoxic effects of two-drug combinations of nelfinavir with the clinically approved antiretroviral therapeutics zidovudine (ZDV), lamivudine (3TC), dideoxycytidine (ddC; zalcitabine), stavudine (d4T), didanosine (ddI), indinavir, saquinavir, and ritona...

Plant Ribonucleases and Nucleases as Antiproliferative Agens Targeting Human Tumors Growing in Mice

Czech Academy of Sciences Publication Activity Database

Matoušek, Jaroslav; Matoušek, Josef

2010-01-01

Roč. 4, č. 1 (2010), s. 29-39 ISSN 1872-2156 R&D Projects: GA ČR GA521/06/1149; GA ČR GA521/09/1214 Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z50450515 Keywords : antiproliferative cytotoxic * effect human * plant nuclease Subject RIV: EB - Genetics ; Molecular Biology

Cytocidal activities of topoisomerase 1 inhibitors and 5-azacytidine against pheochromocytoma/paraganglioma cells in primary human tumor cultures and mouse cell lines.

Directory of Open Access Journals (Sweden)

James F Powers

Full Text Available There is currently no effective treatment for metastatic pheochromocytomas and paragangliomas. A deficiency in current chemotherapy regimens is that the metastases usually grow very slowly. Drugs that target dividing tumor cells have therefore had limited success. To improve treatment, new strategies and valid experimental models are required for pre-clinical testing. However, development of models has itself been hampered by the absence of human pheochromocytoma/paraganglioma cell lines for cultures or xenografts. Topoisomerase 1 (TOP1 inhibitors are drugs that interfere with mechanisms that maintain DNA integrity during transcription in both quiescent and dividing cells. We used primary cultures of representative human tumors to establish the cytotoxicity of camptothecin, a prototypical TOP1 inhibitor, against non-dividing pheochromocytoma/paraganglioma cells, and then employed a mouse pheochromocytoma model (MPC to show that efficacy of low concentrations of camptothecin and other TOP1 inhibitors is increased by intermittent coadministration of sub-toxic concentrations of 5-azacytidine, a DNA methylation inhibitor that modulates transcription. We then tested the same drugs against a clonal MPC derivative that expresses CMV reporter-driven luciferase and GFP, intended for in vivo drug testing. Unexpectedly, luciferase expression, bioluminescence and GFP expression were paradoxically increased by both camptothecin and SN38, the active metabolite of irinotecan, thereby masking cell death. Expression of chromogranin A, a marker for neuroendocrine secretory granules, was not increased, indicating that the drug effects on levels of luciferase and GFP are specific to the GFP-luciferase construct rather than generalized cellular responses. Our findings provide proof of principle for use of TOP1 inhibitors against pheochromocytoma/paraganglioma and suggest novel strategies for enhancing efficacy and reducing toxicity by optimizing the combination and

Nuclear RNA quantification in protoplast cell-cycle phases.

Science.gov (United States)

Bergounioux, C; Perennes, C; Brown, S C; Gadal, P

1988-01-01

Using acridine orange staining and flow cytometry the DNA and RNA levels (arbitrary units) of individual cells may be established. Here, this method has been applied to nuclei isolated from plant protoplasts during culture. The specificity of the technique has been validated for such plant material; ribonuclease markedly reduced nuclear staining without modifying the DNA histogram; ribonuclease inhibitor prevented the action of released cell nucleases; and protoplasts cultivated with actinomycin D did not synthesize RNA. First RNA synthesis was evident 18 h after Petunia hybrida protoplasts had been put into culture. An increase of RNA above a critical level was required for cells to be able to initiate DNA replication from G1, termed G1B. G2 nuclei had an RNA:DNA ratio similar to that of G1 nuclei.

Phosphatase Inhibitors Function as Novel, Broad Spectrum Botulinum Neurotoxin Antagonists in Mouse and Human Embryonic Stem Cell-Derived Motor Neuron-Based Assays.

Directory of Open Access Journals (Sweden)

Erkan Kiris

Full Text Available There is an urgent need to develop novel treatments to counter Botulinum neurotoxin (BoNT poisoning. Currently, the majority of BoNT drug development efforts focus on directly inhibiting the proteolytic components of BoNT, i.e. light chains (LC. Although this is a rational approach, previous research has shown that LCs are extremely difficult drug targets and that inhibiting multi-serotype BoNTs with a single LC inhibitor may not be feasible. An alternative approach would target neuronal pathways involved in intoxication/recovery, rather than the LC itself. Phosphorylation-related mechanisms have been implicated in the intoxication pathway(s of BoNTs. However, the effects of phosphatase inhibitors upon BoNT activity in the physiological target of BoNTs, i.e. motor neurons, have not been investigated. In this study, a small library of phosphatase inhibitors was screened for BoNT antagonism in the context of mouse embryonic stem cell-derived motor neurons (ES-MNs. Four inhibitors were found to function as BoNT/A antagonists. Subsequently, we confirmed that these inhibitors protect against BoNT/A in a dose-dependent manner in human ES-MNs. Additionally, these compounds provide protection when administered in post-intoxication scenario. Importantly, the inhibitors were also effective against BoNT serotypes B and E. To the best of our knowledge, this is the first study showing phosphatase inhibitors as broad-spectrum BoNT antagonists.

Potent human uric acid transporter 1 inhibitors: in vitro and in vivo metabolism and pharmacokinetic studies

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Wempe MF

2012-11-01

Full Text Available Michael F Wempe,1 Janet W Lightner,2 Bettina Miller,1 Timothy J Iwen,1 Peter J Rice,1 Shin Wakui,3 Naohiko Anzai,4 Promsuk Jutabha,4 Hitoshi Endou51Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA; 2Department of Pharmacology, East Tennessee State University, Johnson City, TN, USA; 3Department of Toxicology, Azabu University School of Veterinary Medicine, Chuo Sagamihara, Kanagawa, Japan; 4Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, Mibu, Shimotsuga, Tochigi, Japan; 5Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Mitaka, Tokyo, JapanAbstract: Human uric acid transporter 1 (hURAT1; SLC22A12 is a very important urate anion exchanger. Elevated urate levels are known to play a pivotal role in cardiovascular diseases, chronic renal disease, diabetes, and hypertension. Therefore, the development of potent uric acid transport inhibitors may lead to novel therapeutic agents to combat these human diseases. The current study investigates small molecular weight compounds and their ability to inhibit 14C-urate uptake in oocytes expressing hURAT1. Using the most promising drug candidates generated from our structure–activity relationship findings, we subsequently conducted in vitro hepatic metabolism and pharmacokinetic (PK studies in male Sprague-Dawley rats. Compounds were incubated with rat liver microsomes containing cofactors nicotinamide adenine dinucleotide phosphate and uridine 5'-diphosphoglucuronic acid. In vitro metabolism and PK samples were analyzed using liquid chromatography/mass spectrometry-mass spectrometry methods. Independently, six different inhibitors were orally (capsule dosing or intravenously (orbital sinus administered to fasting male Sprague-Dawley rats. Blood samples were collected and analyzed; these data were used to compare in vitro and in vivo metabolism and to

Complete amino acid sequence of bovine colostrum low-Mr cysteine proteinase inhibitor.

Science.gov (United States)

Hirado, M; Tsunasawa, S; Sakiyama, F; Niinobe, M; Fujii, S

1985-07-01

The complete amino acid sequence of bovine colostrum cysteine proteinase inhibitor was determined by sequencing native inhibitor and peptides obtained by cyanogen bromide degradation, Achromobacter lysylendopeptidase digestion and partial acid hydrolysis of reduced and S-carboxymethylated protein. Achromobacter peptidase digestion was successfully used to isolate two disulfide-containing peptides. The inhibitor consists of 112 amino acids with an Mr of 12787. Two disulfide bonds were established between Cys 66 and Cys 77 and between Cys 90 and Cys 110. A high degree of homology in the sequence was found between the colostrum inhibitor and human gamma-trace, human salivary acidic protein and chicken egg-white cystatin.

Effect of endocrine therapy on growth of T61 human breast cancer xenografts is directly correlated to a specific down-regulation of insulin-like growth factor II (IGF-II)

DEFF Research Database (Denmark)

Brünner, N; Yee, D; Kern, F G

1993-01-01

xenograft. Growth of the T61 tumour is inhibited by treatment with E2 and TAM. Ribonuclease (RNAse) protection assays with human- and mouse-specific IGF-II antisense probes were used to study the regulation of IGF-II mRNA by E2 and TAM in the tumour. IGF-II protein expression was studied by radioimmunoassay......-IR3 resulted in inhibition of tumour growth during treatment.(ABSTRACT TRUNCATED AT 400 WORDS)...

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

OpenAIRE

Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

1988-01-01

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding t...

Differential role of EGF and BFGF in human GBM-TIC proliferation: relationship to EGFR-tyrosine kinase inhibitor sensibility.

Science.gov (United States)

Bajetto, A; Porcile, C; Pattarozzi, A; Scotti, L; Aceto, A; Daga, A; Barbieri, F; Florio, T

2013-01-01

Glioblastoma multiforme (GBM) is among the most devastating human tumors being rapidly fatal despite aggressive surgery, radiation and chemotherapies. It is characterized by extensive dissemination of tumor cells within the brain that hinders complete surgical resection. GBM tumor initiating-cells (TICs) are a rare subpopulation of cells responsible for tumor development, growth, invasiveness and recurrence after chemotherapy. TICs from human GBM can be selected in vitro using the same conditions permissive for the growth of normal neural cells, of which share some features including marker expression, self-renewal capacity, long-term proliferation, and ability to differentiate into neuronal and glial cells. EGFR overexpression and its constitutive activation is one of the most important signaling alteration identified in GBM, and its pharmacological targeting represents an attractive therapeutic goal. We previously demonstrated that human GBM TICs have different sensitivity to the EGFR kinase inhibitors erlotinib and gefitinib, depending on the differential modulation of downstream signaling cascades. In this work we investigated the mechanisms of resistance to erlotinib in two human GBM TIC cultures, analyzing EGF and bFGF individual contribution to proliferation, clonogenicity, and migration. We demonstrated the presence of a small cell subpopulation whose proliferation is supported by EGF and a larger one mainly dependent on bFGF. Thus, insensitivity to EGFR kinase inhibitors as far as TIC proliferation results from a predominant FGFR activation that hides the inhibitory effects induced on EGFR signaling. Conversely, EGF and bFGF induced cell migration with similar efficacy. In addition, unlike neural stem/progenitors cells, the removal of chondroitin sulphate proteoglycans from cell surface was unable to discern EGF- and bFGF-dependent subpopulations in GBM TICs.

Development of Radiosensitizer using farnesyltransferase inhibitors

Energy Technology Data Exchange (ETDEWEB)

Lim, Jong Seok; Choe, Yong Kyung; Han, Mi Young; Kim, Kwang Dong [Korea Research Institute of Bioscience and Biotechnology, Taejon (Korea)

1999-03-01

We selected some compounds that were reported to have an activity of farneyltransferase inhibitor and tested the hypothesis that they might be used to radiosensitize cells transformed by ras oncogenes. The inhibition of ras processing using some, but not all, inhibitors resulted in higher levels of cell death after {gamma}-irradiation and increased radiosensitivity in H-ras-transformed NIH3T3 cells and MCF-10A human tumor cells. They did not induce additional cell death in control cells that doe not have ras mutation. Furthermore, the treatment of inhibitors alone induced a weak G0/G1 block, whereas inhibitors in combination with {gamma}-irradiation induced an additional enrichment in the G2/M phase of the cell cycle that typically represents irradiation-induced growth arrest. At present, the underling mechanism by which the farnesylltransferase inhibitors exert radiosensitizing effect is not known. In summary, our results suggest and lead to the possibility that some of farnesylation inhibitors may prove clinically useful not only as antitumor agents, but also radiosensitizers of tumors whose growth is dependent on ras function. (author). 15 refs., 10 figs., 4 tabs.

VE-821, an ATR inhibitor, causes radiosensitization in human tumor cells irradiated with high LET radiation

International Nuclear Information System (INIS)

Fujisawa, Hiroshi; Nakajima, Nakako Izumi; Sunada, Shigeaki; Lee, Younghyun; Hirakawa, Hirokazu; Yajima, Hirohiko; Fujimori, Akira; Uesaka, Mitsuru; Okayasu, Ryuichi

2015-01-01

High linear energy transfer (LET) radiation such as carbon ion particles is successfully used for treatment of solid tumors. The reason why high LET radiation accomplishes greater tumor-killing than X-rays is still not completely understood. One factor would be the clustered or complex-type DNA damages. We previously reported that complex DNA double-strand breaks produced by high LET radiation enhanced DNA end resection, and this could lead to higher kinase activity of ATR protein recruited to RPA-coated single-stranded DNA. Although the effect of ATR inhibition on cells exposed to low LET gamma-rays has recently been reported, little is known regarding the effect of ATR inhibitor on cells treated with high LET radiation. The purpose of this study is to investigate the effects of the ATR inhibitor VE-821 in human tumor and normal cells irradiated with high LET carbon ions. HeLa, U2OS, and 1BR-hTERT (normal) cells were pre-treated with 1 μM VE-821 for 1 hour and irradiated with either high LET carbon ions or X-rays. Cell survival, cell cycle distribution, cell growth, and micronuclei formation were evaluated. VE-821 caused abrogation of G2/M checkpoint and forced irradiated cells to divide into daughter cells. We also found that carbon ions caused a higher number of multiple micronuclei than X-rays, leading to decreased cell survival in tumor cells when treated with VE-821, while the survival of irradiated normal cells were not significantly affected by this inhibitor. ATR inhibitor would be an effective tumor radiosensitizer with carbon ion irradiation. The online version of this article (doi:10.1186/s13014-015-0464-y) contains supplementary material, which is available to authorized users

Joint bleeds increase the inhibitor response to human factor VIII in a rat model of severe haemophilia A

DEFF Research Database (Denmark)

Löfgren, Karin Maria; Søndergaard, H.; Skov, Søren

2016-01-01

Introduction The most serious complication in haemophilia A (HA) replacement therapy with coagulation factor VIII (FVIII) is neutralizing antibodies, i.e. inhibitors. It has been hypothesized that danger signals generated during a bleed might have an adjuvant effect on the immune response to FVIII...... in on-demand treatment, increasing the inhibitor risk. Aim To compare the antibody response to treatment with recombinant human FVIII (rhFVIII) in relation to induced knee joint bleeds and treatment without concurrent bleeds in a HA rat model. Method HA rats were divided into two groups: one group (n...... = 10) receiving three needle induced knee joint bleeds 14 days apart and a control group (n = 9) receiving three sham procedures. Three hours after each injury/sham 50 IU kg−1 rhFVIII was administrated intravenously. Subsequently, both groups continued rhFVIII treatment for another 9 weeks. Binding...

Development of an on-line high performance liquid chromatography detection system for human cytochrome P450 1A2 inhibitors in extracts of natural products

NARCIS (Netherlands)

Jeurissen, S.M.F.; Claassen, F.W.; Havlik, J.; Bouwmans, E.E.; Cnubben, N.H.P.; Sudhölter, E.J.R.; Rietjens, I.M.C.M.; Beek, T.A. van

2007-01-01

An on-line HPLC screening method for detection of inhibitors of human cytochrome P450 1A2 in extracts was developed. HPLC separation of extracts is connected to a continuous methoxyresorufin-O-demethylation (MROD) assay in which recombinant human P450 1A2 converts methoxyresorufin to its fluorescent

The enhancement of radiosensitivity by celecoxib, selective cyclooxygenase-2 inhibitor, on human cancer cells expressing differential levels of cyclooxygenase-2

International Nuclear Information System (INIS)

Pyo, Hong Ryull; Shin, You Keun; Kim, Hyun Seok; Seong, Jin Sil; Suh, Chang Ok; Kim, Gwi Eon

2003-01-01

To investigate the modulation of radiosensitivity by celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, on cancer cells over- and under-expressing COX-2. A clonogenic radiation survival analysis was performed on A549 human lung and MCF-7 human breast cancer cell lines incubated in both 1 and 10% fetal bovine serum (FBS) containing media. The apoptosis in both cell lines was measured after treatment with radiation and/or celecoxib. Celecoxib enhanced the radiation sensitivity of the A549 cells in the medium containing the 10% FBS, with radiation enhancement ratios of 1.58 and 1.81 respectively, at surviving fractions of 0.1, with 30 μ M and 50 μ M celecoxib. This enhanced radiosensitivity disappeared in the medium containing the 1% FBS. Celecoxib did not change the radiation sensitivity of the MCF-7 cells in either media. The induction of apoptosis by celecoxib and radiation was not synergistic in either cell line. Celecoxib, a selective COX-2 inhibitor, preferentially enhanced the effect of radiation on COX-2 over-expressing cancer cells compared to the cells with a low expression, and this effect disappeared on incubation of the cells during drug treatment in the medium with suboptimal serum concentration. Apoptosis did not appear to be the underlying mechanism of this radiation enhancement effect due to celecoxib on the A549 cells. These findings suggest radiosensitization by a selective COX-2 inhibitor is COX-2 dependent

Translocation of {sup 3}H-DNA, {sup 131}I-ribonuclease and {sup 3}H-DNA {sup 131}I-ribonuclease complexes in germinated barley grains; Translocation des ADN{sup 3}H, RNase{sup 131}I et complexes ADN{sup 3}H - RNase {sup 131}I dans les orges en germination

Energy Technology Data Exchange (ETDEWEB)

Tshitenge, G. [Centre nucléaire TRICO, Kinshasa (Congo, The Democratic Republic of the); Ledoux, L. [Centre d’étude de l' énergie nucléaire Mol (Belgium)

1970-01-15

Barley grains, after germinating for 11 hours in the presence of water, were cut into sections at the end opposite the embryo. They were incubated in solutions of {sup 3}H-DNA, {sup 13I}I-ribonuclease, and {sup 3}H-DNA {sup 131}I-ribonuclease complex for three hours. They were then placed in a water-saturated atmosphere for 24 hours. At this stage the different organs of the seedlings were separated and homogenized in a solution containing 0.15M sodium chloride and 0.1 M sodium ethylenediaminetetraacetate at pH 7. By measuring the radioactivity found in the homogenates one can estimate the penetration of the macromolecules under study. The results show that the quantity found varies from one case to the other and depends both on the nature of the macromolecule and of the organ studied. (author) [French] Des orges qui ont germé pendant 11 h en présence d’eau sont sectionnées au bout opposé à l’embryon. Elles sont incubées avec des solutions d’ADN{sup 3}H, de RNase{sup 131}1 et de complexe ADN{sup 3}H - RNase {sup 131}I, pendant 3 h. Elles sont ensuite placées dans une atmosphère saturée d'eau pendant 24 h. A ce moment, les différents organes des plantules sont séparés et homogénéisés en présence d'une solution 0,15M en NaCl et 0,1M en éthylènediamine-tétracétate de Na à pH 7. La mesure de la radioactivité retrouvée dans les homogénats permet d'évaluer la pénétration des macromolécules considérées. Les résultats montrent que la quantité retrouvée varie d'un cas â l'autre et dépend à la fois de la nature de la macromolécule et de l'organe considéré. (author)

Inhibition of PAF-induced expression of CD11b and shedding of L-selectin on human neutrophils and eosinophils by the type IV selective PDE inhibitor, rolipram

NARCIS (Netherlands)

Dijkhuizen, B; deMonchy, JGR; Dubois, AEJ; Gerritsen, J; Kauffman, HF

We quantitatively determined whether the selective phosphodiesterase (PDE) inhibitor, rolipram, inhibits changes in the adhesion molecules CD11b and L-selectin on platelet-activating factor (PAF)-stimulated human neutrophils and eosinophils in vitro. Incubations were performed in human whole blood

Disease-modifying anti-Alzheimer's drugs: inhibitors of human cholinesterases interfering with β-amyloid aggregation.

Science.gov (United States)

Brogi, Simone; Butini, Stefania; Maramai, Samuele; Colombo, Raffaella; Verga, Laura; Lanni, Cristina; De Lorenzi, Ersilia; Lamponi, Stefania; Andreassi, Marco; Bartolini, Manuela; Andrisano, Vincenza; Novellino, Ettore; Campiani, Giuseppe; Brindisi, Margherita; Gemma, Sandra

2014-07-01

We recently described multifunctional tools (2a-c) as potent inhibitors of human Cholinesterases (ChEs) also able to modulate events correlated with Aβ aggregation. We herein propose a thorough biological and computational analysis aiming at understanding their mechanism of action at the molecular level. We determined the inhibitory potency of 2a-c on Aβ1-42 self-aggregation, the interference of 2a with the toxic Aβ oligomeric species and with the postaggregation states by capillary electrophoresis analysis and transmission electron microscopy. The modulation of Aβ toxicity was assessed for 2a and 2b on human neuroblastoma cells. The key interactions of 2a with Aβ and with the Aβ-preformed fibrils were computationally analyzed. 2a-c toxicity profile was also assessed (human hepatocytes and mouse fibroblasts). Our prototypical pluripotent analogue 2a interferes with Aβ oligomerization process thus reducing Aβ oligomers-mediated toxicity in human neuroblastoma cells. 2a also disrupts preformed fibrils. Computational studies highlighted the bases governing the diversified activities of 2a. Converging analytical, biological, and in silico data explained the mechanism of action of 2a on Aβ1-42 oligomers formation and against Aβ-preformed fibrils. This evidence, combined with toxicity data, will orient the future design of safer analogues. © 2014 John Wiley & Sons Ltd.

Cyclohex-1-ene carboxylic acids: synthesis and biological evaluation of novel inhibitors of human 5 alpha reductase.

Science.gov (United States)

Baston, Eckhard; Salem, Ola I A; Hartmann, Rolf W

2003-03-01

In search of novel nonsteroidal mimics of steroidal inhibitors of 5 alpha reductase, 4-(2-phenylethyl)cyclohex-1-ene carboxylic acids 1-5 were synthesized with different substituents in para position of the phenyl ring (1: N, N-diisopropylcarbamoyl, 2: phenyl, 3: phenoxy, 4: benzoyl, and 5: benzyl). The principal synthetic approach for the desired compounds consisted of a Wittig olefination between 1, 4-dioxaspiro [4.5]-decane-8-carbaldehyde (4g and the appropriate phosphonium salts. The compounds were tested for inhibition of human 5 alpha reductase isozymes 1 and 2 using DU 145 cells and preparations from prostatic tissue, respectively. They turned out to be good inhibitors of the prostatic isozyme 2 with compound 1 being the most potent one (IC(50) = 760 nM). Isozyme 1 was only slightly inhibited. It is concluded that the novel structures are appropriate for being further optimized, aiming at the development of a novel drug for the treatment of benign prostatic hyperplasia.

Fragment-derived inhibitors of human N-myristoyltransferase block capsid assembly and replication of the common cold virus

Science.gov (United States)

Mousnier, Aurélie; Bell, Andrew S.; Swieboda, Dawid P.; Morales-Sanfrutos, Julia; Pérez-Dorado, Inmaculada; Brannigan, James A.; Newman, Joseph; Ritzefeld, Markus; Hutton, Jennie A.; Guedán, Anabel; Asfor, Amin S.; Robinson, Sean W.; Hopkins-Navratilova, Iva; Wilkinson, Anthony J.; Johnston, Sebastian L.; Leatherbarrow, Robin J.; Tuthill, Tobias J.; Solari, Roberto; Tate, Edward W.

2018-06-01

Rhinoviruses (RVs) are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report the discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host-cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. The identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking and conformational control over linker geometry. We show that inhibition of the co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, to deliver a low nanomolar antiviral activity against multiple RV strains, poliovirus and foot and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections.

HTCC: Broad Range Inhibitor of Coronavirus Entry.

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Aleksandra Milewska

Full Text Available To date, six human coronaviruses have been known, all of which are associated with respiratory infections in humans. With the exception of the highly pathogenic SARS and MERS coronaviruses, human coronaviruses (HCoV-NL63, HCoV-OC43, HCoV-229E, and HCoV-HKU1 circulate worldwide and typically cause the common cold. In most cases, infection with these viruses does not lead to severe disease, although acute infections in infants, the elderly, and immunocompromised patients may progress to severe disease requiring hospitalization. Importantly, no drugs against human coronaviruses exist, and only supportive therapy is available. Previously, we proposed the cationically modified chitosan, N-(2-hydroxypropyl-3-trimethylammonium chitosan chloride (HTCC, and its hydrophobically-modified derivative (HM-HTCC as potent inhibitors of the coronavirus HCoV-NL63. Here, we show that HTCC inhibits interaction of a virus with its receptor and thus blocks the entry. Further, we demonstrate that HTCC polymers with different degrees of substitution act as effective inhibitors of all low-pathogenic human coronaviruses.

Acetyl-CoA Carboxylase-α Inhibitor TOFA Induces Human Cancer Cell Apoptosis

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Wang, Chun; Xu, Canxin; Sun, Mingwei; Luo, Dixian; Liao, Duan-fang; Cao, Deliang

2009-01-01

Acetyl-CoA carboxylase-α (ACCA) is a rate-limiting enzyme in long chain fatty acid synthesis, playing a critical role in cellular energy storage and lipid synthesis. ACCA is upregulated in multiple types of human cancers and small interfering RNA-mediated ACCA silencing in human breast and prostate cancer cells results in oxidative stress and apoptosis. This study reports for the first time that TOFA (5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACCA, is cytotoxic to lung cancer cells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15, with an IC50 at approximately 5.0, 5.0, and 4.5 μg/ml, respectively. TOFA at 1.0–20.0 μg/ml effectively blocked fatty acid synthesis and induced cell death in a dose-dependent manner. The cell death was characterized with PARP cleavage, DNA fragmentation, and annexin-V staining, all of which are the features of the apoptosis. Supplementing simultaneously the cells with palmitic acids (100 μM), the end-products of the fatty acid synthesis pathway, prevented the apoptosis induced by TOFA. Taken together, these data suggest that TOFA is a potent cytotoxic agent to lung and colon cancer cells, inducing apoptosis through disturbing their fatty acid synthesis. PMID:19450551

Acetyl-CoA carboxylase-alpha inhibitor TOFA induces human cancer cell apoptosis.

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Wang, Chun; Xu, Canxin; Sun, Mingwei; Luo, Dixian; Liao, Duan-Fang; Cao, Deliang

2009-07-31

Acetyl-CoA carboxylase-alpha (ACCA) is a rate-limiting enzyme in long chain fatty acid synthesis, playing a critical role in cellular energy storage and lipid synthesis. ACCA is upregulated in multiple types of human cancers and small interfering RNA-mediated ACCA silencing in human breast and prostate cancer cells results in oxidative stress and apoptosis. This study reports for the first time that TOFA (5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACCA, is cytotoxic to lung cancer cells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15, with an IC(50) at approximately 5.0, 5.0, and 4.5 microg/ml, respectively. TOFA at 1.0-20.0 microg/ml effectively blocked fatty acid synthesis and induced cell death in a dose-dependent manner. The cell death was characterized with PARP cleavage, DNA fragmentation, and annexin-V staining, all of which are the features of the apoptosis. Supplementing simultaneously the cells with palmitic acids (100 microM), the end-products of the fatty acid synthesis pathway, prevented the apoptosis induced by TOFA. Taken together, these data suggest that TOFA is a potent cytotoxic agent to lung and colon cancer cells, inducing apoptosis through disturbing their fatty acid synthesis.

Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

International Nuclear Information System (INIS)

Gilmour, Peter S.; O'Shea, Patrick J.; Fagura, Malbinder; Pilling, James E.; Sanganee, Hitesh; Wada, Hiroki; Courtney, Paul F.; Kavanagh, Stefan; Hall, Peter A.; Escott, K. Jane

2013-01-01

Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging. GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH 1–34 or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption. - Highlights: • Wnt modulation with 3 novel GSK-3 inhibitors alters bone growth. • Human stem cell osteoblastogenesis and

The "SWOT" of BRAF inhibition in melanoma: RAF inhibitors, MEK inhibitors or both?

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Nissan, Moriah H; Solit, David B

2011-12-01

Activating mutations in the BRAF gene are among the most prevalent kinase mutations in human cancer. BRAF mutations are most frequent in patients with melanoma where they occur in approximately 50% of patients with advanced disease. Remarkable clinical activity has recently been reported with highly selective RAF inhibitors in melanoma patients whose tumors harbor V600E BRAF mutations. The response rates of RAF inhibitors in patients with BRAF-mutant melanomas far exceed the activity level of any prior therapy studied in this disease. The results suggest that we have entered an era of personalized therapy for patients with metastatic melanoma in which treatment selection will be guided by BRAF mutational status. This review will discuss the strengths, weaknesses, opportunities and threats ("SWOT") of developing RAF and MEK selective inhibitors as anti-cancer therapies, recent insights into the mechanisms of intrinsic and acquired resistance to these agents, and current efforts to develop mechanism-based combination therapies.

Phosphodiesterase inhibitors in clinical urology.

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Ückert, Stefan; Kuczyk, Markus A; Oelke, Matthias

2013-05-01

To date, benign diseases of the male and female lower urinary and genital tract, such as erectile dysfunction, bladder overactivity, lower urinary tract symptomatology secondary to benign prostatic hyperplasia and symptoms of female sexual dysfunction (including arousal and orgasmic disorders), can be therapeutically approached by influencing the function of the smooth musculature of the respective tissues. The use of isoenzyme-selective phosphodiesterase (PDE) inhibitors is considered a great opportunity to treat various diseases of the human urogenital tract. PDE inhibitors, in particular the PDE5 (cyclic GMP PDE) inhibitors avanafil, lodenafil, sildenafil, tadalafil, udenafil and vardenafil, are regarded as efficacious, having a fast onset of drug action and an improved effect-to-adverse event ratio, combining a high response rate with the advantage of an on-demand intake. The purpose of this review is to summarize recent as well as potential future indications, namely, erectile dysfunction, Peyronie's disease, overactive bladder, urinary stone disease, lower urinary tract symptomatology secondary to benign prostatic hyperplasia and premature ejaculation, for the use of PDE inhibitors in clinical urology.

Updates in the Development of ImmunoRNases for the Selective Killing of Tumor Cells

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Sandra Jordaan

2018-03-01

Full Text Available Targeted cancer therapy includes, amongst others, antibody-based delivery of toxic payloads to selectively eliminate tumor cells. This payload can be either a synthetic small molecule drug composing an antibody-drug conjugate (ADC or a cytotoxic protein composing an immunotoxin (IT. Non-human cytotoxic proteins, while potent, have limited clinical efficacy due to their immunogenicity and potential off-target toxicity. Humanization of the cytotoxic payload is essential and requires harnessing of potent apoptosis-inducing human proteins with conditional activity, which rely on targeted delivery to contact their substrate. Ribonucleases are attractive candidates, due to their ability to induce apoptosis by abrogating protein biosynthesis via tRNA degradation. In fact, several RNases of the pancreatic RNase A superfamily have shown potential as anti-cancer agents. Coupling of a human RNase to a humanized antibody or antibody derivative putatively eliminates the immunogenicity of an IT (now known as a human cytolytic fusion protein, hCFP. However, RNases are tightly regulated in vivo by endogenous inhibitors, controlling the ribonucleolytic balance subject to the cell’s metabolic requirements. Endogenous inhibition limits the efficacy with which RNase-based hCFPs induce apoptosis. However, abrogating the natural interaction with the natural inhibitors by mutation has been shown to significantly enhance RNase activity, paving the way toward achieving cytolytic potency comparable to that of bacterial immunotoxins. Here, we review the immunoRNases that have undergone preclinical studies as anti-cancer therapeutic agents.

Updates in the Development of ImmunoRNases for the Selective Killing of Tumor Cells.

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Jordaan, Sandra; Akinrinmade, Olusiji A; Nachreiner, Thomas; Cremer, Christian; Naran, Krupa; Chetty, Shivan; Barth, Stefan

2018-03-05

Targeted cancer therapy includes, amongst others, antibody-based delivery of toxic payloads to selectively eliminate tumor cells. This payload can be either a synthetic small molecule drug composing an antibody-drug conjugate (ADC) or a cytotoxic protein composing an immunotoxin (IT). Non-human cytotoxic proteins, while potent, have limited clinical efficacy due to their immunogenicity and potential off-target toxicity. Humanization of the cytotoxic payload is essential and requires harnessing of potent apoptosis-inducing human proteins with conditional activity, which rely on targeted delivery to contact their substrate. Ribonucleases are attractive candidates, due to their ability to induce apoptosis by abrogating protein biosynthesis via tRNA degradation. In fact, several RNases of the pancreatic RNase A superfamily have shown potential as anti-cancer agents. Coupling of a human RNase to a humanized antibody or antibody derivative putatively eliminates the immunogenicity of an IT (now known as a human cytolytic fusion protein, hCFP). However, RNases are tightly regulated in vivo by endogenous inhibitors, controlling the ribonucleolytic balance subject to the cell's metabolic requirements. Endogenous inhibition limits the efficacy with which RNase-based hCFPs induce apoptosis. However, abrogating the natural interaction with the natural inhibitors by mutation has been shown to significantly enhance RNase activity, paving the way toward achieving cytolytic potency comparable to that of bacterial immunotoxins. Here, we review the immunoRNases that have undergone preclinical studies as anti-cancer therapeutic agents.

Small molecule inhibitors uncover synthetic genetic interactions of human flap endonuclease 1 (FEN1 with DNA damage response genes.

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Thomas A Ward

Full Text Available Flap endonuclease 1 (FEN1 is a structure selective endonuclease required for proficient DNA replication and the repair of DNA damage. Cellularly active inhibitors of this enzyme have previously been shown to induce a DNA damage response and, ultimately, cell death. High-throughput screens of human cancer cell-lines identify colorectal and gastric cell-lines with microsatellite instability (MSI as enriched for cellular sensitivity to N-hydroxyurea series inhibitors of FEN1, but not the PARP inhibitor olaparib or other inhibitors of the DNA damage response. This sensitivity is due to a synthetic lethal interaction between FEN1 and MRE11A, which is often mutated in MSI cancers through instabilities at a poly(T microsatellite repeat. Disruption of ATM is similarly synthetic lethal with FEN1 inhibition, suggesting that disruption of FEN1 function leads to the accumulation of DNA double-strand breaks. These are likely a result of the accumulation of aberrant replication forks, that accumulate as a consequence of a failure in Okazaki fragment maturation, as inhibition of FEN1 is toxic in cells disrupted for the Fanconi anemia pathway and post-replication repair. Furthermore, RAD51 foci accumulate as a consequence of FEN1 inhibition and the toxicity of FEN1 inhibitors increases in cells disrupted for the homologous recombination pathway, suggesting a role for homologous recombination in the resolution of damage induced by FEN1 inhibition. Finally, FEN1 appears to be required for the repair of damage induced by olaparib and cisplatin within the Fanconi anemia pathway, and may play a role in the repair of damage associated with its own disruption.

Combined 5-FU and ChoKα inhibitors as a new alternative therapy of colorectal cancer: evidence in human tumor-derived cell lines and mouse xenografts.

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Ana de la Cueva

Full Text Available Colorectal cancer (CRC is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα, an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy.ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS and thymidine kinase (TK1 levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action.Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.

Human pregnane X receptor is activated by dibenzazepine carbamate-based inhibitors of constitutive androstane receptor.

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Jeske, Judith; Windshügel, Björn; Thasler, Wolfgang E; Schwab, Matthias; Burk, Oliver

2017-06-01

Unintentional activation of xenosensing nuclear receptors pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR) by clinical drug use is known to produce severe side effects in patients, which may be overcome by co-administering antagonists. However, especially antagonizing CAR is hampered by the lack of specific inhibitors, which do not activate PXR. Recently, compounds based on a dibenzazepine carbamate scaffold were identified as potent CAR inhibitors. However, their potential to activate PXR was not thoroughly investigated, even if the lead compound was named "CAR inhibitor not PXR activator 1" (CINPA1). Thus, we performed a comprehensive analysis of the interaction of CINPA1 and four analogs with PXR. Cellular assays were used to investigate intra- and intermolecular interactions and transactivation activity of PXR as a function of the compounds. Modulation of PXR target gene expression was analyzed in primary human hepatocytes. Ligand binding to PXR was investigated by molecular docking and limited proteolytic digestion. We show here that CINPA1 induced the assembly of the PXR ligand-binding domain, released co-repressors from and recruited co-activators to the receptor. CINPA1 and its analogs induced the PXR-dependent activation of a CYP3A4 reporter gene and CINPA1 induced the expression of endogenous cytochrome P450 genes in primary hepatocytes, while not consistently inhibiting CAR-mediated induction. Molecular docking revealed favorable binding of CINPA1 and analogs to the PXR ligand-binding pocket, which was confirmed in vitro. Altogether, our data provide consistent evidence that compounds with a dibenzazepine carbamate scaffold, such as CINPA1 and its four analogs, bind to and activate PXR.

Red wine contains a potent inhibitor of phenolsulphotransferase.

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Littlewood, J T; Glover, V; Sandler, M

1985-01-01

Many ethanolic drinks, especially red wine, contain potent inhibitors of phenolsulphotransferase. At a dilution of 1/75 from the original beverage, extracts from six types of red wine inhibited human platelet phenolsulphotransferase P by a mean of 99% and human platelet phenolsulphotransferase M by 12%. Such extracts had no significant effect on rat liver monoamine oxidase A or human platelet monoamine oxidase B. The inhibitors, which have not yet been identified, can be extracted into ethyl acetate at acid or neutral pH. Thus, they are not monoamines. Flavonoid phenols are plausible candidates. As phenolsulphotransferase M and P are involved in the metabolism of many phenols, including drugs, the inhibition of these enzymes could result in the enhancement of pharmacological potency and have important clinical consequences. PMID:3857069

Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells

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Li, Luyuan; Paz, Ana C.; Wilky, Breelyn A.; Johnson, Britt; Galoian, Karina; Rosenberg, Andrew; Hu, Guozhi; Tinoco, Gabriel; Bodamer, Olaf; Trent, Jonathan C.

2015-01-01

Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers including chondrosarcomas. The IDH1 inhibitor AGI-5198 abrogates the ability of mutant IDH1 to produce the oncometabolite D-2 hydroxyglutarate (D-2HG) in gliomas. We sought to determine if treatment with AGI-5198 would similarly inhibit tumorigenic activity and D-2HG production in IDH1-mutant human chondrosarcoma cells. Two human chondrosarcoma cell lines, JJ012 and HT1080 with endogenous IDH1 mutations and a human chondrocyte cell line C28 with wild type IDH1 were employed in our study. Mutation analysis of IDH was performed by PCR-based DNA sequencing, and D-2HG was detected using tandem mass spectrometry. We confirmed that JJ012 and HT1080 harbor IDH1 R132G and R132C mutation, respectively, while C28 has no mutation. D-2HG was detectable in cell pellets and media of JJ012 and HT1080 cells, as well as plasma and urine from an IDH-mutant chondrosarcoma patient, which decreased after tumor resection. AGI-5198 treatment decreased D-2HG levels in JJ012 and HT1080 cells in a dose-dependent manner, and dramatically inhibited colony formation and migration, interrupted cell cycling, and induced apoptosis. In conclusion, our study demonstrates anti-tumor activity of a mutant IDH1 inhibitor in human chondrosarcoma cell lines, and suggests that D-2HG is a potential biomarker for IDH mutations in chondrosarcoma cells. Thus, clinical trials of mutant IDH inhibitors are warranted for patients with IDH-mutant chondrosarcomas. PMID:26368816

Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells.

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Luyuan Li

Full Text Available Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2 were recently described in several cancers including chondrosarcomas. The IDH1 inhibitor AGI-5198 abrogates the ability of mutant IDH1 to produce the oncometabolite D-2 hydroxyglutarate (D-2HG in gliomas. We sought to determine if treatment with AGI-5198 would similarly inhibit tumorigenic activity and D-2HG production in IDH1-mutant human chondrosarcoma cells. Two human chondrosarcoma cell lines, JJ012 and HT1080 with endogenous IDH1 mutations and a human chondrocyte cell line C28 with wild type IDH1 were employed in our study. Mutation analysis of IDH was performed by PCR-based DNA sequencing, and D-2HG was detected using tandem mass spectrometry. We confirmed that JJ012 and HT1080 harbor IDH1 R132G and R132C mutation, respectively, while C28 has no mutation. D-2HG was detectable in cell pellets and media of JJ012 and HT1080 cells, as well as plasma and urine from an IDH-mutant chondrosarcoma patient, which decreased after tumor resection. AGI-5198 treatment decreased D-2HG levels in JJ012 and HT1080 cells in a dose-dependent manner, and dramatically inhibited colony formation and migration, interrupted cell cycling, and induced apoptosis. In conclusion, our study demonstrates anti-tumor activity of a mutant IDH1 inhibitor in human chondrosarcoma cell lines, and suggests that D-2HG is a potential biomarker for IDH mutations in chondrosarcoma cells. Thus, clinical trials of mutant IDH inhibitors are warranted for patients with IDH-mutant chondrosarcomas.

Ribonuclease activity of buckwheat plant (Fagopyrum esculentum cultivars with different sensitivities to buckwheat burn virus

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Y. R. Sindarovska

2014-06-01

Full Text Available Ribonucleases (RNases are present in base-level amounts in intact plants, but this level is able to increase greatly under stress conditions. The possible cause for such an increase is protection against plant RNA-virus attack. Buckwheat burn virus (BBV is a highly virulent pathogen that belongs to Rhabdoviridae family. In our study, we have analyzed the correlation between RNase activity and resistance of different buckwheat cultivars to BBV infection. Two cultivars, Kara-Dag and Roksolana, with different sensitivities to BBV have been used. Kara-Dag is a cultivar with medium sensitivity to virus and Roksolana is a tolerant cultivar. It has been shown that the base level of RNase activity in Roksolana cultivar was in most cases higher than the corresponding parameter in Kara-Dag cultivar. Both infected and uninfected plants of Roksolana cultivar demonstrated high RNase activity during two weeks. Whereas infected plants of Kara-Dag cultivar demonstrated unstable levels of RNase activity. Significant decline in RNase activity was detected on the 7th day post infection with subsequent gradual increase in RNase activity. Decline of the RNase activity during the first week could promote the virus replication and therefore more successful infection of upper leaves of plants. Unstable levels of RNase activity in infected buckwheat plants may be explained by insufficiency of virus-resistant mechanisms that determines the medium sensitivity of the cultivar to BBV. Thus, plants of buckwheat cultivar having less sensitivity to virus, displayed in general higher RNase activity.

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor

International Nuclear Information System (INIS)

Antalis, T.M.; Clark, M.A.; Barnes, T.; Lehrbach, P.R.; Devine, P.L.; Schevzov, G.; Goss, N.H.; Stephens, R.W.; Tolstoshev, P.

1988-01-01

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A) + RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the λ P/sub L/ promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated M/sub r/ of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators

Effects of treatment with Maraviroc a CCR5 inhibitor on a human hepatic stellate cell line.

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Coppola, Nicola; Perna, Angelica; Lucariello, Angela; Martini, Salvatore; Macera, Margherita; Carleo, Maria A; Guerra, Germano; Esposito, Vincenzo; De Luca, Antonio

2018-08-01

After an acute liver damage, tissue regeneration repairs lesions with degradation of deposed fibrotic material, while mechanisms of tissue restoration are persistently activated following several repeated injuries, inducing deposition of extracellular matrix. (ECM). Factors responsible for ECM remodeling have been identified in a pathway involving a family of zinc-dependent enzyme matrix metalloproteinases (MMPs), together with tissue inhibitor of metalloproteinases (TIMPs). Recent experimental models suggested a role of CCR5 receptor in the genesis of liver fibrosis. Drawing from these background we decided to evaluate the effects of the treatment with the CCR5 inhibitor Maraviroc on LX-2, a human hepatic stellate cell line (HSC). Treatment with Maraviroc resulted in a block in S phase of LX-2 cells with increased expression levels of cyclin D1 and p21 while the expression of p53 was reduced. Treatment with Maraviroc was also able to block the accumulation of fibrillar collagens and extracellular matrix proteins (ECM), as demonstrated by the decrease of specific markers as Collagen type I, α-SMA, and TGF-β1. In addition we observed a down regulation of both metalloproteins (MMP-2, MMP-9), used for the degradation of the extracellular matrix and their inhibitors (TIMP-1, TIMP-2). The identification of a compound that may modulate the dynamic of liver fibrosis could be crucial in all chronic liver diseases. Maraviroc could play an important role because, in addition to its own anti-HIV activity, it could reduce the release of pro-inflammatory citokynes implicated in liver fibrogenesis. © 2018 Wiley Periodicals, Inc.

Molecular modeling of human acidic mammalian chitinase in complex with the natural-product cyclopentapeptide chitinase inhibitor argifin.

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Gouda, Hiroaki; Terashima, Shinichi; Iguchi, Kanami; Sugawara, Akihiro; Saito, Yoshifumi; Yamamoto, Tsuyoshi; Hirose, Tomoyasu; Shiomi, Kazuro; Sunazuka, Toshiaki; Omura, Satoshi; Hirono, Shuichi

2009-09-01

Human acidic mammalian chitinase (hAMCase) is an attractive target for developing anti-asthma medications. We used a variety of computational methods to investigate the interaction between hAMCase and the natural-product cyclopentapeptide chitinase inhibitor argifin. The three-dimensional structure of hAMCase was first constructed using homology modeling. The interaction mode and binding free energy between argifin and hAMCase were then examined by the molecular-docking calculation and the molecular mechanics Poisson-Boltzmann surface area method combined with molecular dynamics simulation, respectively. The results suggested that argifin binds to hAMCase in a similar fashion to the interaction mode observed in the crystal structure of argifin-human chitotriosidase complex, and possesses inhibitory activity against hAMCase in the micromolar range. We further designed argifin derivatives expected to be selective for hAMCase.

Molecular docking and 3D-QSAR studies on inhibitors of DNA damage signaling enzyme human PARP-1.

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Fatima, Sabiha; Bathini, Raju; Sivan, Sree Kanth; Manga, Vijjulatha

2012-08-01

Poly (ADP-ribose) polymerase-1 (PARP-1) operates in a DNA damage signaling network. Molecular docking and three dimensional-quantitative structure activity relationship (3D-QSAR) studies were performed on human PARP-1 inhibitors. Docked conformation obtained for each molecule was used as such for 3D-QSAR analysis. Molecules were divided into a training set and a test set randomly in four different ways, partial least square analysis was performed to obtain QSAR models using the comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). Derived models showed good statistical reliability that is evident from their r², q²(loo) and r²(pred) values. To obtain a consensus for predictive ability from all the models, average regression coefficient r²(avg) was calculated. CoMFA and CoMSIA models showed a value of 0.930 and 0.936, respectively. Information obtained from the best 3D-QSAR model was applied for optimization of lead molecule and design of novel potential inhibitors.

Trisubstituted purine inhibitors of PDGFRα and their antileukemic activity in the human eosinophilic cell line EOL-1.

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Malínková, Veronika; Řezníčková, Eva; Jorda, Radek; Gucký, Tomáš; Kryštof, Vladimír

2017-12-15

Inhibition of protein kinases is a validated concept for pharmacological intervention in cancers. Many kinase inhibitors have been approved for clinical use, but their practical application is often limited. Here, we describe a collection of 23 novel 2,6,9-trisubstituted purine derivatives with nanomolar inhibitory activities against PDGFRα, a receptor tyrosine kinase often found constitutively activated in various tumours. The compounds demonstrated strong and selective cytotoxicity in the human eosinophilic leukemia cell line EOL-1, whereas several other cell lines were substantially less sensitive. The cytotoxicity in EOL-1, which is known to express the FIP1L1-PDGFRA fusion gene encoding an oncogenic kinase, correlated significantly with PDGFRα inhibition. EOL-1 cells treated with the compounds also exhibited dose-dependent inhibition of PDGFRα autophosphorylation and suppression of its downstream signaling pathways with concomitant G 1 phase arrest, confirming the proposed mechanism of action. Our results show that substituted purines can be used as platforms for preparing tyrosine kinase inhibitors with specific activity towards eosinophilic leukemia. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Human Tyrosyl-DNA Phosphodiesterase 1 (hTdp1) Inhibitor NSC120686 as an Exploratory Tool to Investigate Plant Tdp1 Genes.

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Macovei, Anca; Pagano, Andrea; Sabatini, Maria Elisa; Grandi, Sofia; Balestrazzi, Alma

2018-03-28

The hTdp1 (human tyrosyl-DNA phosphodiesterase 1) inhibitor NSC120686 has been used, along with topoisomerase inhibitors, as a pharmacophoric model to restrain the Tdp1 activity as part of a synergistic treatment for cancer. While this compound has an end-point application in medical research, in plants, its application has not been considered so far. The originality of our study consists in the use of hTdp1 inhibitor in Medicago truncatula cells, which, unlike human cells, contain two Tdp1 genes. Hence, the purpose of this study was to test the hTdp1 inhibitor NSC120686 as an exploratory tool to investigate the plant Tdp1 genes, since their characterization is still in incipient phases. To do so, M. truncatula calli were exposed to increasing (75, 150, 300 μM) concentrations of NSC120686. The levels of cell mortality and DNA damage, measured via diffusion assay and comet assay, respectively, were significantly increased when the highest doses were used, indicative of a cytotoxic and genotoxic threshold. In addition, the NSC120686-treated calli and untreated MtTdp1α -depleted calli shared a similar response in terms of programmed cell death (PCD)/necrosis and DNA damage. Interestingly, the expression profiles of MtTdp1α and MtTdp1β genes were differently affected by the NSC120686 treatment, as MtTdp1α was upregulated while MtTdp1β was downregulated. The NSC120686 treatment affected not only the MtTdp1 genes but also other genes with roles in alternative DNA repair pathways. Since the expression patterns of these genes were different than what was observed in the MtTdp1α -depleted plants, it could be hypothesized that the NSC120686 treatment exerts a different influence compared to that resulting from the lack of the MtTdp1α gene function.

Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

Energy Technology Data Exchange (ETDEWEB)

Gilmour, Peter S., E-mail: Peter.Gilmour@astrazeneca.com [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); O' Shea, Patrick J.; Fagura, Malbinder [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Pilling, James E. [Discovery Sciences, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Sanganee, Hitesh [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Wada, Hiroki [R and I IMed, AstraZeneca R and D, Molndal (Sweden); Courtney, Paul F. [DMPK, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Kavanagh, Stefan; Hall, Peter A. [Safety Assessment, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Escott, K. Jane [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom)

2013-10-15

Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging. GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH{sub 1–34} or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption. - Highlights: • Wnt modulation with 3 novel GSK-3 inhibitors alters bone growth. • Human stem cell osteoblastogenesis

mTOR inhibitors alone and in combination with JAK2 inhibitors effectively inhibit cells of myeloproliferative neoplasms.

Directory of Open Access Journals (Sweden)

Costanza Bogani

Full Text Available BACKGROUND: Dysregulated signaling of the JAK/STAT pathway is a common feature of chronic myeloproliferative neoplasms (MPN, usually associated with JAK2V617F mutation. Recent clinical trials with JAK2 inhibitors showed significant improvements in splenomegaly and constitutional symptoms in patients with myelofibrosis but meaningful molecular responses were not documented. Accordingly, there remains a need for exploring new treatment strategies of MPN. A potential additional target for treatment is represented by the PI3K/AKT/mammalian target of rapamycin (mTOR pathway that has been found constitutively activated in MPN cells; proof-of-evidence of efficacy of the mTOR inhibitor RAD001 has been obtained recently in a Phase I/II trial in patients with myelofibrosis. The aim of the study was to characterize the effects in vitro of mTOR inhibitors, used alone and in combination with JAK2 inhibitors, against MPN cells. FINDINGS: Mouse and human JAK2V617F mutated cell lines and primary hematopoietic progenitors from MPN patients were challenged with an allosteric (RAD001 and an ATP-competitive (PP242 mTOR inhibitor and two JAK2 inhibitors (AZD1480 and ruxolitinib. mTOR inhibitors effectively reduced proliferation and colony formation of cell lines through a slowed cell division mediated by changes in cell cycle transition to the S-phase. mTOR inhibitors also impaired the proliferation and prevented colony formation from MPN hematopoietic progenitors at doses significantly lower than healthy controls. JAK2 inhibitors produced similar antiproliferative effects in MPN cell lines and primary cells but were more potent inducers of apoptosis, as also supported by differential effects on cyclinD1, PIM1 and BcLxL expression levels. Co-treatment of mTOR inhibitor with JAK2 inhibitor resulted in synergistic activity against the proliferation of JAK2V617F mutated cell lines and significantly reduced erythropoietin-independent colony growth in patients with

Inhibition of potentially lethal radiation damage repair in normal and neoplastic human cells by 3-aminobenzamide: an inhibitor of poly(ADP-ribosylation)

International Nuclear Information System (INIS)

Thraves, P.J.; Mossman, K.L.; Frazier, D.T.; Dritschilo, A.

1986-01-01

The effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase, on potentially lethal damage repair (PLDR) was investigated in normal human fibroblasts and four human tumor cell lines from tumors with varying degrees of radiocurability. The tumor lines selected were: Ewing's sarcoma, a bone tumor considered radiocurable and, human lung adenocarcinoma, osteosarcoma, and melanoma, three tumors considered nonradiocurable. PLDR was measured by comparing cell survival when cells were irradiated in a density-inhibited state and replated at appropriate cell numbers at specified times following irradiation to cell survival when cells were replated immediately following irradiation. 3AB was added to cultures 2 hr prior to irradiation and removed at the time of replating. Different test radiation doses were used for the various cell lines to obtain equivalent levels of cell survival. In the absence of inhibitor, PLDR was similar in all cell lines tested. In the presence of 8 mM 3AB, differential inhibition of PLDR was observed. PLDR was almost completely inhibited in Ewing's sarcoma cells and partially inhibited in normal fibroblast cells and osteosarcoma cells. No inhibition of PLDR was observed in the lung adenocarcinoma or melanoma cells. Except for the osteosarcoma cells, inhibition of PLDR by 3AB correlated well with radiocurability

Growth inhibitory effects of the dual ErbB1/ErbB2 tyrosine kinase inhibitor PKI-166 on human prostate cancer xenografts.

Science.gov (United States)

Mellinghoff, Ingo K; Tran, Chris; Sawyers, Charles L

2002-09-15

Experiments with human prostate cancer cell lines have shown that forced overexpression of the ErbB2-receptor tyrosine kinase (RTK) promotes androgen-independent growth and increases androgen receptor-transcriptional activity in a ligand-independent fashion. To investigate the relationship between ErbB-RTK signaling and androgen in genetically unmanipulated human prostate cancer, we performed biochemical and biological studies with the dual ErbB1/ErbB2 RTK inhibitor PKI-166 using human prostate cancer xenograft models with isogenic sublines reflecting the transition from androgen-dependent to androgen-independent growth. In the presence of low androgen concentrations, PKI-166 showed profound growth-inhibitory effects on tumor growth, which could be partially reversed by androgen add-back. At physiological androgen concentrations, androgen withdrawal greatly enhanced the ability of PKI-166 to retard tumor growth. The level of extracellular signal-regulated kinase activation correlated with the response to PKI-166 treatment, whereas the expression levels of ErbB1 and ErbB2 did not. These results suggest that ErbB1/ErbB2 RTKs play an important role in the biology of androgen-independent prostate cancer and provide a rationale for clinical evaluation of inhibitors targeted to this pathway.

Plasma-derived human C1-esterase inhibitor does not prevent mechanical ventilation-induced pulmonary complement activation in a rat model of Streptococcus pneumoniae pneumonia

NARCIS (Netherlands)

de Beer, F. M.; Aslami, H.; Hoeksma, J.; van Mierlo, G.; Wouters, D.; Zeerleder, S.; Roelofs, J. J. T. H.; Juffermans, N. P.; Schultz, M. J.; Lagrand, W. K.

2014-01-01

Mechanical ventilation has the potential to cause lung injury, and the role of complement activation herein is uncertain. We hypothesized that inhibition of the complement cascade by administration of plasma-derived human C1-esterase inhibitor (C1-INH) prevents ventilation-induced pulmonary

Interaction of amyloid inhibitor proteins with amyloid beta peptides: insight from molecular dynamics simulations.

Directory of Open Access Journals (Sweden)

Payel Das

Full Text Available Knowledge of the detailed mechanism by which proteins such as human αB- crystallin and human lysozyme inhibit amyloid beta (Aβ peptide aggregation is crucial for designing treatment for Alzheimer's disease. Thus, unconstrained, atomistic molecular dynamics simulations in explicit solvent have been performed to characterize the Aβ17-42 assembly in presence of the αB-crystallin core domain and of lysozyme. Simulations reveal that both inhibitor proteins compete with inter-peptide interaction by binding to the peptides during the early stage of aggregation, which is consistent with their inhibitory action reported in experiments. However, the Aβ binding dynamics appear different for each inhibitor. The binding between crystallin and the peptide monomer, dominated by electrostatics, is relatively weak and transient due to the heterogeneous amino acid distribution of the inhibitor surface. The crystallin-bound Aβ oligomers are relatively long-lived, as they form more extensive contact surface with the inhibitor protein. In contrast, a high local density of arginines from lysozyme allows strong binding with Aβ peptide monomers, resulting in stable complexes. Our findings not only illustrate, in atomic detail, how the amyloid inhibitory mechanism of human αB-crystallin, a natural chaperone, is different from that of human lysozyme, but also may aid de novo design of amyloid inhibitors.

Structure-guided approach identifies a novel class of HIV-1 ribonuclease H inhibitors: binding mode insights through magnesium complexation and site-directed mutagenesis studies

DEFF Research Database (Denmark)

Poongavanam, Vasanthanathan; Corona, Angela; Steinmann, Casper

2018-01-01

is a long and expensive process that can be speeded up by in silico methods. In the present study, a structure-guided screening is coupled with a similarity-based search on the Specs database to identify a new class of HIV-1 RNase H inhibitors. Out of the 45 compounds selected for experimental testing, 15...... inhibited the RNase H function below 100 μM with three hits exhibiting IC50 values active compound, AA, inhibits HIV-1 RNase H with an IC50 of 5.1 μM and exhibits a Mg-independent mode of inhibition. Site-directed mutagenesis studies provide valuable insight into the binding mode of newly...

Proton pump inhibitors induce a caspase-independent antitumor effect against human multiple myeloma.

Science.gov (United States)

Canitano, Andrea; Iessi, Elisabetta; Spugnini, Enrico Pierluigi; Federici, Cristina; Fais, Stefano

2016-07-01

Multiple Myeloma (MM) is the second most common hematological malignancy and is responsive to a limited number of drugs. Unfortunately, to date, despite the introduction of novel drugs, no relevant increase in survival rates has been obtained. Proton pump inhibitors (PPIs) have been shown to have significant antitumor action as single agents as well as in combination with chemotherapy. This study investigates the potential anti-tumor effectiveness of two PPIs, Lansoprazole and Omeprazole, against human MM cells. We found that Lansoprazole exerts straightforward efficacy against myeloma cells, even at suboptimal concentrations (50 µM), while Omeprazole has limited cytotoxic action. The Lansoprazole anti-MM effect was mostly mediated by a caspase-independent apoptotic-like cytotoxicity, with only a secondary anti-proliferative action. This study provides clear evidence supporting the use of Lansoprazole in the strive against MM with an efficacy proven much higher than current therapeutical approaches and without reported side effects. It is however conceivable that, consistent with the results obtained in other human tumors, Lansoprazole may well be combined with existing anti-myeloma therapies with the aim to improve the low level of efficacy of the current strategies. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The dipeptidyl peptidase-4 (DPP-4) inhibitor teneligliptin functions as antioxidant on human endothelial cells exposed to chronic hyperglycemia and metabolic high-glucose memory.

Science.gov (United States)

Pujadas, Gemma; De Nigris, Valeria; Prattichizzo, Francesco; La Sala, Lucia; Testa, Roberto; Ceriello, Antonio

2017-06-01

Dipeptidyl peptidase-4 inhibitors are widely used in type 2 diabetes. Endothelium plays a crucial role maintaining vascular integrity and function. Chronic exposure to high glucose drives to endothelial dysfunction generating oxidative stress. Teneligliptin is a novel dipeptidyl peptidase-4 inhibitor with antioxidant properties. This study is aimed to verify a potential protective action of teneligliptin in endothelial cells exposed to high glucose. Human umbilical vein endothelial cells were cultured under normal (5 mmol/L) or high glucose (25 mmol/L) during 21 days, or at high glucose during 14 days followed by 7 days at normal glucose, to reproduce the high-metabolic memory state. During this period, different concentrations of teneligliptin (0.1, 1.0 and 3.0 µmol/L) or sitagliptin (0.5 µmol/L) were added to cells. Ribonucleic acid and protein expression were assessed for antioxidant response, proliferation, apoptosis and endoplasmic reticulum stress markers. Teneligliptin promotes the antioxidant response in human umbilical vein endothelial cells, reducing ROS levels and inducing Nrf2-target genes messenger ribonucleic acid expression. Teneligliptin, but not sitagliptin, reduces the expression of the nicotine amide adenine dinucleotide phosphate oxidase regulatory subunit P22 -phox , however, both blunt the high glucose-induced increase of TXNIP. Teneligliptin improves proliferation rates in human umbilical vein endothelial cells exposed to high glucose, regulating the expression of cell-cycle inhibitors markers (P53, P21 and P27), and reducing proapoptotic genes (BAX and CASP3), while promotes BCL2 expression. Teneligliptin ameliorates high glucose-induced endoplasmic reticulum stress reducing the expression of several markers (BIP, PERK, ATF4, CHOP, IRE1a and ATF6). Teneligliptin has antioxidant properties, ameliorates oxidative stress and apoptotic phenotype and it can overcome the metabolic memory effect, induced by chronic exposure to high

Topoisomerase I inhibitor, camptothecin, induces apoptogenic signaling in human embryonic stem cells

Directory of Open Access Journals (Sweden)

Carolina Paola García

2014-03-01

Full Text Available Embryonic stem cells (ESCs need to maintain their genomic integrity in response to DNA damage to safeguard the integrity of the organism. DNA double strand breaks (DSBs are one of the most lethal forms of DNA damage and, if not repaired correctly, they can lead to cell death, genomic instability and cancer. How human ESCs (hESCs maintain genomic integrity in response to agents that cause DSBs is relatively unclear. In the present study we aim to determine the hESC response to the DSB inducing agent camptothecin (CPT. We find that hESCs are hypersensitive to CPT, as evidenced by high levels of apoptosis. CPT treatment leads to DNA-damage sensor kinase (ATM and DNA-PKcs phosphorylation on serine 1981 and serine 2056, respectively. Activation of ATM and DNA-PKcs was followed by histone H2AX phosphorylation on Ser 139, a sensitive reporter of DNA damage. Nuclear accumulation and ATM-dependent phosphorylation of p53 on serine 15 were also observed. Remarkably, hESC viability was further decreased when ATM or DNA-PKcs kinase activity was impaired by the use of specific inhibitors. The hypersensitivity to CPT treatment was markedly reduced by blocking p53 translocation to mitochondria with pifithrin-μ. Importantly, programmed cell death was achieved in the absence of the cyclin dependent kinase inhibitor, p21Waf1, a bona fide p53 target gene. Conversely, differentiated hESCs were no longer highly sensitive to CPT. This attenuated apoptotic response was accompanied by changes in cell cycle profile and by the presence of p21Waf1. The results presented here suggest that p53 has a key involvement in preventing the propagation of damaged hESCs when genome is threatened. As a whole, our findings support the concept that the phenomenon of apoptosis is a prominent player in normal embryonic development.

Strong synergism between small molecule inhibitors of HER2, PI3K, mTOR and Bcl-2 in human breast cancer cells.

Science.gov (United States)

Hamunyela, Roswita H; Serafin, Antonio M; Akudugu, John M

2017-02-01

Targeting pro-survival cell signaling components has been promising in cancer therapy, but the benefit of targeting with single agents is limited. For malignancies such as triple-negative breast cancer, there is a paucity of targets that are amenable to existing interventions as they are devoid of the human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR), and estrogen receptor (ER). Concurrent targeting of cell signaling entities other than HER2, PR and ER with multiple agents may be more effective. Evaluating modes of interaction between agents can inform efficient selection of agents when used in cocktails. Using clonogenic cell survival, interaction between inhibitors of HER2 (TAK-165), phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) (NVP-BEZ235), and the pro-survival gene (Bcl-2) (ABT-263) in three human breast cell lines (MDA-MB-231, MCF-7 and MCF-12A) ranged from strong to very strong synergism. The strongest synergy was demonstrated in PR and ER negative cells. Inhibition of PI3K, mTOR and Bcl-2 could potentially be effective in the treatment of triple-negative cancers. The very strong synergy observed even at lowest concentrations of inhibitors indicates that these cocktails might be able to be used at a minimised risk of systemic toxicity. Concurrent use of multiple inhibitors can potentiate conventional interventions like radiotherapy and chemotherapy. Copyright © 2016 Elsevier B.V. All rights reserved.

FoxM1 is a general target for proteasome inhibitors.

Directory of Open Access Journals (Sweden)

Uppoor G Bhat

2009-08-01

Full Text Available Proteasome inhibitors are currently in the clinic or in clinical trials, but the mechanism of their anticancer activity is not completely understood. The oncogenic transcription factor FoxM1 is one of the most overexpressed genes in human tumors, while its expression is usually halted in normal non-proliferating cells. Previously, we established that thiazole antibiotics Siomycin A and thiostrepton inhibit FoxM1 and induce apoptosis in human cancer cells. Here, we report that Siomycin A and thiostrepton stabilize the expression of a variety of proteins, such as p21, Mcl-1, p53 and hdm-2 and also act as proteasome inhibitors in vitro. More importantly, we also found that well-known proteasome inhibitors such as MG115, MG132 and bortezomib inhibit FoxM1 transcriptional activity and FoxM1 expression. In addition, overexpression of FoxM1 specifically protects against bortezomib-, but not doxorubicin-induced apoptosis. These data suggest that negative regulation of FoxM1 by proteasome inhibitors is a general feature of these drugs and it may contribute to their anticancer properties.

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

Science.gov (United States)

Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

1988-02-01

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators.

Characterization of the archaeal ribonuclease P proteins from Pyrococcus horikoshii OT3.

Science.gov (United States)

Terada, Atsushi; Honda, Takashi; Fukuhara, Hideo; Hada, Kazumasa; Kimura, Makoto

2006-08-01

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5'-leader sequence of precursor tRNA (pre-tRNA). Our earlier study revealed that RNase P RNA (pRNA) and five proteins (PhoPop5, PhoRpp38, PhoRpp21, PhoRpp29, and PhoRpp30) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 reconstituted RNase P activity that exhibits enzymatic properties like those of the authentic enzyme. In present study, we investigated involvement of the individual proteins in RNase P activity. Two particles (R-3Ps), in which pRNA was mixed with three proteins, PhoPop5, PhoRpp30, and PhoRpp38 or PhoPop5, PhoRpp30, and PhoRpp21 showed a detectable RNase P activity, and five reconstituted particles (R-4Ps) composed of pRNA and four proteins exhibited RNase P activity, albeit at reduced level compared to that of the reconstituted particle (R-5P) composed of pRNA and five proteins. Time-course analysis of the RNase P activities of R-4Ps indicated that the R-4Ps lacking PhoPop5, PhoRpp21, or PhoRpp30 had virtually reduced activity, while omission of PhoRpp29 or PhoRpp38 had a slight effect on the activity. The results indicate that the proteins contribute to RNase P activity in order of PhoPop5 > PhoRpp30 > PhoRpp21 > PhoRpp29 > PhoRpp38. It was further found that R-4Ps showed a characteristic Mg2+ ion dependency approximately identical to that of R-5P. However, R-4Ps had optimum temperature of around at 55 degrees C which is lower than 70 degrees C for R-5P. Together, it is suggested that the P. horikoshii RNase P proteins are predominantly involved in optimization of the pRNA conformation, though they are individually dispensable for RNase P activity in vitro.

Small Molecule Inhibitors That Selectively Block Dengue Virus Methyltransferase*

OpenAIRE

Lim, Siew Pheng; Sonntag, Louis Sebastian; Noble, Christian; Nilar, Shahul H.; Ng, Ru Hui; Zou, Gang; Monaghan, Paul; Chung, Ka Yan; Dong, Hongping; Liu, Boping; Bodenreider, Christophe; Lee, Gladys; Ding, Mei; Chan, Wai Ling; Wang, Gang

2010-01-01

Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crysta...

ROCK inhibitor is not required for embryoid body formation from singularized human embryonic stem cells.

Science.gov (United States)

Pettinato, Giuseppe; Vanden Berg-Foels, Wendy S; Zhang, Ning; Wen, Xuejun

2014-01-01

We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications.

Regulation of collagenase inhibitor production in chondrosarcoma chondrocytes

International Nuclear Information System (INIS)

Harper, J.; Harper, E.

1987-01-01

Swarm rat chondrosarcoma chondrocytes produce an inhibitor of collagenase. This inhibitor is similar to those isolated from normal cartilage tissues. These cells will synthesize proteins in the absence of serum. Since serum contains inhibitors of collagenase, it is necessary to culture cells without serum in order to obtain accurate measurements of enzyme and inhibitor levels. They examined the effect of insulin on inhibitor secretion by cultures of Swarm rat chondrosarcoma chondrocytes. They observed a 2.5 to 3.5 fold stimulation of inhibitory activity in the presence of as little as 10 ng/ml insulin as compared to controls in serum free Dulbecco's modified Eagle's medium supplemented with 4.5 g/l glucose. The units of inhibitor were determined over a 7 day culture period. Medium was harvested daily and assayed for collagenase activity and for inhibition of a known collagenase from rabbit skin or human skin, using the 14 C-glycine peptide release assay. The amount of inhibitor obtained from days 2 through 7 were: 1.4 unit (control), 3.8 units (10 ng/ml insulin), 5.2 units (1 μg/ml insulin). The addition of 1 mM dibutyryl cyclic AMP to these chondrocytes in the presence of 1 μg/ml insulin caused a decrease in the level of inhibitor, suggesting that a dephosphorylation event may be necessary for this stimulation by insulin to occur

Determination of activities of human carbonic anhydrase II inhibitors ...

African Journals Online (AJOL)

Purpose: To evaluate the activities of new curcumin analogs as carbonic anhydrase II (CA-II) inhibitor. Methods: Carbonic anhydrase II (CA-II) inhibition was determined by each ligand capability to inhibit the esterase activity of CA-II using 4-NPA as a substrate in 96-well plates. Dimethyl sulfoxide was used to dissolve each ...

The effect of phosphodiesterase-5 inhibitors on cerebral blood flow in humans

DEFF Research Database (Denmark)

Pauls, Mathilde Mh; Moynihan, Barry; Barrick, Thomas R

2018-01-01

, ED, type 2 diabetes, stroke, pulmonary hypertension, Becker muscular dystrophy and subarachnoid haemorrhage. Most studies used middle cerebral artery flow velocity to estimate CBF. Few studies employed direct measurements of tissue perfusion. Resting CBF velocity was unaffected by phosphodiesterase-5...... inhibitors, but cerebrovascular regulation was improved in ED, pulmonary hypertension, diabetes, Becker's and a group of healthy volunteers. This evidence suggests that phosphodiesterase-5 inhibitors improve responsiveness of the cerebral vasculature, particularly in disease states associated...

Preparation, structural analysis and bioactivity of ribonuclease A-albumin conjugate: tetra-conjugation or PEG as the linker.

Science.gov (United States)

Li, Chunju; Lin, Qixun; Wang, Jun; Shen, Lijuan; Ma, Guanghui; Su, Zhiguo; Hu, Tao

2012-12-31

Ribonuclease A (RNase A) is a therapeutic enzyme with cytotoxic action against tumor cells. Its clinical application is limited by the short half-life and insufficient stability. Conjugation of albumin can overcome the limitation, whereas dramatically decrease the enzymatic activity of RNase A. Here, three strategies were proposed to prepare the RNase A-bovine serum albumin (BSA) conjugates. R-SMCC-B (a conjugate of four RNase A attached with one BSA) and R-PEG-B (a mono-conjugate) were prepared using Sulfo-SMCC (a short bifunctional linker) and mal-PEG-NHS (a bifunctional PEG), respectively. Mal-PEG-NHS and hexadecylamine (HDA) were used to prepare the mono-conjugate, R-HDA-B, where HDA was adopted to bind BSA. The PEG linker can elongate the proximity between RNase A and BSA. In contrast, four RNase A were closely located on BSA in R-SMCC-B. R-SMCC-B showed the lowest K(m) and the highest relative enzymatic activity and k(cat)/K(m) in the three conjugates. Presumably, the tetravalent interaction of RNase A in R-SMCC-B can increase the binding affinity to its substrate. In addition, the slow release of BSA from R-HDA-B may increase the enzymatic activity of R-HDA-B. Our study is expected to provide strategies to develop protein-albumin conjugate with high therapeutic potential. Copyright © 2012 Elsevier B.V. All rights reserved.

Microarray-based screening of heat shock protein inhibitors.

Science.gov (United States)

Schax, Emilia; Walter, Johanna-Gabriela; Märzhäuser, Helene; Stahl, Frank; Scheper, Thomas; Agard, David A; Eichner, Simone; Kirschning, Andreas; Zeilinger, Carsten

2014-06-20

Based on the importance of heat shock proteins (HSPs) in diseases such as cancer, Alzheimer's disease or malaria, inhibitors of these chaperons are needed. Today's state-of-the-art techniques to identify HSP inhibitors are performed in microplate format, requiring large amounts of proteins and potential inhibitors. In contrast, we have developed a miniaturized protein microarray-based assay to identify novel inhibitors, allowing analysis with 300 pmol of protein. The assay is based on competitive binding of fluorescence-labeled ATP and potential inhibitors to the ATP-binding site of HSP. Therefore, the developed microarray enables the parallel analysis of different ATP-binding proteins on a single microarray. We have demonstrated the possibility of multiplexing by immobilizing full-length human HSP90α and HtpG of Helicobacter pylori on microarrays. Fluorescence-labeled ATP was competed by novel geldanamycin/reblastatin derivatives with IC50 values in the range of 0.5 nM to 4 μM and Z(*)-factors between 0.60 and 0.96. Our results demonstrate the potential of a target-oriented multiplexed protein microarray to identify novel inhibitors for different members of the HSP90 family. Copyright © 2014 Elsevier B.V. All rights reserved.

Novel Combinatorial Chemistry-Derived Inhibitors of Oncogenic Phosphatases

National Research Council Canada - National Science Library

Lazo, John

1999-01-01

Our overall goal of this US Army Breast Cancer Grant entitled "Novel Combinatorial Chemistry-Derived Inhibitors of Oncogenic Phosphatases" is to identity and develop novel therapeutic agents for human breast cancer...

Canagliflozin, an SGLT2 inhibitor, attenuates the development of hepatocellular carcinoma in a mouse model of human NASH.

Science.gov (United States)

Shiba, Kumiko; Tsuchiya, Kyoichiro; Komiya, Chikara; Miyachi, Yasutaka; Mori, Kentaro; Shimazu, Noriko; Yamaguchi, Shinobu; Ogasawara, Naomi; Katoh, Makoto; Itoh, Michiko; Suganami, Takayoshi; Ogawa, Yoshihiro

2018-02-05

Sodium glucose cotransporter 2 (SGLT2) inhibitors, an antidiabetic drug, promotes urinary excretion of glucose by blocking its reabsorption in the renal proximal tubules. It is unclear whether SGLT2 inhibition could attenuate nonalcoholic steatohepatitis (NASH) and NASH-associated hepatocellular carcinoma. We examined the preventive effects of an SGLT2 inhibitor canagliflozin (CANA) in Western diet (WD)-fed melanocortin 4 receptor-deficient (MC4R-KO) mice, a mouse model of human NASH. An eight-week CANA treatment attenuated hepatic steatosis in WD-fed MC4R-KO mice, with increased epididymal fat mass without inflammatory changes. CANA treatment for 20 weeks inhibited the development of hepatic fibrosis in WD-fed MC4R-KO mice. After one year of CANA treatment, the number of liver tumors was significantly reduced in WD-fed MC4R-KO mice. In adipose tissue, CANA suppressed the ratio of oxidative to reduced forms of glutathiones (GSSG/GSH) in WD-fed MC4R-KO mice. Treatment with GSH significantly attenuated the H 2 O 2 -induced upregulation of genes related to NADPH oxidase in 3T3-L1 adipocytes, and that of Il6, Tgfb, and Pdgfb in RAW264.7 cells. This study provides evidence that SGLT2 inhibitors represent the unique class of drugs that can attenuate or delay the onset of NASH and eventually hepatocellular carcinoma, at least partly, through "healthy adipose expansion".

Indanones as high-potency reversible inhibitors of monoamine oxidase.

Science.gov (United States)

Mostert, Samantha; Petzer, Anél; Petzer, Jacobus P

2015-05-01

Recent reports document that α-tetralone (3,4-dihydro-2H-naphthalen-1-one) is an appropriate scaffold for the design of high-potency monoamine oxidase (MAO) inhibitors. Based on the structural similarity between α-tetralone and 1-indanone, the present study involved synthesis of 34 1-indanone and related indane derivatives as potential inhibitors of recombinant human MAO-A and MAO-B. The results show that C6-substituted indanones are particularly potent and selective MAO-B inhibitors, with IC50 values ranging from 0.001 to 0.030 μM. C5-Substituted indanone and indane derivatives are comparatively weaker MAO-B inhibitors. Although the 1-indanone and indane derivatives are selective inhibitors of the MAO-B isoform, a number of homologues are also potent MAO-A inhibitors, with three homologues possessing IC50 values 1-indanone as a reversible MAO inhibitor with a competitive mode of inhibition. It may be concluded that 1-indanones are promising leads for the design of therapies for neurodegenerative and neuropsychiatric disorders such as Parkinson's disease and depression. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Exploiting Chemical Libraries, Structure, and Genomics in the Search for Kinase Inhibitors

NARCIS (Netherlands)

Gray, Nathanael S.; Wodicka, Lisa; Thunnissen, Andy-Mark W.H.; Norman, Thea C.; Kwon, Soojin; Espinoza, F. Hernan; Morgan, David O.; Barnes, Georjana; LeClerc, Sophie; Meijer, Laurent; Kim, Sung-Hou; Lockhart, David J.; Schultz, Peter G.

1998-01-01

Selective protein kinase inhibitors were developed on the basis of the unexpected binding mode of 2,6,9-trisubstituted purines to the adenosine triphosphate-binding site of the human cyclin-dependent kinase 2 (CDK2). By iterating chemical library synthesis and biological screening, potent inhibitors

Locostatin, a disrupter of Raf kinase inhibitor protein, inhibits extracellular matrix production, proliferation, and migration in human uterine leiomyoma and myometrial cells.

Science.gov (United States)

Janjusevic, Milijana; Greco, Stefania; Islam, Md Soriful; Castellucci, Clara; Ciavattini, Andrea; Toti, Paolo; Petraglia, Felice; Ciarmela, Pasquapina

2016-11-01

To investigate the presence of Raf kinase inhibitor protein (RKIP) in human myometrium and leiomyoma as well as to determine the effect of locostatin (RKIP inhibitor) on extracellular matrix (ECM) production, proliferation, and migration in human myometrial and leiomyoma cells. Laboratory study. Human myometrium and leiomyoma. Thirty premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Myometrial and leiomyoma tissues were used to investigate the localization and the expression level of RKIP through immunohistochemistry and Western blotting. Myometrial and leiomyoma cells were treated with locostatin (10 μM) to measure ECM expression by real-time polymerase chain reaction, GSK3β expression by Western blotting, cell migration by wound-healing assay, and cell proliferation by MTT assay and immunocytochemistry. The expression of RKIP in human myometrial and leiomyoma tissue; ECM components and GSK3β expression, migration, and proliferation in myometrial and leiomyoma cells. RKIP is expressed in human myometrial and leiomyoma tissue. Locostatin treatment resulted in the activation of the mitogen-activated protein kinase (MAPK) signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, RKIP inhibition by locostatin reduces ECM components. Moreover, the inhibition of RKIP by locostatin impaired cell proliferation and migration in both leiomyoma and myometrial cells. Finally, locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells. Our results indicate that RKIP may be involved in leiomyoma pathophysiology. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Comparative effect of two pan-class I PI3K inhibitors used as anticancer drugs on human T cell function.

Science.gov (United States)

Blanco, Belén; Herrero-Sánchez, Carmen; Rodríguez-Serrano, Concepción; Sánchez-Barba, Mercedes; Del Cañizo, María Consuelo

2015-09-01

The phosphatidylinositol 3-kinase (PI3K) pathway is commonly deregulated in cancer and, thus, PI3K has been recognized as an attractive molecular target for novel anti-cancer therapies. However, the effect of PI3K inhibitors on T-cell function, a key component of antitumor immunity, has been scantly explored. The objective of this study was to investigate the effect on human T-cell activation of two PI3K inhibitors currently being tested in clinical trials: PX-866 and BKM120. Their activity against a leukemic T cell line was also assessed. For that purpose, Jurkat cells or anti-CD3/anti-CD28 stimulated human peripheral blood mononuclear cells were cultured in the presence of different concentrations of PX-866 or BKM120 and their effect on T-cell proliferation, apoptosis, expression of activation markers and cytokine secretion was analyzed by flow cytometry. In addition, Akt and Erk phosphorylation was analyzed by Western blotting. Both PX-866 and BKM120 decreased viability of Jurkat cells and blocked cell cycle progression. Regarding primary T cells, both compounds similarly inhibited expression of activation markers and cytokine secretion, although they did not induce apoptosis of stimulated T cells. Interestingly, we found differences in their ability to block T-cell proliferation and IL-2 secretion, exerting BKM120 a more potent inhibition. These disparate effects could be related to differences observed in PI3K/Akt and RAS/MEK/ERK signaling between PX-866 and BKM120 treated cells. Our results suggest that, when selecting a PI3K inhibitor for cancer therapy, immunosuppressive characteristics should be taken into account in order to minimize detrimental effects on immune function. Copyright © 2015 Elsevier B.V. All rights reserved.

Hepatocyte growth factor inhibitor-2 prevents shedding of matritpase

DEFF Research Database (Denmark)

Larsen, Brian R; Steffensen, Simon D; Nielsen, Nis V L

2013-01-01

Hepatocyte growth factor activator inhibitor-2 (HAI-2) is an inhibitor of many proteases in vitro, including the membrane-bound serine protease, matriptase. Studies of knock-out mice have shown that HAI-2 is essential for placental development only in mice expressing matriptase, suggesting that HAI......-2 is important for regulation of matriptase. Previous studies have shown that recombinant expression of matriptase was unsuccessful unless co-expressed with another HAI, HAI-1. In the present study we show that when human matriptase is recombinantly expressed alone in the canine cell line MDCK......, then human matriptase mRNA can be detected and the human matriptase ectodomain is shed to the media, suggesting that matriptase expressed alone is rapidly transported through the secretory pathway and shed. Whereas matriptase expressed together with HAI-1 or HAI-2 accumulates on the plasma membrane where...

Cellular inhibitor of apoptosis protein 2 (cIAP2) controls human colonic epithelial restitution, migration and Rac1 activation

DEFF Research Database (Denmark)

Seidelin, JB; Larsen, Sylvester; Linnemann, D

2015-01-01

epithelial cells (IECs) was increased at the wound edge after 24 h (P 2 was induced in vitro in regenerating Caco2 IECs after wound infliction (P ...Identification of pathways involved in wound healing is important for understanding the pathogenesis of various intestinal diseases. Cellular inhibitor of apoptosis protein 2 (cIAP2) regulates proliferation and migration in nonepithelial cells and is expressed in human colonocytes. The aim...... of the study was to investigate the role of cIAP2 for wound healing in the normal human colon. Wound tissue was generated by taking rectosigmoidal biopsies across an experimental ulcer in healthy subjects after 5, 24, and 48 h. In experimental ulcers, the expression of cIAP2 in regenerating intestinal...

Identification of putative substrates for cynomolgus monkey cytochrome P450 2C8 by substrate depletion assays with 22 human P450 substrates and inhibitors.

Science.gov (United States)

Hosaka, Shinya; Murayama, Norie; Satsukawa, Masahiro; Uehara, Shotaro; Shimizu, Makiko; Iwasaki, Kazuhide; Iwano, Shunsuke; Uno, Yasuhiro; Yamazaki, Hiroshi

2016-07-01

Cynomolgus monkeys are widely used in drug developmental stages as non-human primate models. Previous studies used 89 compounds to investigate species differences associated with cytochrome P450 (P450 or CYP) function that reported monkey specific CYP2C76 cleared 19 chemicals, and homologous CYP2C9 and CYP2C19 metabolized 17 and 30 human CYP2C9 and/or CYP2C19 substrates/inhibitors, respectively. In the present study, 22 compounds selected from viewpoints of global drug interaction guidances and guidelines were further evaluated to seek potential substrates for monkey CYP2C8, which is highly homologous to human CYP2C8 (92%). Amodiaquine, montelukast, quercetin and rosiglitazone, known as substrates or competitive inhibitors of human CYP2C8, were metabolically depleted by recombinant monkey CYP2C8 at relatively high rates. Taken together with our reported findings of the slow eliminations of amodiaquine and montelukast by monkey CYP2C9, CYP2C19 and CYP2C76, the present results suggest that these at least four chemicals may be good marker substrates for monkey CYP2C8. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Histone deacetylase inhibitors for the treatment of cancer stem cells

Czech Academy of Sciences Publication Activity Database

Dvořáková, Marcela; Vaněk, Tomáš

2016-01-01

Roč. 7, č. 12 (2016), s. 2217-2231 ISSN 2040-2503 R&D Projects: GA MŠk LD14128 Institutional support: RVO:61389030 Keywords : acute myeloid-leukemia * epithelial-mesenchymal transition * acute myelogenous leukemia * tumor-initiating cells * human aml cells * breast-cancer * hdac inhibitors * sirtuin inhibitors * colorectal-cancer * anticancer agents Subject RIV: CC - Organic Chemistry Impact factor: 2.608, year: 2016

Serine proteinase inhibitors from nematodes and the arms race between host and pathogen.

Science.gov (United States)

Zang, X; Maizels, R M

2001-03-01

Serine proteinase inhibitors are encoded by a large gene family of long evolutionary standing. Recent discoveries of parasite proteins that inhibit human serine proteinases, together with the complete genomic sequence from Caenorhabditis elegans, have provided a set of new serine proteinase inhibitors from more primitive metazoan animals such as nematodes. The structural features (e.g. reactive centre residues), gene organization (including intron arrangements) and inhibitory function and targets (e.g. inflammatory and coagulation pathway proteinase) all contribute important new insights into proteinase inhibitor evolution. Some parasite products have evolved that block enzymes in the mammalian host, but the human host responds with a significant immune response to the parasite inhibitors. Thus, infection produces a finely balanced conflict between host and pathogen at the molecular level, and this might have accelerated the evolution of these proteins in parasitic species as well as their hosts.

Activities of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor nelfinavir mesylate in combination with reverse transcriptase and protease inhibitors against acute HIV-1 infection in vitro.

Science.gov (United States)

Patick, A K; Boritzki, T J; Bloom, L A

1997-10-01

Nelfinavir mesylate (formerly AG1343) is a potent and selective, nonpeptidic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease that was discovered by protein structure-based design methodologies. We evaluated the antiviral and cytotoxic effects of two-drug combinations of nelfinavir with the clinically approved antiretroviral therapeutics zidovudine (ZDV), lamivudine (3TC), dideoxycytidine (ddC; zalcitabine), stavudine (d4T), didanosine (ddI), indinavir, saquinavir, and ritonavir and a three-drug combination of nelfinavir with ZDV and 3TC against an acute HIV-1 strain RF infection of CEM-SS cells in vitro. Quantitative assessment of drug interaction was evaluated by a universal response surface approach (W. R. Greco, G. Bravo, and J. C. Parsons, Pharm. Rev. 47:331-385, 1995) and by the method of M. N. Prichard and C. Shipman (Antiviral Res. 14:181-206, 1990). Both analytical methods yielded similar results and showed that the two-drug combinations of nelfinavir with the reverse transcriptase inhibitors ZDV, 3TC, ddI, d4T, and ddC and the three-drug combination with ZDV and 3TC resulted in additive to statistically significant synergistic interactions. In a similar manner, the combination of nelfinavir with the three protease inhibitors resulted in additive (ritonavir and saquinavir) to slightly antagonistic (indinavir) interactions. In all combinations, minimal cellular cytotoxicity was observed with any drug alone and in combination. These results suggest that administration of combinations of the appropriate doses of nelfinavir with other currently approved antiretroviral therapeutic agents in vivo may result in enhanced antiviral activity with no associated increase in cellular cytotoxicity.

High resolution crystal structure of rat long chain hydroxy acid oxidase in complex with the inhibitor 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1, 2, 3-thiadiazole. Implications for inhibitor specificity and drug design

Energy Technology Data Exchange (ETDEWEB)

Chen, Zhi-wei; Vignaud, Caroline; Jaafar, Adil; Lévy, Bernard; Guéritte, Françoise; Guénard, Daniel; Lederer, Florence; Mathews, F. Scott (CNRS-UMR); (WU-MED)

2012-05-24

Long chain hydroxy acid oxidase (LCHAO) is responsible for the formation of methylguanidine, a toxic compound with elevated serum levels in patients with chronic renal failure. Its isozyme glycolate oxidase (GOX), has a role in the formation of oxalate, which can lead to pathological deposits of calcium oxalate, in particular in the disease primary hyperoxaluria. Inhibitors of these two enzymes may have therapeutic value. These enzymes are the only human members of the family of FMN-dependent L-2-hydroxy acid-oxidizing enzymes, with yeast flavocytochrome b{sub 2} (Fcb2) among its well studied members. We screened a chemical library for inhibitors, using in parallel rat LCHAO, human GOX and the Fcb2 flavodehydrogenase domain (FDH). Among the hits was an inhibitor, CCPST, with an IC{sub 50} in the micromolar range for all three enzymes. We report here the crystal structure of a complex between this compound and LCHAO at 1.3 {angstrom} resolution. In comparison with a lower resolution structure of this enzyme, binding of the inhibitor induces a conformational change in part of the TIM barrel loop 4, as well as protonation of the active site histidine. The CCPST interactions are compared with those it forms with human GOX and those formed by two other inhibitors with human GOX and spinach GOX. These compounds differ from CCPST in having the sulfur replaced with a nitrogen in the five-membered ring as well as different hydrophobic substituents. The possible reason for the {approx}100-fold difference in affinity between these two series of inhibitors is discussed. The present results indicate that specificity is an issue in the quest for therapeutic inhibitors of either LCHAO or GOX, but they may give leads for this quest.

Frequent expression loss of Inter-alpha-trypsin inhibitor heavy chain (ITIH genes in multiple human solid tumors: A systematic expression analysis

Directory of Open Access Journals (Sweden)

Werbowetski-Ogilvie Tamra

2008-01-01

Full Text Available Abstract Background The inter-alpha-trypsin inhibitors (ITI are a family of plasma protease inhibitors, assembled from a light chain – bikunin, encoded by AMBP – and five homologous heavy chains (encoded by ITIH1, ITIH2, ITIH3, ITIH4, and ITIH5, contributing to extracellular matrix stability by covalent linkage to hyaluronan. So far, ITIH molecules have been shown to play a particularly important role in inflammation and carcinogenesis. Methods We systematically investigated differential gene expression of the ITIH gene family, as well as AMBP and the interacting partner TNFAIP6 in 13 different human tumor entities (of breast, endometrium, ovary, cervix, stomach, small intestine, colon, rectum, lung, thyroid, prostate, kidney, and pancreas using cDNA dot blot analysis (Cancer Profiling Array, CPA, semiquantitative RT-PCR and immunohistochemistry. Results We found that ITIH genes are clearly downregulated in multiple human solid tumors, including breast, colon and lung cancer. Thus, ITIH genes may represent a family of putative tumor suppressor genes that should be analyzed in greater detail in the future. For an initial detailed analysis we chose ITIH2 expression in human breast cancer. Loss of ITIH2 expression in 70% of cases (n = 50, CPA could be confirmed by real-time PCR in an additional set of breast cancers (n = 36. Next we studied ITIH2 expression on the protein level by analyzing a comprehensive tissue micro array including 185 invasive breast cancer specimens. We found a strong correlation (p Conclusion Altogether, this is the first systematic analysis on the differential expression of ITIH genes in human cancer, showing frequent downregulation that may be associated with initiation and/or progression of these malignancies.

High-throughput screening to identify inhibitors of lysine demethylases.

Science.gov (United States)

Gale, Molly; Yan, Qin

2015-01-01

Lysine demethylases (KDMs) are epigenetic regulators whose dysfunction is implicated in the pathology of many human diseases including various types of cancer, inflammation and X-linked intellectual disability. Particular demethylases have been identified as promising therapeutic targets, and tremendous efforts are being devoted toward developing suitable small-molecule inhibitors for clinical and research use. Several High-throughput screening strategies have been developed to screen for small-molecule inhibitors of KDMs, each with advantages and disadvantages in terms of time, cost, effort, reliability and sensitivity. In this Special Report, we review and evaluate the High-throughput screening methods utilized for discovery of novel small-molecule KDM inhibitors.

Suppression of human T cell proliferation by the caspase inhibitors, z-VAD-FMK and z-IETD-FMK is independent of their caspase inhibition properties

Energy Technology Data Exchange (ETDEWEB)

Lawrence, C.P. [Medical Research Council Toxicology Unit, Hodgkin Building, Lancaster Road, University of Leicester, Leicester LE1 9HN (United Kingdom); Chow, S.C., E-mail: chow.sek.chuen@monash.edu [School of Science, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, 46150 Selangor Darul Ehsan (Malaysia)

2012-11-15

The caspase inhibitors, benzyloxycarbony (Cbz)-l-Val-Ala-Asp (OMe)-fluoromethylketone (z-VAD-FMK) and benzyloxycarbonyl (Cbz)-Ile-Glu (OMe)-Thr-Asp (OMe)-FMK (z-IETD-FMK) at non-toxic doses were found to be immunosuppressive and inhibit human T cell proliferation induced by mitogens and IL-2 in vitro. Both caspase inhibitors were shown to block NF-κB in activated primary T cells, but have little inhibitory effect on the secretion of IL-2 and IFN-γ during T cell activation. However, the expression of IL-2 receptor α-chain (CD25) in activated T cells was inhibited by both z-VAD-FMK and z-IETD-FMK, whereas the expression of the early activated T cell marker, CD69 was unaffected. During primary T cell activation via the antigen receptor, both caspase-8 and caspase-3 were activated and processed to their respective subunits, but neither caspase inhibitors had any effect on the processing of these two caspases. In sharp contrast both caspase inhibitors readily blocked apoptosis and the activation of caspases during FasL-induced apoptosis in activated primary T cells and Jurkat T cells. Collectively, the results demonstrate that both z-VAD-FMK and z-IETD-FMK are immunosuppressive in vitro and inhibit T cell proliferation without blocking the processing of caspase-8 and caspase-3. -- Highlights: ► Caspase-8 and caspase-3 were activated during T cell activation and proliferation. ► T cell proliferation was blocked by caspase inhibitors. ► Caspase activation during T cell proliferation was not block by caspase inhibitors.

MCPIP1 contributes to the toxicity of proteasome inhibitor MG-132 in HeLa cells by the inhibition of NF-κB.

Science.gov (United States)

Skalniak, Lukasz; Dziendziel, Monika; Jura, Jolanta

2014-10-01

Recently, we have shown that the treatment of cells with proteasome inhibitor MG-132 results in the induction of expression of monocyte chemotactic protein-1 induced protein 1 (MCPIP1). MCPIP1 is a ribonuclease, responsible for the degradation of transcripts encoding certain pro-inflammatory cytokines. The protein is also known as an inhibitor of NF-κB transcription factor. Thanks to its molecular properties, MCPIP1 is considered as a regulator of inflammation, differentiation, and survival. Using siRNA technology, we show here that MCPIP1 expression contributes to the toxic properties of MG-132 in HeLa cells. The inhibition of proteasome by MG-132 and epoxomicin markedly increased MCPIP1 expression. While MG-132 induces HeLa cell death, down-regulation of MCPIP1 expression by siRNA partially protects HeLa cells from MG-132 toxicity and restores Nuclear factor-κB (NF-κB) activity, inhibited by MG-132 treatment. Inversely, overexpression of MCPIP1 decreased constitutive activity of NF-κB and limited the survival of HeLa cells, as we have shown in the previous study. Interestingly, although MG-132 decreased the expression of IκBα and increased p65 phosphorylation, the inhibition of constitutive NF-κB activity was observed in MG-132-treated cells. Since the elevated constitutive activity of NF-κB is one of the mechanisms providing increased survival of cancer cells, including HeLa cells, we propose that death-promoting properties of MCPIP1 in MG-132-treated HeLa cells may, at least partially, derive from the negative effect on the constitutive NF-κB activity.

Purification and characterization of bioactive his₆-tagged recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) protein expressed at high yields in mammalian cells

DEFF Research Database (Denmark)

Jensen, Lena Vinther; Lademann, Ulrik Axel; Andersen, Elisabeth Veyhe

2014-01-01

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is an endogenous inhibitor of matrix metalloproteinases (MMPs) with reported tumor promoting, as well as inhibitory, effects. These paradoxical properties are presumably mediated by different biological functions, MMP-dependent as well as -indepen...... TIMP-1, which structurally and functionally is similar to endogenous human TIMP-1, while using an expression system that is adaptable to most biochemical and biomedical laboratories including those that do not perform protein purifications routinely.......Tissue inhibitor of metalloproteinases-1 (TIMP-1) is an endogenous inhibitor of matrix metalloproteinases (MMPs) with reported tumor promoting, as well as inhibitory, effects. These paradoxical properties are presumably mediated by different biological functions, MMP-dependent as well...... as -independent, and probably related to TIMP-1 levels of protein expression, post-translational modifications, and cellular localization. TIMP-1 is an N-glycosylated protein that folds into two functional domains, a C- and an N-terminal domain, with six disulfide bonds. Furthermore, TIMP-1 is processed in the N...

Guanfu base A, an antiarrhythmic alkaloid of Aconitum coreanum, Is a CYP2D6 inhibitor of human, monkey, and dog isoforms.

Science.gov (United States)

Sun, Jianguo; Peng, Ying; Wu, Hui; Zhang, Xueyuan; Zhong, Yunxi; Xiao, Yanan; Zhang, Fengyi; Qi, Huanhuan; Shang, Lili; Zhu, Jianping; Sun, Yue; Liu, Ke; Liu, Jinghan; A, Jiye; Ho, Rodney J Y; Wang, Guangji

2015-05-01

Guanfu base A (GFA) is a novel heterocyclic antiarrhythmic drug isolated from Aconitum coreanum (Lèvl.) rapaics and is currently in a phase IV clinical trial in China. However, no study has investigated the influence of GFA on cytochrome P450 (P450) drug metabolism. We characterized the potency and specificity of GFA CYP2D inhibition based on dextromethorphan O-demethylation, a CYP2D6 probe substrate of activity in human, mouse, rat, dog, and monkey liver microsomes. In addition, (+)-bufuralol 1'-hydroxylation was used as a CYP2D6 probe for the recombinant form (rCYP2D6), 2D1 (rCYP2D1), and 2D2 (rCYP2D2) activities. Results show that GFA is a potent noncompetitive inhibitor of CYP2D6, with inhibition constant Ki = 1.20 ± 0.33 μM in human liver microsomes (HLMs) and Ki = 0.37 ± 0.16 μM for the human recombinant form (rCYP2D6). GFA is also a potent competitive inhibitor of CYP2D in monkey (Ki = 0.38 ± 0.12 μM) and dog (Ki = 2.4 ± 1.3 μM) microsomes. However, GFA has no inhibitory activity on mouse or rat CYP2Ds. GFA did not exhibit any inhibition activity on human recombinant CYP1A2, 2A6, 2C8, 2C19, 3A4, or 3A5, but showed slight inhibition of 2B6 and 2E1. Preincubation of HLMs and rCYP2D6 resulted in the inactivation of the enzyme, which was attenuated by GFA or quinidine. Beagle dogs treated intravenously with dextromethorphan (2 mg/ml) after pretreatment with GFA injection showed reduced CYP2D metabolic activity, with the Cmax of dextrorphan being one-third that of the saline-treated group and area under the plasma concentration-time curve half that of the saline-treated group. This study suggests that GFA is a specific CYP2D6 inhibitor that might play a role in CYP2D6 medicated drug-drug interaction. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models

Science.gov (United States)

Wen, Li; Voronina, Svetlana; Javed, Muhammad A.; Awais, Muhammad; Szatmary, Peter; Latawiec, Diane; Chvanov, Michael; Collier, David; Huang, Wei; Barrett, John; Begg, Malcolm; Stauderman, Ken; Roos, Jack; Grigoryev, Sergey; Ramos, Stephanie; Rogers, Evan; Whitten, Jeff; Velicelebi, Gonul; Dunn, Michael; Tepikin, Alexei V.; Criddle, David N.; Sutton, Robert

2015-01-01

Background & Aims Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release–activated calcium modulator ORAI1 is the most abundant Ca2+ entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice. Methods Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects. Results GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca2+ currents after Ca2+ release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis. Conclusions Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed

In Vitro Evaluation of Nonnucleoside Reverse Transcriptase Inhibitors UC-781 and TMC120-R147681 as Human Immunodeficiency Virus Microbicides†

Science.gov (United States)

Van Herrewege, Yven; Michiels, Jo; Van Roey, Jens; Fransen, Katrien; Kestens, Luc; Balzarini, Jan; Lewi, Paul; Vanham, Guido; Janssen, Paul

2004-01-01

The nonnucleoside reverse transcriptase inhibitors UC-781 and TMC120-R147681 (Dapivirine) effectively prevented human immunodeficiency virus (HIV) infection in cocultures of monocyte-derived dendritic cells and T cells, representing primary targets in sexual transmission. Both drugs had a favorable therapeutic index. A 24-h treatment with 1,000 nM UC-781 or 100 nM TMC120-R147681 prevented cell-free HIV infection, whereas 10-fold-higher concentrations blocked cell-associated HIV. PMID:14693562

Secretion of intact proteins and peptide fragments by lysosomal pathways of protein degradation

International Nuclear Information System (INIS)

Isenman, L.D.; Dice, J.F.

1989-01-01

We report that degradation of proteins microinjected into human fibroblasts is accompanied by release into the culture medium of peptide fragments and intact proteins as well as single amino acids. For the nine proteins and polypeptides microinjected, acid-precipitable radioactivity, i.e. peptide fragments and/or intact proteins, ranged from 10 to 67% of the total released radioactivity. Peptide fragments and/or intact protein accounted for 60% of the radioactivity released into the medium by cells microinjected with ribonuclease A. Two major radiolabeled peptide fragments were found, and one was of an appropriate size to function as an antigen in antigen-presenting cells. The peptides released from microinjected ribonuclease A were derived from lysosomal pathways of proteolysis based on several lines of evidence. Previous studies have shown that microinjected ribonuclease A is degraded to single amino acids entirely within lysosomes. We show that release of free amino acids and peptide fragments and/or intact protein was equivalently stimulated by serum deprivation and equivalently inhibited by NH4Cl. We also show that lysosomal degradation of endocytosed [3H]ribonuclease A was accompanied by the release of two peptide fragments similar in size and charge to those from microinjected [ 3 H]ribonuclease A. These findings demonstrate that degradation within lysosomes occurs in a manner that spares specific peptides; they also suggest a previously unsuspected pathway by which cells can secrete cytosol-derived polypeptides

Preclinical evaluation of sunitinib, a multi-tyrosine kinase inhibitor, as a radiosensitizer for human prostate cancer

International Nuclear Information System (INIS)

Brooks, Colin; Sheu, Tommy; Bridges, Kathleen; Mason, Kathy; Kuban, Deborah; Mathew, Paul; Meyn, Raymond

2012-01-01

Many prostate cancers demonstrate an increased expression of growth factor receptors such as vascular endothelial growth factor receptor (VEGFR) and platelet derived growth factor receptor (PDGFR) which have been correlated with increased resistance to radiotherapy and poor prognosis in other tumors. Therefore, response to radiation could potentially be improved by using inhibitors of these abnormally activated pathways. We have investigated the radiosensitizing effects of sunitinib, a potent, multi-tyrosine kinase inhibitor of the VEGFR and PDGFR receptors, on human prostate cancer cells. The radiosensitizing effects of sunitinib were assessed on human prostate cancer cell lines DU145, PC3 and LNCaP by clonogenic assay. Sunitinib’s ability to inhibit the activities of its key targets was determined by immunoblot analysis. The radiosensitizing effects of sunitinib in vivo were tested on human tumor xenografts growing in nude mice where response was assessed by tumor growth delay. Clonogenic survival curve assays for both DU145 and PC3 cells showed that the surviving fraction at 2 Gy was reduced from 0.70 and 0.52 in controls to 0.44 and 0.38, respectively, by a 24 hr pretreatment with 100 nM sunitinib. LNCaP cells were not radiosensitized by sunitinib. Dose dependent decreases in VEGFR and PDGFR activation were also observed following sunitinib in both DU145 and PC3 cells. We assessed the ability of sunitinib to radiosensitize PC3 xenograft tumors growing in the hind limb of nude mice. Sunitinib given concurrently with radiation did not prolong tumor growth delay. However, when animals were treated with sunitinib commencing the day after fractionated radiation was complete, tumor growth delay was enhanced compared to radiation alone. We conclude, based on the in vivo results, that sunitinib and radiation do not interact directly to radiosensitize the PC3 tumor cells in vivo as they did in vitro. The fact that tumor growth delay was enhanced when sunitinib was

Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI)

DEFF Research Database (Denmark)

Valnickova, Zuzana; Thaysen-Andersen, Morten; Højrup, Peter

2009-01-01

-activatable fibrinolysis inhibitor (TAFI), and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI. RESULTS: The four N-linked glycosylation sequons within the activation peptide were all occupied...

Effects of histone deacetylase inhibitors on regenerative cell responses in human dental pulp cells.

Science.gov (United States)

Luo, Z; Wang, Z; He, X; Liu, N; Liu, B; Sun, L; Wang, J; Ma, F; Duncan, H; He, W; Cooper, P

2017-04-04

To investigate the growth, migratory and adhesive effects of trichostatin A (TSA) and valproic acid (VPA), two histone deacetylase inhibitors (HDACis), on human dental pulp stem cells (hDPSCs). To verify that TSA or VPA functions as an HDAC inhibitor, the expressions of histones H3 and H4 were examined using Western blotting analysis. hDPSC growth and metabolic activity was evaluated by MTT viability analysis at different time-points and by cell count experiments. The expression of cell cycle regulatory proteins and apoptosis-associated proteins was examined by Western blot analysis. Migration effects were investigated using wound healing and transwell migration assays. An adhesion assay was also performed in the presence and absence of HDACis. The levels of chemokines and adhesion molecules relevant to repair in hDPSCs were also assessed by qRT-PCR and Western blot analysis. The data were analysed, where appropriate, using Student's t-test or one-way anova followed by the Student-Newman-Keuls test using SPSS software. Trichostatin A and VPA enhanced acetylation of histones H3 and H4 (P  0.05). At the same time, the expression of Cdx2 and cyclin A was upregulated by 2 nmol L -1 TSA and 1 mmol L -1 VPA (P < 0.05). Higher TSA or VPA concentrations induced apoptosis in hDPSCs in the cell count and apoptosis experiments (P < 0.05). Moreover, TSA and VPA significantly depressed the expression of Cdx2 and cyclin A (P < 0.05), whilst it significantly improved the level of p21 (P < 0.05). TSA and VPA promoted migration and adhesion of hDPSCs (P < 0.05). The levels of chemokines and adhesion molecules were significantly upregulated after exposure of hDPSCs to 20 nmol L -1 TSA or 1 mmol L -1 VPA (P < 0.05). Histone deacetylase inhibitors at specific concentrations promoted proliferation, migration and adhesion of hDPSCs, which may contribute to novel regenerative therapies for pulpal disease treatment. © 2017 International Endodontic Journal. Published

The binding mechanism of a peptidic cyclic serine protease inhibitor

DEFF Research Database (Denmark)

Jiang, Longguang; Svane, Anna Sigrid P.; Sørensen, Hans Peter

2011-01-01

Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries......, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical...... inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding...

Gene expression levels of matrix metalloproteinases in human atherosclerotic plaques and evaluation of radiolabeled inhibitors as imaging agents for plaque vulnerability

International Nuclear Information System (INIS)

Müller, Adrienne; Krämer, Stefanie D.; Meletta, Romana; Beck, Katharina; Selivanova, Svetlana V.; Rancic, Zoran; Kaufmann, Philipp A.; Vos, Bernhard; Meding, Jörg; Stellfeld, Timo; Heinrich, Tobias K.; Bauser, Marcus; Hütter, Joachim; Dinkelborg, Ludger M.; Schibli, Roger; Ametamey, Simon M.

2014-01-01

Introduction: Atherosclerotic plaque rupture is the primary cause for myocardial infarction and stroke. During plaque progression macrophages and mast cells secrete matrix-degrading proteolytic enzymes, such as matrix metalloproteinases (MMPs). We studied levels of MMPs and tissue inhibitor of metalloproteinases-3 (TIMP-3) in relation to the characteristics of carotid plaques. We evaluated in vitro two radiolabeled probes targeting active MMPs towards non-invasive imaging of rupture-prone plaques. Methods: Human carotid plaques obtained from endarterectomy were classified into stable and vulnerable by visual and histological analysis. MMP-1, MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MMP-14, TIMP-3, and CD68 levels were investigated by quantitative polymerase chain reaction. Immunohistochemistry was used to localize MMP-2 and MMP-9 with respect to CD68-expressing macrophages. Western blotting was applied to detect their active forms. A fluorine-18-labeled MMP-2/MMP-9 inhibitor and a tritiated selective MMP-9 inhibitor were evaluated by in vitro autoradiography as potential lead structures for non-invasive imaging. Results: Gene expression levels of all MMPs and CD68 were elevated in plaques. MMP-1, MMP-9, MMP-12 and MMP-14 were significantly higher in vulnerable than stable plaques. TIMP-3 expression was highest in stable and low in vulnerable plaques. Immunohistochemistry revealed intensive staining of MMP-9 in vulnerable plaques. Western blotting confirmed presence of the active form in plaque lysates. In vitro autoradiography showed binding of both inhibitors to stable and vulnerable plaques. Conclusions: MMPs differed in their expression patterns among plaque phenotypes, providing possible imaging targets. The two tested MMP-2/MMP-9 and MMP-9 inhibitors may be useful to detect atherosclerotic plaques, but not the vulnerable lesions selectively

Homologous Recombination in Protozoan Parasites and Recombinase Inhibitors

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Andrew A. Kelso

2017-09-01

Full Text Available Homologous recombination (HR is a DNA double-strand break (DSB repair pathway that utilizes a homologous template to fully repair the damaged DNA. HR is critical to maintain genome stability and to ensure genetic diversity during meiosis. A specialized class of enzymes known as recombinases facilitate the exchange of genetic information between sister chromatids or homologous chromosomes with the help of numerous protein accessory factors. The majority of the HR machinery is highly conserved among eukaryotes. In many protozoan parasites, HR is an essential DSB repair pathway that allows these organisms to adapt to environmental conditions and evade host immune systems through genetic recombination. Therefore, small molecule inhibitors, capable of disrupting HR in protozoan parasites, represent potential therapeutic options. A number of small molecule inhibitors were identified that disrupt the activities of the human recombinase RAD51. Recent studies have examined the effect of two of these molecules on the Entamoeba recombinases. Here, we discuss the current understandings of HR in the protozoan parasites Trypanosoma, Leishmania, Plasmodium, and Entamoeba, and we review the small molecule inhibitors known to disrupt human RAD51 activity.

A Novel Ras Inhibitor (MDC-1016 Reduces Human Pancreatic Tumor Growth in Mice

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Gerardo G Mackenzie

2013-10-01

Full Text Available Pancreatic cancer has one of the poorest prognoses among all cancers partly because of its persistent resistance to chemotherapy. The currently limited treatment options for pancreatic cancer underscore the need for more efficient agents. Because activating Kras mutations initiate and maintain pancreatic cancer, inhibition of this pathway should have a major therapeutic impact. We synthesized phospho-farnesylthiosalicylic acid (PFTS; MDC-1016 and evaluated its efficacy, safety, and metabolism in preclinical models of pancreatic cancer. PFTS inhibited the growth of human pancreatic cancer cells in culture in a concentration- and time-dependent manner. In an MIA PaCa-2 xenograft mouse model, PFTS at a dose of 50 and 100 mg/kg significantly reduced tumor growth by 62% and 65% (P < .05 vs vehicle control. Furthermore, PFTS prevented pancreatitis-accelerated acinar-to-ductal metaplasia in mice with activated Kras. PFTS appeared to be safe, with the animals showing no signs of toxicity during treatment. Following oral administration, PFTS was rapidly absorbed, metabolized to FTS and FTS glucuronide, and distributed through the blood to body organs. Mechanistically, PFTS inhibited Ras-GTP, the active form of Ras, both in vitro and in vivo, leading to the inhibition of downstream effector pathways c-RAF/mitogen-activated protein-extracellular signal-regulated kinase (ERK kinase (MEK/ERK1/2 kinase and phosphatidylinositol 3-kinase/AKT. In addition, PFTS proved to be a strong combination partner with phospho-valproic acid, a novel signal transducer and activator of transcription 3 (STAT3 inhibitor, displaying synergy in the inhibition of pancreatic cancer growth. In conclusion, PFTS, a direct Ras inhibitor, is an efficacious agent for the treatment of pancreatic cancer in preclinical models, deserving further evaluation.

Obatoclax, a Pan-BCL-2 Inhibitor, Targets Cyclin D1 for Degradation to Induce Antiproliferation in Human Colorectal Carcinoma Cells.

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Or, Chi-Hung R; Chang, Yachu; Lin, Wei-Cheng; Lee, Wee-Chyan; Su, Hong-Lin; Cheung, Muk-Wing; Huang, Chang-Po; Ho, Cheesang; Chang, Chia-Che

2016-12-27

Colorectal cancer is the third most common cancer worldwide. Aberrant overexpression of antiapoptotic BCL-2 (B-cell lymphoma 2) family proteins is closely linked to tumorigenesis and poor prognosis in colorectal cancer. Obatoclax is an inhibitor targeting all antiapoptotic BCL-2 proteins. A previous study has described the antiproliferative action of obatoclax in one human colorectal cancer cell line without elucidating the underlying mechanisms. We herein reported that, in a panel of human colorectal cancer cell lines, obatoclax inhibits cell proliferation, suppresses clonogenicity, and induces G₁-phase cell cycle arrest, along with cyclin D1 downregulation. Notably, ectopic cyclin D1 overexpression abrogated clonogenicity suppression but also G₁-phase arrest elicited by obatoclax. Mechanistically, pre-treatment with the proteasome inhibitor MG-132 restored cyclin D1 levels in all obatoclax-treated cell lines. Cycloheximide chase analyses further revealed an evident reduction in the half-life of cyclin D1 protein by obatoclax, confirming that obatoclax downregulates cyclin D1 through induction of cyclin D1 proteasomal degradation. Lastly, threonine 286 phosphorylation of cyclin D1, which is essential for initiating cyclin D1 proteasomal degradation, was induced by obatoclax in one cell line but not others. Collectively, we reveal a novel anticancer mechanism of obatoclax by validating that obatoclax targets cyclin D1 for proteasomal degradation to downregulate cyclin D1 for inducing antiproliferation.

Preparation, description and properties of {sup 3}H- DNA - {sup 131}I-ribonuclease complexes; Preparation, caracterisation et proprietes des complexes ADN{sup 3}H - RNase {sup 131}I

Energy Technology Data Exchange (ETDEWEB)

Tshitenge, G. [Centre nucléaire TRICO, Kinshasa (Congo, The Democratic Republic of the); Ledoux, L. [Centre d’étude de l' énergie nucléaire Mol (Belgium)

1970-01-15

Bacterial DNA, both radioactive and non-radioactive, and pancreatic ribonuclease labelled with iodine-131 are mixed together at a high ion strength and neutral pH. They are then dialysed against 0.009M sodium chloride. During dialysis a precipitate is formed, which is then separated with a centrifuge and redissolved at a neutral pH in a 0.1M phosphate buffer solution, or in 0.15M sodium chloride and 0.015M sodium citrate solution adjusted to pH 7. This solution is then analysed: (1) by DEAE-cellulose paper chromatography using a centrifuge; (2) by sedimentation of caesium chloride along a gradient; (3) by electrophoresis in agar gel. The results obtained show that a stable complex is formed between the DNA and ribonuclease. The properties of this complex are such that it can be compared with natural nucleoproteins. (author) [French] Des ADN bactériens radioactifs ou non et de la ribonucléase pancréatique marquée a l'iode-131 sont mélangés, à force ionique élevée et à pH neutre. Ils sont ensuite dialyses contre du NaCl 0.009M. Au cours de cette dialyse, un précipité se forme. Ce précipité est séparé par centrifugation et redissous à pH neutre dans du tampon phosphate 0,1M, ou dans une solution 0.015M en citrate de Na et 0,15M en NaCl ajustée à pH 7. Cette solution a été analysée: 1) par Chromatographie centrifugée sur pulpe de papier de DEAE- cellulose; 2) par sédimentation en gradient de CsCl; 3) par électrophorèse en gel d'agar. Les résultats obtenus montrent qu'un complexe stable sfest formé entre l'ADN et la RNase. Les propriétés de ce complexe permettent de le comparer aux nucléoprotéines naturelles. (author)

Identification of small molecule inhibitors of Pseudomonas aeruginosa exoenzyme S using a yeast phenotypic screen.

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Anthony Arnoldo

2008-02-01

Full Text Available Pseudomonas aeruginosa is an opportunistic human pathogen that is a key factor in the mortality of cystic fibrosis patients, and infection represents an increased threat for human health worldwide. Because resistance of Pseudomonas aeruginosa to antibiotics is increasing, new inhibitors of pharmacologically validated targets of this bacterium are needed. Here we demonstrate that a cell-based yeast phenotypic assay, combined with a large-scale inhibitor screen, identified small molecule inhibitors that can suppress the toxicity caused by heterologous expression of selected Pseudomonas aeruginosa ORFs. We identified the first small molecule inhibitor of Exoenzyme S (ExoS, a toxin involved in Type III secretion. We show that this inhibitor, exosin, modulates ExoS ADP-ribosyltransferase activity in vitro, suggesting the inhibition is direct. Moreover, exosin and two of its analogues display a significant protective effect against Pseudomonas infection in vivo. Furthermore, because the assay was performed in yeast, we were able to demonstrate that several yeast homologues of the known human ExoS targets are likely ADP-ribosylated by the toxin. For example, using an in vitro enzymatic assay, we demonstrate that yeast Ras2p is directly modified by ExoS. Lastly, by surveying a collection of yeast deletion mutants, we identified Bmh1p, a yeast homologue of the human FAS, as an ExoS cofactor, revealing that portions of the bacterial toxin mode of action are conserved from yeast to human. Taken together, our integrated cell-based, chemical-genetic approach demonstrates that such screens can augment traditional drug screening approaches and facilitate the discovery of new compounds against a broad range of human pathogens.

Molecular insights into human monoamine oxidase (MAO) inhibition by 1,4-naphthoquinone: evidences for menadione (vitamin K3) acting as a competitive and reversible inhibitor of MAO.

Science.gov (United States)

Coelho Cerqueira, Eduardo; Netz, Paulo Augusto; Diniz, Cristiane; Petry do Canto, Vanessa; Follmer, Cristian

2011-12-15

Monoamine oxidase (MAO) catalyzes the oxidative deamination of biogenic and exogenous amines and its inhibitors have therapeutic value for several conditions including affective disorders, stroke, neurodegenerative diseases and aging. The discovery of 2,3,6-trimethyl-1,4-naphthoquinone (TMN) as a nonselective and reversible inhibitor of MAO, has suggested 1,4-naphthoquinone (1,4-NQ) as a potential scaffold for designing new MAO inhibitors. Combining molecular modeling tools and biochemical assays we evaluate the kinetic and molecular details of the inhibition of human MAO by 1,4-NQ, comparing it with TMN and menadione. Menadione (2-methyl-1,4-naphthoquinone) is a multitarget drug that acts as a precursor of vitamin K and an inducer of mitochondrial permeability transition. Herein we show that MAO-B was inhibited competitively by 1,4-NQ (K(i)=1.4 μM) whereas MAO-A was inhibited by non-competitive mechanism (K(i)=7.7 μM). Contrasting with TMN and 1,4-NQ, menadione exhibited a 60-fold selectivity for MAO-B (K(i)=0.4 μM) in comparison with MAO-A (K(i)=26 μM), which makes it as selective as rasagiline. Fluorescence and molecular modeling data indicated that these inhibitors interact with the flavin moiety at the active site of the enzyme. Additionally, docking studies suggest the phenyl side groups of Tyr407 and Tyr444 (for MAO-A) or Tyr398 and Tyr435 (for MAO-B) play an important role in the interaction of the enzyme with 1,4-NQ scaffold through forces of dispersion as verified for menadione, TMN and 1,4-NQ. Taken together, our findings reveal the molecular details of MAO inhibition by 1,4-NQ scaffold and show for the first time that menadione acts as a competitive and reversible inhibitor of human MAO. Copyright © 2011 Elsevier Ltd. All rights reserved.

The Metabolism of Separase Inhibitor Sepin-1 in Human, Mouse, and Rat Liver Microsomes

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Feng Li

2018-05-01

Full Text Available Separase, a known oncogene, is widely overexpressed in numerous human tumors of breast, bone, brain, blood, and prostate. Separase is an emerging target for cancer therapy, and separase enzymatic inhibitors such as sepin-1 are currently being developed to treat separase-overexpressed tumors. Drug metabolism plays a critical role in the efficacy and safety of drug development, as well as possible drug–drug interactions. In this study, we investigated the in vitro metabolism of sepin-1 in human, mouse, and rat liver microsomes (RLM using metabolomic approaches. In human liver microsomes (HLM, we identified seven metabolites including one cysteine–sepin-1 adduct and one glutathione–sepin-1 adduct. All the sepin-1 metabolites in HLM were also found in both mouse and RLM. Using recombinant CYP450 isoenzymes, we demonstrated that multiple enzymes contributed to the metabolism of sepin-1, including CYP2D6 and CYP3A4 as the major metabolizing enzymes. Inhibitory effects of sepin-1 on seven major CYP450s were also evaluated using the corresponding substrates recommended by the US Food and Drug Administration. Our studies indicated that sepin-1 moderately inhibits CYP1A2, CYP2C19, and CYP3A4 with IC50 < 10 μM but weakly inhibits CYP2B6, CYP2C8/9, and CYP2D6 with IC50 > 10 μM. This information can be used to optimize the structures of sepin-1 for more suitable pharmacological properties and to predict the possible sepin-1 interactions with other chemotherapeutic drugs.

A Ribonuclease Isolated from Wild Ganoderma Lucidum Suppressed Autophagy and Triggered Apoptosis in Colorectal Cancer Cells

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Xiuli Dan

2016-07-01

Full Text Available The mushroom Ganoderma lucidum (G. lucidum has been consumed in China as a medicine for promoting health and longevity for thousands of years. Due to its paramount and multiple pharmaceutical effects, G. lucidum has received considerable attention from researchers and its chemical constituents as well as their respective functions were gradually unveiled by using modern research methods. Herein, we reported the isolation of a protein (GLR with anti-colorectal cancer activities from G. lucidum. This protein is a 17.4-kDa RNA degrading enzyme (ribonuclease and was purified by using liquid chromatography procedures. GLR manifested potent anti-proliferative and anti-colony formation activities on HT29 and HCT116 colorectal cancer cells by inducing cell cycle arrest in G1 phase through the regulation of cyclin D1 and P53 expression. GLR was demonstrated to induce cell apoptosis in HCT116 cells by activating unfolded protein response and caspase-9 regulated pathways. Besides, the ability to undergo autophagy which is a stress adaption mechanism to cope with metabolic crisis was significantly suppressed by GLR treatment in HCT116 cells. The activation of apoptosis in GLR-treated HT29 cells was, however, independent of caspase-9 and the suppression of autophagy was also relatively minor. Thus the apoptosis of HT29 cells triggered by GLR was much milder than that in HCT116 cells. Our findings show that the RNase from G. lucidum may be one of the bioactive components that contribute to the anti-colorectal cancer activity of G. lucidum.

The tyrosine kinase inhibitor dasatinib induces a marked adipogenic differentiation of human multipotent mesenchymal stromal cells.

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Adriana Borriello

Full Text Available BACKGROUND: The introduction of specific BCR-ABL inhibitors in chronic myelogenous leukemia therapy has entirely mutated the prognosis of this hematologic cancer from being a fatal disorder to becoming a chronic disease. Due to the probable long lasting treatment with tyrosine-kinase inhibitors (TKIs, the knowledge of their effects on normal cells is of pivotal importance. DESIGN AND METHODS: We investigated the effects of dasatinib treatment on human bone marrow-derived mesenchymal stromal cells (MSCs. RESULTS: Our findings demonstrate, for the first time, that dasatinib induces MSCs adipocytic differentiation. Particularly, when the TKI is added to the medium inducing osteogenic differentiation, a high MSCs percentage acquires adipocytic morphology and overexpresses adipocytic specific genes, including PPARγ, CEBPα, LPL and SREBP1c. Dasatinib also inhibits the activity of alkaline phosphatase, an osteogenic marker, and remarkably reduces matrix mineralization. The increase of PPARγ is also confirmed at protein level. The component of osteogenic medium required for dasatinib-induced adipogenesis is dexamethasone. Intriguingly, the increase of adipocytic markers is also observed in MSCs treated with dasatinib alone. The TKI effect is phenotype-specific, since fibroblasts do not undergo adipocytic differentiation or PPARγ increase. CONCLUSIONS: Our data demonstrate that dasatinib treatment affects bone marrow MSCs commitment and suggest that TKIs therapy might modify normal phenotypes with potential significant negative consequences.

Inhibitin: a specific inhibitor of sodium/sodium exchange in erythrocytes.

OpenAIRE

Morgan, K; Brown, R C; Spurlock, G; Southgate, K; Mir, M A

1986-01-01

An inhibitor of ouabain-insensitive sodium/sodium exchange in erythrocytes has been isolated from leukemic promyelocytes. To explore the specific effects of this inhibitor, named inhibitin, sodium transport experiments were carried out in human erythrocytes. Inhibitin reduced ouabain-insensitive bidirectional sodium transport. It did not change net sodium fluxes, had no significant effect on rubidium influx, and did not inhibit sodium-potassium-ATPase activity. The inhibitory effect of inhibi...

Designing Second Generation Anti-Alzheimer Compounds as Inhibitors of Human Acetylcholinesterase: Computational Screening of Synthetic Molecules and Dietary Phytochemicals.

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Hafsa Amat-Ur-Rasool

Full Text Available Alzheimer's disease (AD, a big cause of memory loss, is a progressive neurodegenerative disorder. The disease leads to irreversible loss of neurons that result in reduced level of acetylcholine neurotransmitter (ACh. The reduction of ACh level impairs brain functioning. One aspect of AD therapy is to maintain ACh level up to a safe limit, by blocking acetylcholinesterase (AChE, an enzyme that is naturally responsible for its degradation. This research presents an in-silico screening and designing of hAChE inhibitors as potential anti-Alzheimer drugs. Molecular docking results of the database retrieved (synthetic chemicals and dietary phytochemicals and self-drawn ligands were compared with Food and Drug Administration (FDA approved drugs against AD as controls. Furthermore, computational ADME studies were performed on the hits to assess their safety. Human AChE was found to be most approptiate target site as compared to commonly used Torpedo AChE. Among the tested dietry phytochemicals, berberastine, berberine, yohimbine, sanguinarine, elemol and naringenin are the worth mentioning phytochemicals as potential anti-Alzheimer drugs The synthetic leads were mostly dual binding site inhibitors with two binding subunits linked by a carbon chain i.e. second generation AD drugs. Fifteen new heterodimers were designed that were computationally more efficient inhibitors than previously reported compounds. Using computational methods, compounds present in online chemical databases can be screened to design more efficient and safer drugs against cognitive symptoms of AD.

Designing Second Generation Anti-Alzheimer Compounds as Inhibitors of Human Acetylcholinesterase: Computational Screening of Synthetic Molecules and Dietary Phytochemicals.

Science.gov (United States)

Amat-Ur-Rasool, Hafsa; Ahmed, Mehboob

2015-01-01

Alzheimer's disease (AD), a big cause of memory loss, is a progressive neurodegenerative disorder. The disease leads to irreversible loss of neurons that result in reduced level of acetylcholine neurotransmitter (ACh). The reduction of ACh level impairs brain functioning. One aspect of AD therapy is to maintain ACh level up to a safe limit, by blocking acetylcholinesterase (AChE), an enzyme that is naturally responsible for its degradation. This research presents an in-silico screening and designing of hAChE inhibitors as potential anti-Alzheimer drugs. Molecular docking results of the database retrieved (synthetic chemicals and dietary phytochemicals) and self-drawn ligands were compared with Food and Drug Administration (FDA) approved drugs against AD as controls. Furthermore, computational ADME studies were performed on the hits to assess their safety. Human AChE was found to be most approptiate target site as compared to commonly used Torpedo AChE. Among the tested dietry phytochemicals, berberastine, berberine, yohimbine, sanguinarine, elemol and naringenin are the worth mentioning phytochemicals as potential anti-Alzheimer drugs The synthetic leads were mostly dual binding site inhibitors with two binding subunits linked by a carbon chain i.e. second generation AD drugs. Fifteen new heterodimers were designed that were computationally more efficient inhibitors than previously reported compounds. Using computational methods, compounds present in online chemical databases can be screened to design more efficient and safer drugs against cognitive symptoms of AD.

Vorinostat, an HDAC inhibitor attenuates epidermoid squamous cell carcinoma growth by dampening mTOR signaling pathway in a human xenograft murine model.

Science.gov (United States)

Kurundkar, Deepali; Srivastava, Ritesh K; Chaudhary, Sandeep C; Ballestas, Mary E; Kopelovich, Levy; Elmets, Craig A; Athar, Mohammad

2013-01-15

Histone deacetylase (HDAC) inhibitors are potent anticancer agents and show efficacy against various human neoplasms. Vorinostat is a potent HDAC inhibitor and has shown potential to inhibit growth of human xenograft tumors. However, its effect on the growth of skin neoplasm remains undefined. In this study, we show that vorinostat (2 μM) reduced expression of HDAC1, 2, 3, and 7 in epidermoid carcinoma A431 cells. Consistently, it increased acetylation of histone H3 and p53. Vorinostat (100mg/kg body weight, IP) treatment reduced human xenograft tumor growth in highly immunosuppressed nu/nu mice. Histologically, the vorinostat-treated tumor showed features of well-differentiation with large necrotic areas. Based on proliferating cell nuclear antigen (PCNA) staining and expression of cyclins D1, D2, E, and A, vorinostat seems to impair proliferation by down-regulating the expression of these proteins. However, it also induced apoptosis. The mechanism by which vorinostat blocks proliferation and makes tumor cells prone to apoptosis, involved inhibition of mTOR signaling which was accompanied by reduction in cell survival AKT and extracellular-signal regulated kinase (ERK) signaling pathways. Our data provide a novel mechanism-based therapeutic intervention for cutaneous squamous cell carcinoma (SCC). Vorinostat may be utilized to cure skin neoplasms in organ transplant recipient (OTR). These patients have high morbidity and surgical removal of these lesions which frequently develop in these patients, is difficult. Copyright © 2012 Elsevier Inc. All rights reserved.

Frequent expression loss of Inter-alpha-trypsin inhibitor heavy chain (ITIH) genes in multiple human solid tumors: A systematic expression analysis

International Nuclear Information System (INIS)

Hamm, Alexander; Knuechel, Ruth; Dahl, Edgar; Veeck, Juergen; Bektas, Nuran; Wild, Peter J; Hartmann, Arndt; Heindrichs, Uwe; Kristiansen, Glen; Werbowetski-Ogilvie, Tamra; Del Maestro, Rolando

2008-01-01

The inter-alpha-trypsin inhibitors (ITI) are a family of plasma protease inhibitors, assembled from a light chain – bikunin, encoded by AMBP – and five homologous heavy chains (encoded by ITIH1, ITIH2, ITIH3, ITIH4, and ITIH5), contributing to extracellular matrix stability by covalent linkage to hyaluronan. So far, ITIH molecules have been shown to play a particularly important role in inflammation and carcinogenesis. We systematically investigated differential gene expression of the ITIH gene family, as well as AMBP and the interacting partner TNFAIP6 in 13 different human tumor entities (of breast, endometrium, ovary, cervix, stomach, small intestine, colon, rectum, lung, thyroid, prostate, kidney, and pancreas) using cDNA dot blot analysis (Cancer Profiling Array, CPA), semiquantitative RT-PCR and immunohistochemistry. We found that ITIH genes are clearly downregulated in multiple human solid tumors, including breast, colon and lung cancer. Thus, ITIH genes may represent a family of putative tumor suppressor genes that should be analyzed in greater detail in the future. For an initial detailed analysis we chose ITIH2 expression in human breast cancer. Loss of ITIH2 expression in 70% of cases (n = 50, CPA) could be confirmed by real-time PCR in an additional set of breast cancers (n = 36). Next we studied ITIH2 expression on the protein level by analyzing a comprehensive tissue micro array including 185 invasive breast cancer specimens. We found a strong correlation (p < 0.001) between ITIH2 expression and estrogen receptor (ER) expression indicating that ER may be involved in the regulation of this ECM molecule. Altogether, this is the first systematic analysis on the differential expression of ITIH genes in human cancer, showing frequent downregulation that may be associated with initiation and/or progression of these malignancies

Exploration of natural product ingredients as inhibitors of human HMG-CoA reductase through structure-based virtual screening.

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Lin, Shih-Hung; Huang, Kao-Jean; Weng, Ching-Feng; Shiuan, David

2015-01-01

Cholesterol plays an important role in living cells. However, a very high level of cholesterol may lead to atherosclerosis. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase is the key enzyme in the cholesterol biosynthesis pathway, and the statin-like drugs are inhibitors of human HMG-CoA reductase (hHMGR). The present study aimed to virtually screen for potential hHMGR inhibitors from natural product to discover hypolipidemic drug candidates with fewer side effects and lesser toxicities. We used the 3D structure 1HWK from the PDB (Protein Data Bank) database of hHMGR as the target to screen for the strongly bound compounds from the traditional Chinese medicine database. Many interesting molecules including polyphenolic compounds, polisubstituted heterocyclics, and linear lipophilic alcohols were identified and their ADMET (absorption, disrtibution, metabolism, excretion, toxicity) properties were predicted. Finally, four compounds were obtained for the in vitro validation experiments. The results indicated that curcumin and salvianolic acid C can effectively inhibit hHMGR, with IC50 (half maximal inhibitory concentration) values of 4.3 µM and 8 µM, respectively. The present study also demonstrated the feasibility of discovering new drug candidates through structure-based virtual screening.

Crystal structure of a novel cysteinless plant Kunitz-type protease inhibitor

International Nuclear Information System (INIS)

Hansen, Daiane; Macedo-Ribeiro, Sandra; Verissimo, Paula; Yoo Im, Sonia; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela

2007-01-01

Bauhinia bauhinioides Cruzipain Inhibitor (BbCI) is a cysteine protease inhibitor highly homologous to plant Kunitz-type inhibitors. However, in contrast to classical Kunitz family inhibitors it lacks cysteine residues and therefore disulfide bridges. BbCI is also distinct in the ability to inactivate enzymes belonging to two different classes, cysteine and serine proteases. Besides inhibiting the cysteine protease cruzipain, BbCI also inhibits cathepsin L and the serine proteases HNE (human neutrophil elastase) and PPE (porcine pancreatic elastase). Monoclinic crystals of the recombinant inhibitor that diffract to 1.7 A resolution were obtained using hanging drop method by vapor diffusion at 18 o C. The refined structure shows the conservative β-trefoil fold features of the Kunitz inhibitors. In BbCI, one of the two characteristic S-S bonds is replaced by the water-mediated interaction between Tyr125 and Gly132. In this work we explore the structural differences between Kunitz-type inhibitors and analyze the essential interactions that maintain the protein structural stability preserving its biological function

Comparison of responses of human melanoma cell lines to MEK and BRAF inhibitors

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Clare Judith Stones

2013-05-01

Full Text Available The NRAS and BRAF genes are frequently mutated in melanoma, suggesting that the NRAS-BRAF-MEK-ERK signalling pathway is an important target for therapy. Two classes of drugs, one targeting activated BRAF and one targeting MEK, are currently undergoing clinical evaluation. We have analysed the NRAS and BRAF mutational status of a series of 44 early passage lines developed from New Zealand patients with metastatic melanoma. 41% of the lines analysed had BRAF mutations, 23% had NRAS mutations and 36% had neither. We then determined IC50 values (drug concentrations for 50% growth inhibition for CI-1040, a commonly used inhibitor of MEK kinase; trametinib, a clinical agent targeting MEK kinase; and vemurafenib, an inhibitor of mutant BRAF kinase. Cell lines with activating BRAF mutations were significantly more sensitive to vemurafenib than lines with NRAS mutations or lines lacking either mutation (p < 0.001. IC50 values for CI-1040 and trametinib were strongly correlated (r = 0.98 with trametinib showing ~100-fold greater potency. Cell lines sensitive to vemurafenib were also sensitive to CI-1040 and trametinib, but there was no relationship between IC50 values and NRAS mutation status. A small number of lines lacking a BRAF mutation were sensitive to CI-1040 but resistant to vemurafenib. We used western blotting to investigate the effect on ERK phosphorylation of CI-1040 in four lines, of vemurafenib in two lines and of trametinib in two lines. The results support the view that MEK inhibitors might be combined with BRAF inhibitors in the treatment melanomas of with activated BRAF. The high sensitivity to trametinib of some lines with wild-type BRAF status also suggests that MEK inhibitors could have a therapeutic effect against some melanomas as single agents.

Identification of a human protein-derived HIV-1 fusion inhibitor targeting the gp41 fusion core structure.

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Lijun Chao

Full Text Available The HIV-1 envelope glycoprotein (Env gp41 plays a crucial role in the viral fusion process. The peptides derived from the C-terminal heptad repeat (CHR of gp41 are potent HIV fusion inhibitors. However, the activity of these anti-HIV-1 peptides in vivo may be attenuated by their induction of anti-gp41 antibodies. Thus, it is essential to identify antiviral peptides or proteins with low, or no, immunogenicity to humans. Here, we found that the C-terminal fragment (aa 462-521 of the human POB1 (the partner of RalBP1, designated C60, is an HIV-1 fusion inhibitor. It bound to N36, the peptide derived from the N-terminal heptad repeat (NHR of gp41, and to the six-helix bundle (6-HB formed by N36 and C34, a CHR-peptide, but it did not bind to C34. Unlike the CHR-peptides, C60 did not block gp41 6-HB formation. Rather, results suggest that C60 inhibits HIV-1 fusion by binding to the 6-HB, in particular, the residues in the gp41 NHR domain that are exposed on the surface of 6-HB. Since 6-HB plays a crucial role in the late stage of fusion between the viral envelope and endosomal membrane during the endocytic process of HIV-1, C60 may serve as a host restriction factor to suppress HIV-1 entry into CD4+ T lymphocytes. Taken together, it can be concluded from these results that C60 can be used as a lead for the development of anti-HIV-1 therapeutics or microbicides for the treatment and prevention of HIV-1 infection, as well as a molecular probe to study the fusogenic mechanism of HIV-1.

Design and synthesis of novel chalcones as potent selective monoamine oxidase-B inhibitors.

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Hammuda, Arwa; Shalaby, Raed; Rovida, Stefano; Edmondson, Dale E; Binda, Claudia; Khalil, Ashraf

2016-05-23

A novel series of substituted chalcones were designed and synthesized to be evaluated as selective human MAO-B inhibitors. A combination of either methylsulfonyl or trifluoromethyl substituents on the aromatic ketone moiety with a benzodioxol ring on the other end of the chalcone scaffold was investigated. The compounds were tested for their inhibitory activities on both human MAO-A and B. All compounds appeared to be selective MAO-B inhibitors with Ki values in the micromolar to submicromolar range. Molecular modeling studies have been performed to get insight into the binding mode of the synthesized compounds to human MAO-B active site. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells.

Science.gov (United States)

Liu, Quan; Liu, Juan; Roschmann, Kristina Irene Lisolette; van Egmond, Danielle; Golebski, Korneliusz; Fokkens, Wytske Johanna; Wang, Dehui; van Drunen, Cornelis Maria

2013-04-11

HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL37 in human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells(PNEC) in response to HDAC inhibitors with or without poly (I:C) stimulation was assessed using real-time PCR and western blot. In parallel, IL-6 expression was evaluated by ELISA. Our results showed that HDAC inhibitors up-regulated LL-37 gene expression independent of poly (I:C) stimulation in PNEC as well as in NCI-H292 cells. HDAC inhibitors increased LL37 protein expression in NCI-H292 cells but not in PNEC. In addition, HDAC inhibitors significantly inhibited poly (I:C)-induced IL-6 production in both of the epithelial cells. In conclusion, HDAC inhibitors directly up-regulated LL-37 gene expression in human airway epithelial cells.

Checkpoint inhibitors in cancer immunotherapy: Cross reactivity of a CTLA-4 antibody and IDO-inhibitor L-1MT in pigs

DEFF Research Database (Denmark)

Al-Shatrawi, Zina Adil; Frøsig, Thomas Mørch; Jungersen, Gregers

a non-specific activation of porcine T cells. This will be further investigated to provide the basis for in vivo studies investigating checkpoint inhibitor blockade in combination with other cancer immunotherapies. Eventually our goal is to establish pigs as an alternative large animal model......Blockade of checkpoint inhibitors has recently shown very convincing results in the treatment of cancer. One key target is CTLA-4, which has been demonstrated to be a potent negative regulator of lymphocyte activation. The treatment with the FDA-approved fully human CTLA-4 monoclonal antibody...... Ipilimumab increases anticancer T-cell reactivity and overall survival of metastatic cancer patients. Indole-amine 2,3-dioxygenase (IDO) is another checkpoint inhibitor which suppresses T-cell immunity by the depletion of tryptophan in the T-cell microenvironment, and also inhibition of IDO by L-1...

Selective Serotonin Reuptake Inhibitors in Human Pregnancy: To Treat or Not to Treat?

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Orna Diav-Citrin

2012-01-01

Full Text Available Selective serotonin reuptake inhibitors (SSRIs are increasingly prescribed during pregnancy. The purpose of the present paper is to summarize and evaluate the current evidence for the risk/benefit analysis of SSRI use in human pregnancy. The literature has been inconsistent. Although most studies have not shown an increase in the overall risk of major malformations, several studies have suggested that SSRIs may be associated with a small increased risk for cardiovascular malformations. Others have noted associations between SSRIs and specific types of rare major malformations. In some studies, there appears to be a small increased risk for miscarriages, which may be associated with the underlying maternal condition. Neonatal effects have been described in up to 30% of neonates exposed to SSRIs late in pregnancy. Persistent pulmonary hypertension of the newborn has also been described with an absolute risk of <1%. The risk associated with treatment discontinuation, for example, higher frequency of relapse and increased risk of preterm delivery, should also be considered. The overall benefit of treatment seems to outweigh the potential risks.

Herpes simplex virus downregulation of secretory leukocyte protease inhibitor enhances human papillomavirus type 16 infection.

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Skeate, Joseph G; Porras, Tania B; Woodham, Andrew W; Jang, Julie K; Taylor, Julia R; Brand, Heike E; Kelly, Thomas J; Jung, Jae U; Da Silva, Diane M; Yuan, Weiming; Kast, W Martin

2016-02-01

Herpes simplex virus (HSV) was originally implicated in the aetiology of cervical cancer, and although high-risk human papillomavirus (HPV) is now the accepted causative agent, the epidemiological link between HSV and HPV-associated cancers persists. The annexin A2 heterotetramer (A2t) has been shown to mediate infectious HPV type 16 (HPV16) uptake by human keratinocytes, and secretory leukocyte protease inhibitor (SLPI), an endogenous A2t ligand, inhibits HPV16 uptake and infection. Interestingly, HSV infection induces a sustained downregulation of SLPI in epithelial cells, which we hypothesized promotes HPV16 infection through A2t. Here, we show that in vitro infection of human keratinocytes with HSV-1 or HSV-2, but not with an HSV-1 ICP4 deletion mutant that does not downregulate SLPI, leads to a >70% reduction of SLPI mRNA and a >60% decrease in secreted SLPI protein. Consequently, we observed a significant increase in the uptake of HPV16 virus-like particles and gene transduction by HPV16 pseudovirions (two- and 2.5-fold, respectively) in HSV-1- and HSV-2-infected human keratinocyte cell cultures compared with uninfected cells, whereas exogenously added SLPI reversed this effect. Using a SiMPull (single-molecule pulldown) assay, we demonstrated that endogenously secreted SLPI interacts with A2t on epithelial cells in an autocrine/paracrine manner. These results suggested that ongoing HSV infection and resultant downregulation of local levels of SLPI may impart a greater susceptibility for keratinocytes to HPV16 infection through the host cell receptor A2t, providing a mechanism that may, in part, provide an explanation for the aetiological link between HSV and HPV-associated cancers.

Selective kallikrein inhibitors alter human neutrophil elastase release during extracorporeal circulation

NARCIS (Netherlands)

Wachtfogel, Y.T.; Hack, C.E.; Nuijens, J.H; Kettner, C.; Reilly, T.M.; Knabb, R.M.; Bischoff, Rainer; Tschesche, H.; Wenzel, H.; Kucich, U.

1995-01-01

Cardiopulmonary bypass causes hemorrhagic complications and initiates a biochemical and cellular "whole body inflammatory response." This study investigates whether a variety of selective inhibitors of the contact pathway of intrinsic coagulation modulate complement and neutrophil activation during

Janus Associated Kinases Inhibitors in the Pharmacological Thera

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Daniela Santos1

2017-01-01

Full Text Available Janus associated kinases inhibitors are a new strategy for the treatment of different clinical conditions like immunologic, inflammatory and oncology disorders. The aim of this study was to perform a review of all Janus associated kinases inhibitors available in national and international pharmaceutical market, their therapeutic indications and adverse effects, and the potential indications for investigation of those already available in the pharmaceutical market. It was also performed a review of the main new Janus associated kinases inhibitors that are still in clinical research. A literature review was conducted by consulting the summary of product characteristics of Janus associated kinases inhibitors available in the pharmaceutical market and a research in the bibliographic database PubMed using the terms «JAK inhibitors», «Janus associated kinases inhibitors» and «Janus kinases inhibitors». Ninety-five publications were included in the present review, published from January 2014 to January 2015. Drug databases of the European Medicines Agency and United States Food and Drug Administration were also consulted to search for Janus associated kinases inhibitors authorized in clinical practice. Currently, ruxolitinib and tofacitinib are available in the pharmaceutical market and oclatinib is approved as a veterinary medicinal product. Both drugs approved for human use have major adverse effects at hematological and immunological levels, which enhance the importance of the pharmacist’s role in the monitoring of patients involved in these treatments. However, several molecules are in pre-clinical and clinical studies trying to prove its potential in the treatment of several immunologic, inflammatory and oncology disorders. Thus, it is still necessary to deepen the knowledge in this area in order to overcome the risks of therapy with these agents. These risks weighed against the benefits of its clinical use have compromised the progress of

Expression and localization of tissue factor pathway inhibitor-2 in normal and atherosclerotic human vessels

NARCIS (Netherlands)

Crawley, James T. B.; Goulding, David A.; Ferreira, Valérie; Severs, Nicholas J.; Lupu, Florea

2002-01-01

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type, serine protease inhibitor with inhibitory activity toward activated factor XI, plasma kallikrein, plasmin, certain matrix metalloproteinases, and the tissue factor:activated factor VII complex. In this study, we investigated TFPI-2

Reversible dual inhibitor against G9a and DNMT1 improves human iPSC derivation enhancing MET and facilitating transcription factor engagement to the genome.

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Juan Roberto Rodriguez-Madoz

Full Text Available The combination of defined factors with small molecules targeting epigenetic factors is a strategy that has been shown to enhance optimal derivation of iPSCs and could be used for disease modelling, high throughput screenings and/or regenerative medicine applications. In this study, we showed that a new first-in-class reversible dual G9a/DNMT1 inhibitor compound (CM272 improves the efficiency of human cell reprogramming and iPSC generation from primary cells of healthy donors and patient samples, using both integrative and non-integrative methods. Moreover, CM272 facilitates the generation of human iPSC with only two factors allowing the removal of the most potent oncogenic factor cMYC. Furthermore, we demonstrated that mechanistically, treatment with CM272 induces heterochromatin relaxation, facilitates the engagement of OCT4 and SOX2 transcription factors to OSKM refractory binding regions that are required for iPSC establishment, and enhances mesenchymal to epithelial transition during the early phase of cell reprogramming. Thus, the use of this new G9a/DNMT reversible dual inhibitor compound may represent an interesting alternative for improving cell reprogramming and human iPSC derivation for many different applications while providing interesting insights into reprogramming mechanisms.

Reversible dual inhibitor against G9a and DNMT1 improves human iPSC derivation enhancing MET and facilitating transcription factor engagement to the genome.

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Rodriguez-Madoz, Juan Roberto; San Jose-Eneriz, Edurne; Rabal, Obdulia; Zapata-Linares, Natalia; Miranda, Estibaliz; Rodriguez, Saray; Porciuncula, Angelo; Vilas-Zornoza, Amaia; Garate, Leire; Segura, Victor; Guruceaga, Elizabeth; Agirre, Xabier; Oyarzabal, Julen; Prosper, Felipe

2017-01-01

The combination of defined factors with small molecules targeting epigenetic factors is a strategy that has been shown to enhance optimal derivation of iPSCs and could be used for disease modelling, high throughput screenings and/or regenerative medicine applications. In this study, we showed that a new first-in-class reversible dual G9a/DNMT1 inhibitor compound (CM272) improves the efficiency of human cell reprogramming and iPSC generation from primary cells of healthy donors and patient samples, using both integrative and non-integrative methods. Moreover, CM272 facilitates the generation of human iPSC with only two factors allowing the removal of the most potent oncogenic factor cMYC. Furthermore, we demonstrated that mechanistically, treatment with CM272 induces heterochromatin relaxation, facilitates the engagement of OCT4 and SOX2 transcription factors to OSKM refractory binding regions that are required for iPSC establishment, and enhances mesenchymal to epithelial transition during the early phase of cell reprogramming. Thus, the use of this new G9a/DNMT reversible dual inhibitor compound may represent an interesting alternative for improving cell reprogramming and human iPSC derivation for many different applications while providing interesting insights into reprogramming mechanisms.

Methods Of Using Chemical Libraries To Search For New Kinase Inhibitors

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Gray, Nathanael S. , Schultz, Peter , Wodicka, Lisa , Meijer, Laurent , Lockhart, David J.

2003-06-03

The generation of selective inhibitors for specific protein kinases would provide new tools for analyzing signal transduction pathways and possibly new therapeutic agents. We have invented an approach to the development of selective protein kinase inhibitors based on the unexpected binding mode of 2,6,9-trisubstituted purines to the ATP binding site of human CDK2. The most potent inhibitor, purvalanol B (IC.sub.50 =6 nM), binds with a 30-fold greater affinity than the known CDK2 inhibitor, flavopiridol. The cellular effects of this class of compounds were examined and compared to those of flavopiridol by monitoring changes in mRNA expression levels for all genes in treated cells of Saccharomyces cerevisiae using high-density oligonucleotide probe arrays.

Molecularly imprinted nanoparticles for inhibiting ribonuclease in reverse transcriptase polymerase chain reaction

DEFF Research Database (Denmark)

Feng, Xiaotong; Ashley, Jon; Zhou, Tongchang

2018-01-01

Molecularly imprinted nanoparticles (nanoMIPs) are synthesized via a solid-phase approach using RNase as the template. The feasibility of employing the nanoMIPs as RNase inhibitor is successfully demonstrated in reverse transcriptase polymerase chain reaction (RT-PCR) assays, suggesting the tailor...

A preclinical orthotopic model for glioblastoma recapitulates key features of human tumors and demonstrates sensitivity to a combination of MEK and PI3K pathway inhibitors.

Science.gov (United States)

El Meskini, Rajaa; Iacovelli, Anthony J; Kulaga, Alan; Gumprecht, Michelle; Martin, Philip L; Baran, Maureen; Householder, Deborah B; Van Dyke, Terry; Weaver Ohler, Zoë

2015-01-01

Current therapies for glioblastoma multiforme (GBM), the highest grade malignant brain tumor, are mostly ineffective, and better preclinical model systems are needed to increase the successful translation of drug discovery efforts into the clinic. Previous work describes a genetically engineered mouse (GEM) model that contains perturbations in the most frequently dysregulated networks in GBM (driven by RB, KRAS and/or PI3K signaling and PTEN) that induce development of Grade IV astrocytoma with properties of the human disease. Here, we developed and characterized an orthotopic mouse model derived from the GEM that retains the features of the GEM model in an immunocompetent background; however, this model is also tractable and efficient for preclinical evaluation of candidate therapeutic regimens. Orthotopic brain tumors are highly proliferative, invasive and vascular, and express histology markers characteristic of human GBM. Primary tumor cells were examined for sensitivity to chemotherapeutics and targeted drugs. PI3K and MAPK pathway inhibitors, when used as single agents, inhibited cell proliferation but did not result in significant apoptosis. However, in combination, these inhibitors resulted in a substantial increase in cell death. Moreover, these findings translated into the in vivo orthotopic model: PI3K or MAPK inhibitor treatment regimens resulted in incomplete pathway suppression and feedback loops, whereas dual treatment delayed tumor growth through increased apoptosis and decreased tumor cell proliferation. Analysis of downstream pathway components revealed a cooperative effect on target downregulation. These concordant results, together with the morphologic similarities to the human GBM disease characteristics of the model, validate it as a new platform for the evaluation of GBM treatment. © 2015. Published by The Company of Biologists Ltd.

A preclinical orthotopic model for glioblastoma recapitulates key features of human tumors and demonstrates sensitivity to a combination of MEK and PI3K pathway inhibitors

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Rajaa El Meskini

2015-01-01

Full Text Available Current therapies for glioblastoma multiforme (GBM, the highest grade malignant brain tumor, are mostly ineffective, and better preclinical model systems are needed to increase the successful translation of drug discovery efforts into the clinic. Previous work describes a genetically engineered mouse (GEM model that contains perturbations in the most frequently dysregulated networks in GBM (driven by RB, KRAS and/or PI3K signaling and PTEN that induce development of Grade IV astrocytoma with properties of the human disease. Here, we developed and characterized an orthotopic mouse model derived from the GEM that retains the features of the GEM model in an immunocompetent background; however, this model is also tractable and efficient for preclinical evaluation of candidate therapeutic regimens. Orthotopic brain tumors are highly proliferative, invasive and vascular, and express histology markers characteristic of human GBM. Primary tumor cells were examined for sensitivity to chemotherapeutics and targeted drugs. PI3K and MAPK pathway inhibitors, when used as single agents, inhibited cell proliferation but did not result in significant apoptosis. However, in combination, these inhibitors resulted in a substantial increase in cell death. Moreover, these findings translated into the in vivo orthotopic model: PI3K or MAPK inhibitor treatment regimens resulted in incomplete pathway suppression and feedback loops, whereas dual treatment delayed tumor growth through increased apoptosis and decreased tumor cell proliferation. Analysis of downstream pathway components revealed a cooperative effect on target downregulation. These concordant results, together with the morphologic similarities to the human GBM disease characteristics of the model, validate it as a new platform for the evaluation of GBM treatment.

Discovery of amido-benzisoxazoles as potent c-Kit inhibitors

Energy Technology Data Exchange (ETDEWEB)

Kunz, Roxanne K.; Rumfelt, Shannon; Chen, Ning; Zhang, Dawei; Tasker, Andrew S.; Bürli, Roland; Hungate, Randall; Yu, Violeta; Nguyen, Yen; Whittington, Douglas A.; Meagher, Kristin L.; Plant, Matthew; Tudor, Yanyan; Schrag, Michael; Xu, Yang; Ng, Gordon Y.; Hu, Essa (Amgen)

2010-01-12

Deregulation of the receptor tyrosine kinase c-Kit is associated with an increasing number of human diseases, including certain cancers and mast cell diseases. Interference of c-Kit signaling with multi-kinase inhibitors has been shown clinically to successfully treat gastrointestinal stromal tumors and mastocytosis. Targeted therapy of c-Kit activity may provide therapeutic advantages against off-target effects for non-oncology applications. A new structural class of c-Kit inhibitors is described, including in vitro c-Kit potency, kinase selectivity, and the observed binding mode.

Are SGLT2 inhibitors reasonable antihypertensive drugs and renoprotective?

Science.gov (United States)

Lovshin, J A; Gilbert, R E

2015-06-01

By eliminating glucose in the urine, the sodium-glucose-linked cotransporter-2 (SGLT2) inhibitors act as osmotic diuretics to lower blood pressure in addition to reducing plasma glucose and assisting with weight loss. While not approved as antihypertensive agents, the ability of this new class of antihyperglycemic agents to lower blood pressure is not insubstantial, and while not used primarily for this indication, they may assist diabetic individuals in attaining currently recommended blood pressure targets. In addition to lowering systemic pressure, preclinical and exploratory human studies suggest that SGLT2 inhibitors may also lower intraglomerular pressure, potentially reducing the rate of GFR decline in patients with diabetic nephropathy. However, given the lack of clinically meaningful endpoint data, the use of SGLT2 inhibitors, primarily, as either antihypertensive or renoprotective agents would, at present, be premature. Fortunately, further insight will be garnered from large, randomized controlled trials that will assess the effects of various SGLT2 inhibitors on cardiovascular and renal outcomes.

A Pan-GTPase Inhibitor as a Molecular Probe.

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Lin Hong

Full Text Available Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed.

Urinary trypsin inhibitor - an experimental and clinical study

International Nuclear Information System (INIS)

Berling, B.M.

1991-01-01

The urinary trypsin inhibitor (UTI) is an acid stable proteinase inhibitor present in blood and urine. It was purified from urine using affinity chromatography, ion exchange chromatography and gel filtration. Two forms of UTI were present in urine, A and B. A radioimmunoassay for measurement of UTI in urine and plasma was performed. The normal level of UTI in plasma and serum was about 2 mg/l. The normal excretion in urine was about 8 mg per 24 hours. The plasma and urine levels of UTI were studied in patients with acute pancreatitis and in patients undergoing cholecystectomy. Uremic patients had a marked increase of UTI in plasma compatible with decreased glomerular filtration. In samples from healthy persons as well as from patients only inhibitor A was found. Inhibitor B has recently been renamed bikunin because of its two Kunitz-type inhibiting domains. Inhibitor A might be called tetrakunin. Radioactively labeled UTI (inhibitor A) was injected intravenously in three male volunteers. The plasma half-life of 125 I UTI was 2 hours. Free biologically active inhibitor was found in the urine during the first four hours after injection. The organ distribution of intravenously injected 125 I UTI was studied in rats. Fifteen minutes after injection the major part of the radioactivity was found in the kidneys, suggesting that the kidneys are the primary site of UTI metabolism. Using immunohistochemical techniques UTI was found in the proximal tubules of the normal human kidney further indicating the tubular reabsorption and methabolisms of UTI

A novel class of small molecule inhibitors of HDAC6.

Science.gov (United States)

Inks, Elizabeth S; Josey, Benjamin J; Jesinkey, Sean R; Chou, C James

2012-02-17

Histone deacetylases (HDACs) are a family of enzymes that play significant roles in numerous biological processes and diseases. HDACs are best known for their repressive influence on gene transcription through histone deacetylation. Mapping of nonhistone acetylated proteins and acetylation-modifying enzymes involved in various cellular pathways has shown protein acetylation/deacetylation also plays key roles in a variety of cellular processes including RNA splicing, nuclear transport, and cytoskeletal remodeling. Studies of HDACs have accelerated due to the availability of small molecule HDAC inhibitors, most of which contain a canonical hydroxamic acid or benzamide that chelates the metal catalytic site. To increase the pool of unique and novel HDAC inhibitor pharmacophores, a pharmacological active compound screen was performed. Several unique HDAC inhibitor pharmacophores were identified in vitro. One class of novel HDAC inhibitors, with a central naphthoquinone structure, displayed a selective inhibition profile against HDAC6. Here we present the results of a unique class of HDAC6 inhibitors identified using this compound library screen. In addition, we demonstrated that treatment of human acute myeloid leukemia cell line MV4-11 with the selective HDAC6 inhibitors decreases levels of mutant FLT-3 and constitutively active STAT5 and attenuates Erk phosphorylation, all of which are associated with the inhibitor's selective toxicity against leukemia.

A miR-21 inhibitor enhances apoptosis and reduces G2-M accumulation induced by ionizing radiation in human glioblastoma U251 cells

International Nuclear Information System (INIS)

Li, Yi; Li, Qiang; Asai, Akio; Kawamoto, Keiji; Zhao Shiguang; Zhen Yunbo; Teng Lei

2011-01-01

MicroRNAs (miRNAs) are small noncoding RNAs that take part in diverse biological processes by suppressing target gene expression. Elevated expression of miR-21 has been reported in many types of human cancers. Radiotherapy is a standard adjuvant treatment for patients with glioblastoma. However, the resistance of glioblastoma cells to radiation limits the success of this treatment. In this study, we found that miR-21 expression was upregulated in response to ionizing radiation (IR) in U251 cells, which suggested that miR-21 could be involved in the response of U251 cells to radiation. We showed that a miR-21 inhibitor enhanced IR-induced glioblastoma cell growth arrest and increased the level of apoptosis, which was probably caused by abrogation of the G 2 -M arrest induced by IR. Further research demonstrated that the miR-21 inhibitor induced the upregulation of Cdc25A. Taken together, these findings suggest that miR-21 inhibitor can increase IR-induced growth arrest and apoptosis in U251 glioblastoma cells, at least in part by abrogating G 2 -M arrest, and that Cdc25A is a potential target of miR-21. (author)

Structure-Activity Relationships of the Human Immunodeficiency Virus Type 1 Maturation Inhibitor PF-46396.

Science.gov (United States)

Murgatroyd, Christopher; Pirrie, Lisa; Tran, Fanny; Smith, Terry K; Westwood, Nicholas J; Adamson, Catherine S

2016-09-15

HIV-1 maturation inhibitors are a novel class of antiretroviral compounds that consist of two structurally distinct chemical classes: betulinic acid derivatives and the pyridone-based compound PF-46396. It is currently believed that both classes act by similar modes of action to generate aberrant noninfectious particles via inhibition of CA-SP1 cleavage during Gag proteolytic processing. In this study, we utilized a series of novel analogues with decreasing similarity to PF-46396 to determine the chemical groups within PF-46396 that contribute to antiviral activity, Gag binding, and the relationship between these essential properties. A spectrum of antiviral activity (active, intermediate, and inactive) was observed across the analogue series with respect to CA-SP1 cleavage and HIV-1 (NL4-3) replication kinetics in Jurkat T cells. We demonstrate that selected inactive analogues are incorporated into wild-type (WT) immature particles and that one inactive analogue is capable of interfering with PF-46396 inhibition of CA-SP1 cleavage. Mutations that confer PF-46396 resistance can impose a defective phenotype on HIV-1 that can be rescued in a compound-dependent manner. Some inactive analogues retained the capacity to rescue PF-46396-dependent mutants (SP1-A3V, SP1-A3T, and CA-P157S), implying that they can also interact with mutant Gag. The structure-activity relationships observed in this study demonstrate that (i) the tert-butyl group is essential for antiviral activity but is not an absolute requirement for Gag binding, (ii) the trifluoromethyl group is optimal but not essential for antiviral activity, and (iii) the 2-aminoindan group is important for antiviral activity and Gag binding but is not essential, as its replacement is tolerated. Combinations of antiretroviral drugs successfully treat HIV/AIDS patients; however, drug resistance problems make the development of new mechanistic drug classes an ongoing priority. HIV-1 maturation inhibitors are novel as they

Galectin-1 Inhibitor OTX008 Induces Tumor Vessel Normalization and Tumor Growth Inhibition in Human Head and Neck Squamous Cell Carcinoma Models.

Science.gov (United States)

Koonce, Nathan A; Griffin, Robert J; Dings, Ruud P M

2017-12-09

Galectin-1 is a hypoxia-regulated protein and a prognostic marker in head and neck squamous cell carcinomas (HNSCC). Here we assessed the ability of non-peptidic galectin-1 inhibitor OTX008 to improve tumor oxygenation levels via tumor vessel normalization as well as tumor growth inhibition in two human HNSCC tumor models, the human laryngeal squamous carcinoma SQ20B and the human epithelial type 2 HEp-2. Tumor-bearing mice were treated with OTX008, Anginex, or Avastin and oxygen levels were determined by fiber-optics and molecular marker pimonidazole binding. Immuno-fluorescence was used to determine vessel normalization status. Continued OTX008 treatment caused a transient reoxygenation in SQ20B tumors peaking on day 14, while a steady increase in tumor oxygenation was observed over 21 days in the HEp-2 model. A >50% decrease in immunohistochemical staining for tumor hypoxia verified the oxygenation data measured using a partial pressure of oxygen (pO₂) probe. Additionally, OTX008 induced tumor vessel normalization as tumor pericyte coverage increased by approximately 40% without inducing any toxicity. Moreover, OTX008 inhibited tumor growth as effectively as Anginex and Avastin, except in the HEp-2 model where Avastin was found to suspend tumor growth. Galectin-1 inhibitor OTX008 transiently increased overall tumor oxygenation via vessel normalization to various degrees in both HNSCC models. These findings suggest that targeting galectin-1-e.g., by OTX008-may be an effective approach to treat cancer patients as stand-alone therapy or in combination with other standards of care.

Inhibitors

Science.gov (United States)

... JM, and the Hemophilia Inhibitor Research Study Investigators. Validation of Nijmegen-Bethesda assay modifications to allow inhibitor ... webinars on blood disorders Language: English (US) Español (Spanish) File Formats Help: How do I view different ...

The heat shock protein 90 inhibitor 17-AAG suppresses growth and induces apoptosis in human cholangiocarcinoma cells.

Science.gov (United States)

Zhang, Jianjun; Zheng, Zhichao; Zhao, Yan; Zhang, Tao; Gu, Xiaohu; Yang, Wei

2013-11-01

The aim of this study was to investigate the effects of 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (HSP90) inhibitor, on the proliferation, cell cycle, and apoptosis of human cholangiocarcinoma (CCA) cells. Cell proliferation and cell cycle distribution were measured by the MTT assay and flow cytometry analysis, respectively. Induction of apoptosis was determined by flow cytometry and Hoechst staining. The expressions of cleaved poly ADP-ribose polymerase (PARP), Bcl-2, Survivin, and Cyclin B1 were detected by Western blot analysis. The activity of caspase-3 was also examined. We found that 17-AAG inhibited cell growth and induced G2/M cell cycle arrest and apoptosis in CCA cells together with the down-regulation of Bcl-2, Survivin and Cyclin B1, and the up-regulation of cleaved PARP. Moreover, increased caspase-3 activity was also observed in CCA cells treated with 17-AAG. In conclusion, our data suggest that the inhibition of HSP90 function by 17-AAG may provide a promising therapeutic strategy for the treatment of human CCA.

Efficacy of c-Met inhibitor for advanced prostate cancer

International Nuclear Information System (INIS)

Tu, William H; Zhu, Chunfang; Clark, Curtis; Christensen, James G; Sun, Zijie

2010-01-01

Aberrant expression of HGF/SF and its receptor, c-Met, often correlates with advanced prostate cancer. Our previous study showed that expression of c-Met in prostate cancer cells was increased after attenuation of androgen receptor (AR) signalling. This suggested that current androgen ablation therapy for prostate cancer activates c-Met expression and may contribute to development of more aggressive, castration resistant prostate cancer (CRPC). Therefore, we directly assessed the efficacy of c-Met inhibition during androgen ablation on the growth and progression of prostate cancer. We tested two c-Met small molecule inhibitors, PHA-665752 and PF-2341066, for anti-proliferative activity by MTS assay and cell proliferation assay on human prostate cancer cell lines with different levels of androgen sensitivity. We also used renal subcapsular and castrated orthotopic xenograft mouse models to assess the effect of the inhibitors on prostate tumor formation and progression. We demonstrated a dose-dependent inhibitory effect of PHA-665752 and PF-2341066 on the proliferation of human prostate cancer cells and the phosphorylation of c-Met. The effect on cell proliferation was stronger in androgen insensitive cells. The c-Met inhibitor, PF-2341066, significantly reduced growth of prostate tumor cells in the renal subcapsular mouse model and the castrated orthotopic mouse model. The effect on cell proliferation was greater following castration. The c-Met inhibitors demonstrated anti-proliferative efficacy when combined with androgen ablation therapy for advanced prostate cancer

Wheat Subtilisin/Chymotrypsin Inhibitor (WSCI) as a scaffold for novel serine protease inhibitors with a given specificity.

Science.gov (United States)

Tedeschi, Francesca; Di Maro, Antimo; Facchiano, Angelo; Costantini, Susan; Chambery, Angela; Bruni, Natalia; Capuzzi, Valeria; Ficca, Anna Grazia; Poerio, Elia

2012-10-30

WSCI (Wheat Subtilisin/Chymotrypsin Inhibitor) is a small protein belonging to the Potato inhibitor I family exhibiting a high content of essential amino acid. In addition to bacterial subtilisins and mammalian chymotrypsins, WSCI inhibits chymotrypsin-like activities isolated from digestive traits of a number of insect larvae. In vivo, as suggested for many plant proteinase inhibitors, WSCI seems to play a role of natural defence against attacks of pests and pathogens. The functional region of WSCI, containing the inhibitor reactive site (Met48-Glu49), corresponds to an extended flexible loop (Val42-Asp53) whose architecture is somehow stabilized by a number of secondary interactions established with a small β-sheet located underneath. The aim of this study was to employ a WSCI molecule as a stable scaffold to obtain recombinant inhibitors with new acquired anti-proteinase activity or, alternatively, inactive WSCI variants. A gene sequence coding for the native WSCI, along with genes coding for muteins with different specficities, could be exploited to obtain transformed non-food use plants with improved insect resistance. On the other hand, the genetic transformation of cereal plants over-expressing inactive WSCI muteins could represent a possible strategy to improve the nutritional quality of cereal-based foods, without risk of interference with human or animal digestive enzymes. Here, we described the characterization of four muteins containing single/multiple amino acid substitutions at the WSCI reactive site and/or at its proximity. Modalities of interaction of these muteins with proteinases (subtilisin, trypsin and chymotrypsin) were investigated by time course hydrolysis and molecular simulations studies.

In vitro differentiation of human monocytes to macrophages: change of PDE profile and its relationship to suppression of tumour necrosis factor-α release by PDE inhibitors

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Gantner, Florian; Kupferschmidt, Rochus; Schudt, Christian; Wendel, Albrecht; Hatzelmann, Armin

1997-01-01

During in vitro culture in 10% human AB serum, human peripheral blood monocytes acquire a macrophage-like phenotype. The underlying differentiation was characterized by increased activities of the macrophage marker enzymes unspecific esterase (NaF-insensitive form) and acid phosphatase, as well as by a down-regulation in surface CD14 expression. In parallel, a dramatic change in the phosphodiesterase (PDE) profile became evident within a few days that strongly resembled that previously described for human alveolar macrophages. Whereas PDE1 and PDE3 activities were augmented, PDE4 activity, which represented the major cyclic AMP-hydrolysing activity of peripheral blood monocytes, rapidly declined. Monocytes and monocyte-derived macrophages responded to lipopolysaccharide (LPS) with the release of tumour necrosis factor-α (TNF). In line with the change in CD14 expression, the EC50 value of LPS for induction of TNF release increased from approximately 0.1 ng ml−1 in peripheral blood monocytes to about 2 ng ml−1 in macrophages. Both populations of cells were equally susceptible towards inhibition of TNF release by cyclic AMP elevating agents such as dibutyryl cyclic AMP, prostaglandin E2 (PGE2) or forskolin, which all led to a complete abrogation of TNF production in a concentration-dependent manner and which were more efficient than the glucocorticoid dexamethasone. In monocytes, PDE4 selective inhibitors (rolipram, RP73401) suppressed TNF formation by 80%, whereas motapizone, a PDE3 selective compound, exerted a comparatively weak effect (10–15% inhibition). Combined use of PDE3 plus PDE4 inhibitors resulted in an additive effect and fully abrogated LPS-induced TNF release as did the mixed PDE3/4 inhibitor tolafentrine. In monocyte-derived macrophages, neither PDE3- nor PDE4-selective drugs markedly affected TNF generation when used alone (<15% inhibition), whereas in combination, they led to a maximal inhibition of TNF formation by about 40–50

Apoptosis-related molecular differences for response to tyrosin kinase inhibitors in drug-sensitive and drug-resistant human bladder cancer cells

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Jixia Li

2013-01-01

Full Text Available Context: The epidermal growth factor receptor (EGFR family is reportedly overexpressed in bladder cancer, and tyrosine kinaseinhibitors (TKIs have been suggested as treatment. Gefitinib is a selective inhibitor of the EGFR and lapatinib is a dual inhibitor of both the EGFR and HER2 (human EGFR type 2 receptor. Both compounds compete with the binding of adenosine triphosphate (ATP to the tyrosine kinase domain of the respective receptors to inhibit receptor autophosphorylation causing suppression of signal transduction. Unfortunately, resistance to these inhibitors is a major clinical problem. Aims: To compare the apoptosis signaling pathway(s induced by gefitinib and lapatinib, in UM-UC-5 (drug-sensitive and UM-UC-14 (drug-resistant bladder cancer cells and to identify molecular differences that might be useful predictors of their efficacy. Materials and Methods: Cell proliferation, cell cycle and apoptosis assay were used to detect the effect of TKIs on UM-UC-5 and UM-UC-14 cells. Molecular differences for response to TKIs were examined by protein array. Results: TKIs strongly inhibited cell proliferation and induced cell cycle G1 arrest and apoptosis in UM-UC-5 cells. Most notable apoptosis molecular differences included decreased claspin, trail, and survivin by TKIs in the sensitive cells. In contrast, TKIs had no effect on resistant cells. Conclusions: Claspin, trail, and survivin might be used to determine the sensitivity of bladder cancers to TKIs.

Effects of inhibitors of DNA repair on the frequencies of chromosomal aberrations induced by x-rays or alkylating agents in cultured human lymphocytes

International Nuclear Information System (INIS)

Kihlman, B.A.; Andersson, H.C.

1986-01-01

In the first part of this presentation the authors give examples of the synergistic enhancements that are obtained with various inhibitor combinations in G/sub 2/. The second part of the presentation deals with the effects of two agents, also well known for their capacity to potentiate the frequency of chromosomal aberrations induced by physical and chemical agents, but with a different mechanism of action. These agents are caffeine and 3-aminobenzamide (3AB). Caffeine has for decades been used as an inhibitor of DNA repair although its mechanism of action has not been fully understood. 3AB has more recently come into focus as an efficient inhibitor of the synthesis of poly-(ADP-ribose), a substance believed to be of importance in connection with the repair of certain types of DNA damage. The results presented do not quite fit in with the general idea about the mode of action of these agents. All experiments were carried out with whole-blood cultures of human lymphocytes. When inhibitors were used as post-treatments, chromosomal aberrations were induced by X-rays or by the alkylating agents thiotepa (TT) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). X-rays were generated by a Siemens Stabilipan 200 apparatus, at a dose rate of 0.5 Gy/min. The tube (TR 200f) was operated at 180 kV, 10 mA and the radiation filtered through 4 mm Al

Finding Potent Sirt Inhibitor in Coffee: Isolation, Confirmation and Synthesis of Javamide-II (N-Caffeoyltryptophan as Sirt1/2 Inhibitor.

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Jae B Park

Full Text Available Recent studies suggest that Sirt inhibition may have beneficial effects on several human diseases such as neurodegenerative diseases and cancer. Coffee is one of most popular beverages with several positive health effects. Therefore, in this paper, potential Sirt inhibitors were screened using coffee extract. First, HPLC was utilized to fractionate coffee extract, then screened using a Sirt1/2 inhibition assay. The screening led to the isolation of a potent Sirt1/2 inhibitor, whose structure was determined as javamide-II (N-caffeoyltryptophan by NMR. For confirmation, the amide was chemically synthesized and its capacity of inhibiting Sirt1/2 was also compared with the isolated amide. Javamide-II inhibited Sirt2 (IC50; 8.7 μM better than Sirt1(IC50; 34μM. Since javamide-II is a stronger inhibitor for Sirt2 than Sirt1. The kinetic study was performed against Sirt2. The amide exhibited noncompetitive Sirt2 inhibition against the NAD+ (Ki = 9.8 μM and showed competitive inhibition against the peptide substrate (Ki = 5.3 μM. Also, a docking simulation showed stronger binding pose of javamide-II to Sirt2 than AGK2. In cellular levels, javamide-II was able to increase the acetylation of total lysine, cortactin and histone H3 in neuronal NG108-15 cells. In the same cells, the amide also increased the acetylation of lysine (K382 in p53, but not (K305. This study suggests that Javamide-II found in coffee may be a potent Sirt1/2 inhibitor, probably with potential use in some conditions of human diseases.

Cloning, purification, and functional characterization of Carocin S2, a ribonuclease bacteriocin produced by Pectobacterium carotovorum

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Tzeng Kuo-Ching

2011-05-01

Full Text Available Abstract Background Most isolates of Pectobacterium carotovorum subsp. carotovorum (Pcc produce bacteriocins. In this study, we have determined that Pcc strain F-rif-18 has a chromosomal gene encoding the low-molecular-weight bacteriocin, Carocin S2, and that this bacteriocin inhibits the growth of a closely related strain. Carocin S2 is inducible by ultraviolet radiation but not by mutagenic agents such as mitomycin C. Results A carocin S2-defective mutant, TF1-2, was obtained by Tn5 insertional mutagenesis using F-rif-18. A 5706-bp DNA fragment was detected by Southern blotting, selected from a genomic DNA library, and cloned to the vector, pMS2KI. Two adjacent complete open reading frames within pMS2KI were sequenced, characterized, and identified as caroS2K and caroS2I, which respectively encode the killing protein and immunity protein. Notably, carocin S2 could be expressed not only in the mutant TF1-2 but also in Escherichia coli DH5α after entry of the plasmid pMS2KI. Furthermore, the C-terminal domain of CaroS2K was homologous to the nuclease domains of colicin D and klebicin D. Moreover, SDS-PAGE analysis showed that the relative mass of CaroS2K was 85 kDa and that of CaroS2I was 10 kDa. Conclusion This study shown that another nuclease type of bacteriocin was found in Pectobacterium carotovorum. This new type of bacteriocin, Carocin S2, has the ribonuclease activity of CaroS2K and the immunity protein activity of CaroS2I.

6-Substituted 3,4-dihydro-naphthalene-2-carboxylic acids: synthesis and structure-activity studies in a novel class of human 5alpha reductase inhibitors.

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Baston, Eckhard; Salem, Ola I A; Hartmann, Rolf W

2002-10-01

Novel 3,4-dihydro-naphthalene-2-carboxylic acids were synthesized and evaluated for 5alpha reductase inhibitory activity. This enzyme exists in two isoforms and is a pharmacological target for the treatment of benign prostatic hyperplasia, male pattern baldness and acne. In the present study non-steroidal compounds capable of mimicking the transition state of the steroidal substrates were prepared. The synthetic strategy for the preparation of compounds 1-6 consisted of triflation followed by subsequent Heck-type carboxylation or methoxy carbonylation for 6-phenyl-3,4-dihydronaphthalen-2(1H)-one 1c. A Negishi-type coupling reaction between 6-(trifluoro-methanesulfonyloxy)-3,4-dihydro-naphthalene-2-carboxylic acid methyl ester 7b and various aryl bromides led, after further transformations, to 6-substituted 3,4-dihydro-naphthalene-2-carboxylic acids 7-15. In a similar way the corresponding naphthalene-2-carboxylic acids 16 and 17 were obtained. The DU 145 cell line and prostate homogenates served as enzyme sources for the human type 1 and type 2 isozymes, whereas ventral prostate was employed to evaluate rat isozyme inhibitory potency. The most active inhibitors identified in this study were 6-[4-(N,N-dicyclohexylaminocarbonyl)phenyl]-3,4-dihydro-naphthalene-2-carboxylic acid (3) (IC50 = 0.09 microM, rat type 1), 6-[3-(N,N-dicyclohexylaminocarbonyl)phenyl]-3,4-dihydro-naphthalene-2-carboxylic acid (13) (IC50 = 0.75 microM, human type 2; IC50 = 0.81 microM, human type 1) and 6-[4-(N,N-diisopropylamino-carbonyl)phenyl]naphthalene-2-carboxylic acid (16) (IC50 = 0.2 microM, human type 2). The latter compound was shown to deactivate the enzyme in an uncompetitive manner (Ki = 90 nM; Km, Testosterone = 0.8-1.0 microM) similar to the steroidal inhibitor Epristeride. Select inhibitors (13 and 16) were tested in vivo using testosterone propionate-treated, juvenile, orchiectomized SD-rats. None of the compounds was active at a dose of 25 mg/kg. This result might in part be

Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

DEFF Research Database (Denmark)

Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter

2003-01-01

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosyla......The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous...... with the glycosylation sites could be excluded as explanation for the differential reactivity. The latency transition of non-glycosylated, but not of glycosylated PAI-1, was strongly accelerated by a non-ionic detergent. The different biochemical properties of glycosylated and non-glycosylated PAI-1 depended...

A novel method for screening the glutathione transferase inhibitors

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Węgrzyn Grzegorz

2009-03-01

Full Text Available Abstract Background Glutathione transferases (GSTs belong to the family of Phase II detoxification enzymes. GSTs catalyze the conjugation of glutathione to different endogenous and exogenous electrophilic compounds. Over-expression of GSTs was demonstrated in a number of different human cancer cells. It has been found that the resistance to many anticancer chemotherapeutics is directly correlated with the over-expression of GSTs. Therefore, it appears to be important to find new GST inhibitors to prevent the resistance of cells to anticancer drugs. In order to search for glutathione transferase (GST inhibitors, a novel method was designed. Results Our results showed that two fragments of GST, named F1 peptide (GYWKIKGLV and F2 peptide (KWRNKKFELGLEFPNL, can significantly inhibit the GST activity. When these two fragments were compared with several known potent GST inhibitors, the order of inhibition efficiency (measured in reactions with 2,4-dinitrochlorobenzene (CDNB and glutathione as substrates was determined as follows: tannic acid > cibacron blue > F2 peptide > hematin > F1 peptide > ethacrynic acid. Moreover, the F1 peptide appeared to be a noncompetitive inhibitor of the GST-catalyzed reaction, while the F2 peptide was determined as a competitive inhibitor of this reaction. Conclusion It appears that the F2 peptide can be used as a new potent specific GST inhibitor. It is proposed that the novel method, described in this report, might be useful for screening the inhibitors of not only GST but also other enzymes.

Synergism of antifungal activity between mitochondrial respiration inhibitors and kojic acid.

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Kim, Jong H; Campbell, Bruce C; Chan, Kathleen L; Mahoney, Noreen; Haff, Ronald P

2013-01-25

Co-application of certain types of compounds to conventional antimicrobial drugs can enhance the efficacy of the drugs through a process termed chemosensitization. We show that kojic acid (KA), a natural pyrone, is a potent chemosensitizing agent of complex III inhibitors disrupting the mitochondrial respiratory chain in fungi. Addition of KA greatly lowered the minimum inhibitory concentrations of complex III inhibitors tested against certain filamentous fungi. Efficacy of KA synergism in decreasing order was pyraclostrobin > kresoxim-methyl > antimycin A. KA was also found to be a chemosensitizer of cells to hydrogen peroxide (H₂O₂), tested as a mimic of reactive oxygen species involved in host defense during infection, against several human fungal pathogens and Penicillium strains infecting crops. In comparison, KA-mediated chemosensitization to complex III inhibitors/H₂O₂ was undetectable in other types of fungi, including Aspergillus flavus, A. parasiticus, and P. griseofulvum, among others. Of note, KA was found to function as an antioxidant, but not as an antifungal chemosensitizer in yeasts. In summary, KA could serve as an antifungal chemosensitizer to complex III inhibitors or H₂O₂ against selected human pathogens or Penicillium species. KA-mediated chemosensitization to H₂O₂ seemed specific for filamentous fungi. Thus, results indicate strain- and/or drug-specificity exist during KA chemosensitization.

Structural investigation of HIV-1 nonnucleoside reverse transcriptase inhibitors: 2-Aryl-substituted benzimidazoles

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Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

2009-11-01

Acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) is one of the most destructive epidemics in history. Inhibitors of HIV enzymes are the main targets to develop drugs against that disease. Nonnucleoside reverse transcriptase inhibitors of HIV-1 (NNRTIs) are potentially effective and nontoxic. Structural studies provide information necessary to design more active compounds. The crystal structures of four NNRTI derivatives of 2-aryl-substituted N-benzyl-benzimidazole are presented here. Analysis of the geometrical parameters shows that the structures of the investigated inhibitors are rigid. The important geometrical parameter is the dihedral angle between the planes of the π-electron systems of the benzymidazole and benzyl moieties. The values of these dihedral angles are in a narrow range for all investigated inhibitors. There is no significant difference between the structure of the free inhibitor and the inhibitor in the complex with RT HIV-1. X-ray structures of the investigated inhibitors are a good basis for modeling enzyme-inhibitor interactions in rational drug design.

Replication and Inhibitors of Enteroviruses and Parechoviruses

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Lonneke van der Linden

2015-08-01

Full Text Available The Enterovirus (EV and Parechovirus genera of the picornavirus family include many important human pathogens, including poliovirus, rhinovirus, EV-A71, EV-D68, and human parechoviruses (HPeV. They cause a wide variety of diseases, ranging from a simple common cold to life-threatening diseases such as encephalitis and myocarditis. At the moment, no antiviral therapy is available against these viruses and it is not feasible to develop vaccines against all EVs and HPeVs due to the great number of serotypes. Therefore, a lot of effort is being invested in the development of antiviral drugs. Both viral proteins and host proteins essential for virus replication can be used as targets for virus inhibitors. As such, a good understanding of the complex process of virus replication is pivotal in the design of antiviral strategies goes hand in hand with a good understanding of the complex process of virus replication. In this review, we will give an overview of the current state of knowledge of EV and HPeV replication and how this can be inhibited by small-molecule inhibitors.

Can Inhibitors of Snake Venom Phospholipases A₂ Lead to New Insights into Anti-Inflammatory Therapy in Humans? A Theoretical Study.

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Sales, Thaís A; Marcussi, Silvana; da Cunha, Elaine F F; Kuca, Kamil; Ramalho, Teodorico C

2017-10-25

Human phospholipase A₂ ( h PLA₂) of the IIA group (HGIIA) catalyzes the hydrolysis of membrane phospholipids, producing arachidonic acid and originating potent inflammatory mediators. Therefore, molecules that can inhibit this enzyme are a source of potential anti-inflammatory drugs, with different action mechanisms of known anti-inflammatory agents. For the study and development of new anti-inflammatory drugs with this action mechanism, snake venom PLA₂ ( sv PLA₂) can be employed, since the sv PLA₂ has high similarity with the human PLA₂ HGIIA. Despite the high similarity between these secretory PLA₂s , it is still not clear if these toxins can really be employed as an experimental model to predict the interactions that occur with the human PLA₂ HGIIA and its inhibitors. Thus, the present study aims to compare and evaluate, by means of theoretical calculations, docking and molecular dynamics simulations, as well as experimental studies, the interactions of human PLA₂ HGIIA and two sv PLA₂s , Bothrops toxin II and Crotoxin B (BthTX-II and CB, respectively). Our theoretical findings corroborate experimental data and point out that the human PLA₂ HGIIA and sv PLA₂ BthTX-II lead to similar interactions with the studied compounds. From our results, the sv PLA₂ BthTX-II can be used as an experimental model for the development of anti-inflammatory drugs for therapy in humans.

Thermodynamic parameters for binding of some halogenated inhibitors of human protein kinase CK2

International Nuclear Information System (INIS)

Winiewska, Maria; Makowska, Małgorzata; Maj, Piotr; Wielechowska, Monika; Bretner, Maria; Poznański, Jarosław; Shugar, David

2015-01-01

Highlights: • Two new compounds being potential human CK2a inhibitors are studied. • Their IC50 values were determined in vitro. • The heats of binding and kbind were estimated using DSC. • The increased stability of protein–ligand complexes was followed by fluorescence. • Methylated TBBt derivative (MeBr3Br) is almost as active as TBBt. - Abstract: The interaction of human CK2α with a series of tetrabromobenzotriazole (TBBt) and tetrabromobenzimidazole (TBBz) analogs, in which one of the bromine atoms proximal to the triazole/imidazole ring is replaced by a methyl group, was studied by biochemical (IC 50 ) and biophysical methods (thermal stability of protein–ligand complex monitored by DSC and fluorescence). Two newly synthesized tri-bromo derivatives display inhibitory activity comparable to that of the reference compounds, TBBt and TBBz, respectively. DSC analysis of the stability of protein–ligand complexes shows that the heat of ligand binding (H bind ) is driven by intermolecular electrostatic interactions involving the triazole/imidazole ring, as indicated by a strong correlation between H bind and ligand pK a . Screening, based on fluorescence-monitored thermal unfolding of protein–ligand complexes, gave comparable results, clearly identifying ligands that most strongly bind to the protein. Overall results, additionally supported by molecular modeling, confirm that a balance of hydrophobic and electrostatic interactions contribute predominantly, relative to possible intermolecular halogen bonding, in binding of the ligands to the CK2α ATP-binding site

Thermodynamic parameters for binding of some halogenated inhibitors of human protein kinase CK2

Energy Technology Data Exchange (ETDEWEB)

Winiewska, Maria; Makowska, Małgorzata [Institute of Biochemistry and Biophysics PAS, Warszawa (Poland); Maj, Piotr [Institute of Biochemistry and Biophysics PAS, Warszawa (Poland); Nencki Institute of Experimental Biology PAS, Warszawa (Poland); Wielechowska, Monika; Bretner, Maria [Warsaw University of Technology, Faculty of Chemistry, Warszawa (Poland); Poznański, Jarosław, E-mail: jarek@ibb.waw.pl [Institute of Biochemistry and Biophysics PAS, Warszawa (Poland); Shugar, David [Institute of Biochemistry and Biophysics PAS, Warszawa (Poland)

2015-01-02

Highlights: • Two new compounds being potential human CK2a inhibitors are studied. • Their IC50 values were determined in vitro. • The heats of binding and kbind were estimated using DSC. • The increased stability of protein–ligand complexes was followed by fluorescence. • Methylated TBBt derivative (MeBr3Br) is almost as active as TBBt. - Abstract: The interaction of human CK2α with a series of tetrabromobenzotriazole (TBBt) and tetrabromobenzimidazole (TBBz) analogs, in which one of the bromine atoms proximal to the triazole/imidazole ring is replaced by a methyl group, was studied by biochemical (IC{sub 50}) and biophysical methods (thermal stability of protein–ligand complex monitored by DSC and fluorescence). Two newly synthesized tri-bromo derivatives display inhibitory activity comparable to that of the reference compounds, TBBt and TBBz, respectively. DSC analysis of the stability of protein–ligand complexes shows that the heat of ligand binding (H{sub bind}) is driven by intermolecular electrostatic interactions involving the triazole/imidazole ring, as indicated by a strong correlation between H{sub bind} and ligand pK{sub a}. Screening, based on fluorescence-monitored thermal unfolding of protein–ligand complexes, gave comparable results, clearly identifying ligands that most strongly bind to the protein. Overall results, additionally supported by molecular modeling, confirm that a balance of hydrophobic and electrostatic interactions contribute predominantly, relative to possible intermolecular halogen bonding, in binding of the ligands to the CK2α ATP-binding site.

Crystal structure of the N domain of human somatic angiotensin I-converting enzyme provides a structural basis for domain-specific inhibitor design.

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Corradi, Hazel R; Schwager, Sylva L U; Nchinda, Aloysius T; Sturrock, Edward D; Acharya, K Ravi

2006-03-31

Human somatic angiotensin I-converting enzyme (sACE) is a key regulator of blood pressure and an important drug target for combating cardiovascular and renal disease. sACE comprises two homologous metallopeptidase domains, N and C, joined by an inter-domain linker. Both domains are capable of cleaving the two hemoregulatory peptides angiotensin I and bradykinin, but differ in their affinities for a range of other substrates and inhibitors. Previously we determined the structure of testis ACE (C domain); here we present the crystal structure of the N domain of sACE (both in the presence and absence of the antihypertensive drug lisinopril) in order to aid the understanding of how these two domains differ in specificity and function. In addition, the structure of most of the inter-domain linker allows us to propose relative domain positions for sACE that may contribute to the domain cooperativity. The structure now provides a platform for the design of "domain-specific" second-generation ACE inhibitors.

Ebselen Reversibly Inhibits Human Glutamate Dehydrogenase at the Catalytic Site.

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Jin, Yanhong; Li, Di; Lu, Shiying; Zhao, Han; Chen, Zhao; Hou, Wei; Ruan, Benfang Helen

Human glutamate dehydrogenase (GDH) plays an important role in neurological diseases, tumor metabolism, and hyperinsulinism-hyperammonemia syndrome (HHS). However, there are very few inhibitors known for human GDH. Recently, Ebselen was reported to crosslink with Escherichia coli GDH at the active site cysteine residue (Cys321), but the sequence alignment showed that the corresponding residue is Ala329 in human GDH. To investigate whether Ebselen could be an inhibitor for human GDH, we cloned and expressed an N-terminal His-tagged human GDH in E. coli. The recombinant human GDH enzyme showed expected properties such as adenosine diphosphate activation and nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate dual recognition. Further, we developed a 2-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl) benzenesulfonate sodium salt (EZMTT)-based assay for human GDH, which was highly sensitive and is suitable for high-throughput screening for potent GDH inhibitors. In addition, ForteBio binding assays demonstrated that Ebselen is a reversible active site inhibitor for human GDH. Since Ebselen is a multifunctional organoselenium compound in Phase III clinical trials for inflammation, an Ebselen-based GDH inhibitor might be valuable for future drug discovery for HHS patients.

High frequencies of chromatid aberrations produced during G/sub 2/ in human lymphocytes by very low doses (0. 025-0. 4 Gy) of X-rays in combination with inhibitors of DNA synthesis

Energy Technology Data Exchange (ETDEWEB)

Andersson, H.C.; Kihlman, B.A. (Uppsala Univ. (Sweden). Dept. of Genetics)

1984-09-01

Whole-blood cultures of human lymphocytes were exposed in the G/sub 2/-phase (3.5 h before harvesting) to various doses of X-rays and post-treated for 3 h with inhibitors of DNA synthesis. The inhibitors used were 2'-deoxyadenosine (dAdo), hydroxyurea (HU) and 1-..beta..-D-arabinofuranosylcytosine (ara-C). To prevent deamination of dAdo by adenosine deaminase (ADA), the dAdo treatments were carried out in the presence of the ADA inhibitor coformycin. HU and ara-C were used either alone or in combination. After the 3-h inhibitor treatments, the cultures were harvested and slides prepared and analyzed for chromatid aberrations in metaphase. When the inhibitors were used at concentrations high enough to cause marked chromosome damage by themselves, very low doses of X-rays (0.025-0.2 Gy) were sufficient to produce a dramatic increase in the frequency of chromatid aberrations. High frequencies of chromatid aberrations were also obtained when cultures that had received moderate doses of X-rays (0.4-0.8 Gy) were post-treated with low inhibitor concentrations that produce no or only a few aberrations by themselves.

Resorcinol-, catechol- and saligenin-based bronchodilating β2-agonists as inhibitors of human cholinesterase activity.

Science.gov (United States)

Bosak, Anita; Knežević, Anamarija; Gazić Smilović, Ivana; Šinko, Goran; Kovarik, Zrinka

2017-12-01

We investigated the influence of bronchodilating β2-agonists on the activity of human acetylcholinesterase (AChE) and usual, atypical and fluoride-resistant butyrylcholinesterase (BChE). We determined the inhibition potency of racemate and enantiomers of fenoterol as a resorcinol derivative, isoetharine and epinephrine as catechol derivatives and salbutamol and salmeterol as saligenin derivatives. All of the tested compounds reversibly inhibited cholinesterases with K i constants ranging from 9.4 μM to 6.4 mM and had the highest inhibition potency towards usual BChE, but generally none of the cholinesterases displayed any stereoselectivity. Kinetic and docking results revealed that the inhibition potency of the studied compounds could be related to the size of the hydroxyaminoethyl chain on the benzene ring. The additional π-π interaction of salmeterol's benzene ring and Trp286 and hydrogen bond with His447 probably enhanced inhibition by salmeterol which was singled out as the most potent inhibitor of all the cholinesterases.

Expression of ODC Antizyme Inhibitor 2 (AZIN2 in Human Secretory Cells and Tissues.

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Tiina Rasila

Full Text Available Ornithine decarboxylase (ODC antizyme inhibitor 2 (AZIN2, originally called ODCp, is a regulator of polyamine synthesis that we originally identified and cloned. High expression of ODCp mRNA was found in brain and testis. We reported that AZIN2 is involved in regulation of cellular vesicle transport and / or secretion, but the ultimate physiological role(s of AZIN2 is still poorly understood. In this study we used a peptide antibody (K3 to human AZIN2 and by immunohistochemistry mapped its expression in various normal tissues. We found high expression in the nervous system, in type 2 pneumocytes in the lung, in megakaryocytes, in gastric parietal cells co-localized with H,K-ATPase beta subunit, in selected enteroendocrine cells, in acinar cells of sweat glands, in podocytes, in macula densa cells and epithelium of collecting ducts in the kidney. The high expression of AZIN2 in various cells with secretory or vesicle transport activity indicates that the polyamine metabolism regulated by AZIN2 is more significantly involved in these events than previously appreciated.

ORF Alignment: NC_002655 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available ... Ribonuclease I precursor (EC 3.1.27.6) (enterobacter ... ribonuclease) (RNase I). [Escherichi...a coli K12] ... dbj|BAA35240.1| Ribonuclease I precursor (EC 3.1.27.6) ... (enterobacter ribon

ORF Alignment: NC_004431 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available ... Ribonuclease I precursor (EC 3.1.27.6) (enterobacter ... ribonuclease) (RNase I). [Escherichi...a coli K12] ... dbj|BAA35240.1| Ribonuclease I precursor (EC 3.1.27.6) ... (enterobacter ribon

ORF Alignment: NC_000913 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available ... Ribonuclease I precursor (EC 3.1.27.6) (enterobacter ... ribonuclease) (RNase I). [Escherichi...a coli K12] ... dbj|BAA35240.1| Ribonuclease I precursor (EC 3.1.27.6) ... (enterobacter ribon

Histone Deacetylase Inhibitor Induced Radiation Sensitization Effects on Human Cancer Cells after Photon and Hadron Radiation Exposure

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Ariungerel Gerelchuluun

2018-02-01

Full Text Available Suberoylanilide hydroxamic acid (SAHA is a histone deacetylase inhibitor, which has been widely utilized throughout the cancer research field. SAHA-induced radiosensitization in normal human fibroblasts AG1522 and lung carcinoma cells A549 were evaluated with a combination of γ-rays, proton, and carbon ion exposure. Growth delay was observed in both cell lines during SAHA treatment; 2 μM SAHA treatment decreased clonogenicity and induced cell cycle block in G1 phase but 0.2 μM SAHA treatment did not show either of them. Low LET (Linear Energy Transfer irradiated A549 cells showed radiosensitization effects on cell killing in cycling and G1 phase with 0.2 or 2 μM SAHA pretreatment. In contrast, minimal sensitization was observed in normal human cells after low and high LET radiation exposure. The potentially lethal damage repair was not affected by SAHA treatment. SAHA treatment reduced the rate of γ-H2AX foci disappearance and suppressed RAD51 and RPA (Replication Protein A focus formation. Suppression of DNA double strand break repair by SAHA did not result in the differences of SAHA-induced radiosensitization between human cancer cells and normal cells. In conclusion, our results suggest SAHA treatment will sensitize cancer cells to low and high LET radiation with minimum effects to normal cells.

Translational and rotational motions of proteins in a protein crowded environment

NARCIS (Netherlands)

Zorilla, S.; Hink, M.A.; Visser, A.J.W.G.; Lillo, M.P.

2007-01-01

Fluorescence correlation spectroscopy (FCS) was used to measure the translational diffusion of labeled apomyoglobin (tracer) in concentrated solutions of ribonuclease A and human serum albumin (crowders), as a quantitative model system of protein diffusive motions in crowded physiological

Novel structural hybrids of pyrazolobenzothiazines with benzimidazoles as cholinesterase inhibitors.

Science.gov (United States)

Aslam, Sana; Zaib, Sumera; Ahmad, Matloob; Gardiner, John M; Ahmad, Aqeel; Hameed, Abdul; Furtmann, Norbert; Gütschow, Michael; Bajorath, Jürgen; Iqbal, Jamshed

2014-05-06

Two series of novel pyrazolobenzothiazine-based hybrid compounds were efficiently synthesized starting from saccharin sodium salt. Pyrazolo[4,3-c][1,2]benzothiazine scaffolds were N-arylated by using p-fluorobenzaldehyde, followed by the incorporation of a benzimidazole or similar ring systems by treatment with arylenediamines. These phenylene-connected hybrid compounds were investigated as potential inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Compounds 12d and 12k were the most potent AChE inhibitors with IC50 values of 11 and 13 nM, respectively, while 6j (IC50 = 17 nM) proved to be the most active inhibitor against BuChE with remarkable selectivity for BuChE over AChE. Molecular docking studies were also performed on human AChE and BuChE to suggest possible binding modes in which the inhibitor's extended structure is accommodated along the active site gorge of both enzymes. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

N- and O-glycosylation Analysis of Human C1-inhibitor Reveals Extensive Mucin-type O-Glycosylation.

Science.gov (United States)

Stavenhagen, Kathrin; Kayili, H Mehmet; Holst, Stephanie; Koeleman, Carolien A M; Engel, Ruchira; Wouters, Diana; Zeerleder, Sacha; Salih, Bekir; Wuhrer, Manfred

2018-06-01

Human C1-inhibitor (C1-Inh) is a serine protease inhibitor and the major regulator of the contact activation pathway as well as the classical and lectin complement pathways. It is known to be a highly glycosylated plasma glycoprotein. However, both the structural features and biological role of C1-Inh glycosylation are largely unknown. Here, we performed for the first time an in-depth site-specific N - and O -glycosylation analysis of C1-Inh combining various mass spectrometric approaches, including C18-porous graphitized carbon (PGC)-LC-ESI-QTOF-MS/MS applying stepping-energy collision-induced dissociation (CID) and electron-transfer dissociation (ETD). Various proteases were applied, partly in combination with PNGase F and exoglycosidase treatment, in order to analyze the (glyco)peptides. The analysis revealed an extensively O -glycosylated N-terminal region. Five novel and five known O -glycosylation sites were identified, carrying mainly core1-type O -glycans. In addition, we detected a heavily O -glycosylated portion spanning from Thr 82 -Ser 121 with up to 16 O -glycans attached. Likewise, all known six N -glycosylation sites were covered and confirmed by this site-specific glycosylation analysis. The glycoforms were in accordance with results on released N -glycans by MALDI-TOF/TOF-MS/MS. The comprehensive characterization of C1-Inh glycosylation described in this study will form the basis for further functional studies on the role of these glycan modifications. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Acetylation site specificities of lysine deacetylase inhibitors in human cells

DEFF Research Database (Denmark)

Schölz, Christian; Weinert, Brian Tate; Wagner, Sebastian A

2015-01-01

Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acety......1-α, providing a possible mechanistic explanation of its adverse, pro-inflammatory effects. Our results offer a systems view of KDACI specificities, providing a framework for studying function of acetylation and deacetylases....

Isotope-edited proton NMR study on the structure of a pepsin/inhibitor complex

International Nuclear Information System (INIS)

Fesik, S.W.; Luly, J.R.; Erickson, J.W.; Abad-Zapatero, C.

1988-01-01

A general approach is illustrated for providing detailed structural information on large enzyme/inhibitor complexes using NMR spectroscopy. The method involves the use of isotopically labeled ligands to simplify two-dimensional NOE spectra of large molecular complexes by isotope-editing techniques. With this approach, the backbone and side-chain conformations (at the P 2 and P 3 sites) of a tightly bound inhibitor of porcine pepsin have bene determined. In addition, structural information on the active site of pepsin has been obtained. Due to the sequence homology between porcine pepsin and human renin, this structural information may prove useful for modeling renin/inhibitor complexes with the ultimate goal of designing more effective renin inhibitors. Moreover, this general approach can be applied to study other biological systems of interest such as other enzyme/inhibitor complexes, ligands bound to soluble receptors, and enzyme/substrate interactions

Proton pump inhibitors inhibit pancreatic secretion

DEFF Research Database (Denmark)

Wang, Jing; Barbuskaite, Dagne; Tozzi, Marco

2015-01-01

+/K+-ATPases are expressed and functional in human pancreatic ducts and whether proton pump inhibitors (PPIs) have effect on those. Here we show that the gastric HKα1 and HKβ subunits (ATP4A; ATP4B) and non-gastric HKα2 subunits (ATP12A) of H+/K+-ATPases are expressed in human pancreatic cells. Pumps have similar...... of major ions in secretion follow similar excretory curves in control and PPI treated animals. In addition to HCO3-, pancreas also secretes K+. In conclusion, this study calls for a revision of the basic model for HCO3- secretion. We propose that proton transport is driving secretion, and that in addition...

5 alpha-reductase inhibitors and prostatic disease.

Science.gov (United States)

Schröder, F H

1994-08-01

5 alpha-Reductase inhibitors are a new class of substances with very specific effects on type I and type II 5 alpha R which may be of use in the treatment of skin disease, such as male pattern baldness, male acne and hirsutism, as well as prostatic hyperplasia and prostate cancer. At least two types of 5 alpha R inhibitors with a different pH optimum have been described. cDNA encoding for both the type I and the type II enzyme has been cloned. Most of the orally effective 5 alpha R inhibitors belong to the class of 4-azasteroids. The radical substituted in the 17 position of the steroid ring seems to be related to species specific variations and to the types of 5 alpha R enzymes in different species and organ systems. 5 alpha R inhibitors lead to a decrease of plasma DHT by about 65% while there is a slight rise in plasma testosterone. The decrease of tissue DHT in the ventral prostate of the intact rat, the dog and in humans is more pronounced and amounts to about 85%. There is a reciprocal rise of tissue T in these systems. The application of an inhibitor of 5 alpha R type II leads to a shrinkage of BPH in men by about 30%. In the rat a similar shrinkage accompanied by a significant decrease of total organ DNA occurs. This decrease, however, is not as pronounced as can be achieved with castration.(ABSTRACT TRUNCATED AT 250 WORDS)

Glyceroneogenesis is inhibited through HIV protease inhibitor-induced inflammation in human subcutaneous but not visceral adipose tissue

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Leroyer, Stéphanie; Vatier, Camille; Kadiri, Sarah; Quette, Joëlle; Chapron, Charles; Capeau, Jacqueline; Antoine, Bénédicte

2011-01-01

Glyceroneogenesis, a metabolic pathway that participates during lipolysis in the recycling of free fatty acids to triglycerides into adipocytes, contributes to the lipid-buffering function of adipose tissue. We investigated whether glyceroneogenesis could be affected by human immunodeficiency virus (HIV) protease inhibitors (PIs) responsible or not for dyslipidemia in HIV-infected patients. We treated explants obtained from subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) depots from lean individuals. We observed that the dyslipidemic PIs nelfinavir, lopinavir and ritonavir, but not the lipid-neutral PI atazanavir, increased lipolysis and decreased glyceroneogenesis, leading to an increased release of fatty acids from SAT but not from VAT. At the same time, dyslipidemic PIs decreased the amount of perilipin and increased interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) secretion in SAT but not in VAT. Parthenolide, an inhibitor of the NFκB pathway, counteracted PI-induced increased inflammation and decreased glyceroneogenesis. IL-6 (100 ng) inhibited the activity of phosphoenolpyruvate carboxykinase, the key enzyme of glyceroneogenesis, in SAT but not in VAT. Our data show that dyslipidemic but not lipid-neutral PIs decreased glyceroneogenesis as a consequence of PI-induced increased inflammation in SAT that could have an affect on adipocytes and/or macrophages. These results add a new link between fat inflammation and increased fatty acids release and suggest a greater sensitivity of SAT than VAT to PI-induced inflammation. PMID:21068005

Synergism of Antifungal Activity between Mitochondrial Respiration Inhibitors and Kojic Acid

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Ronald P. Haff

2013-01-01

Full Text Available Co-application of certain types of compounds to conventional antimicrobial drugs can enhance the efficacy of the drugs through a process termed chemosensitization. We show that kojic acid (KA, a natural pyrone, is a potent chemosensitizing agent of complex III inhibitors disrupting the mitochondrial respiratory chain in fungi. Addition of KA greatly lowered the minimum inhibitory concentrations of complex III inhibitors tested against certain filamentous fungi. Efficacy of KA synergism in decreasing order was pyraclostrobin > kresoxim-methyl > antimycin A. KA was also found to be a chemosensitizer of cells to hydrogen peroxide (H2O2, tested as a mimic of reactive oxygen species involved in host defense during infection, against several human fungal pathogens and Penicillium strains infecting crops. In comparison, KA-mediated chemosensitization to complex III inhibitors/H2O2 was undetectable in other types of fungi, including Aspergillus flavus, A. parasiticus, and P. griseofulvum, among others. Of note, KA was found to function as an antioxidant, but not as an antifungal chemosensitizer in yeasts. In summary, KA could serve as an antifungal chemosensitizer to complex III inhibitors or H2O2 against selected human pathogens or Penicillium species. KA-mediated chemosensitization to H2O2 seemed specific for filamentous fungi. Thus, results indicate strain- and/or drug-specificity exist during KA chemosensitization.

Probing the Inhibitor versus Chaperone Properties of sp2-Iminosugars towards Human β-Glucocerebrosidase: A Picomolar Chaperone for Gaucher Disease

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Teresa Mena-Barragán

2018-04-01

Full Text Available A series of sp2-iminosugar glycomimetics differing in the reducing or nonreducing character, the configurational pattern (d-gluco or l-ido, the architecture of the glycone skeleton, and the nature of the nonglycone substituent has been synthesized and assayed for their inhibition properties towards commercial glycosidases. On the basis of their affinity and selectivity towards GH1 β-glucosidases, reducing and nonreducing bicyclic derivatives having a hydroxylation profile of structural complementarity with d-glucose and incorporating an N′-octyl-isourea or -isothiourea segment were selected for further evaluation of their inhibitory/chaperoning potential against human glucocerebrosidase (GCase. The 1-deoxynojirimycin (DNJ-related nonreducing conjugates behaved as stronger GCase inhibitors than the reducing counterparts and exhibited potent chaperoning capabilities in Gaucher fibroblasts hosting the neuronopathic G188S/G183W mutation, the isothiourea derivative being indeed one of the most efficient chaperone candidates reported up to date (70% activity enhancement at 20 pM. At their optimal concentration, the four selected compounds promoted mutant GCase activity enhancements over 3-fold; yet, the inhibitor/chaperoning balance became unfavorable at much lower concentration for nonreducing as compared to reducing derivatives.

6-(1-Benzyl-1H-pyrrol-2-yl)-2,4-dioxo-5-hexenoic acids as dual inhibitors of recombinant HIV-1 integrase and ribonuclease H, synthesized by a parallel synthesis approach.

Science.gov (United States)

Costi, Roberta; Métifiot, Mathieu; Esposito, Francesca; Cuzzucoli Crucitti, Giuliana; Pescatori, Luca; Messore, Antonella; Scipione, Luigi; Tortorella, Silvano; Zinzula, Luca; Novellino, Ettore; Pommier, Yves; Tramontano, Enzo; Marchand, Christophe; Di Santo, Roberto

2013-11-14

The increasing efficiency of HAART has helped to transform HIV/AIDS into a chronic disease. Still, resistance and drug-drug interactions warrant the development of new anti-HIV agents. We previously discovered hit 6, active against HIV-1 replication and targeting RNase H in vitro. Because of its diketo-acid moiety, we speculated that this chemotype could serve to develop dual inhibitors of both RNase H and integrase. Here, we describe a new series of 1-benzyl-pyrrolyl diketohexenoic derivatives, 7a-y and 8a-y, synthesized following a parallel solution-phase approach. Those 50 analogues have been tested on recombinant enzymes (RNase H and integrase) and in cell-based assays. Approximately half (22) exibited inhibition of HIV replication. Compounds 7b, 7u, and 8g were the most active against the RNase H activity of reverse-transcriptase, with IC50 values of 3, 3, and 2.5 μM, respectively. Compound 8g was also the most potent integrase inhibitor with an IC50 value of 26 nM.

Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

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Sanne H. Olesen

2015-01-01

Full Text Available The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein–protein interaction (PPI inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2 did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM, while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.

Isomeric mono-, di-, and tri-bromobenzo-1H-triazoles as inhibitors of human protein kinase CK2α.

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Romualda Wąsik

Full Text Available To further clarify the role of the individual bromine atoms of 4,5,6,7-tetrabromotriazole (TBBt, a relatively selective inhibitor of protein kinase CK2, we have examined the inhibition (IC(50 of human CK2α by the two mono-, the four di-, and the two tri- bromobenzotriazoles relative to that of TBBt. Halogenation of the central vicinal C(5/C(6 atoms proved to be a key factor in enhancing inhibitory activity, in that 5,6-di-Br(2Bt and 4,5,6-Br(3Bt were almost as effective inhibitors as TBBt, notwithstanding their marked differences in pK(a for dissociation of the triazole proton. The decrease in pK(a on halogenation of the peripheral C(4/C(7 atoms virtually nullifies the gain due to hydrophobic interactions, and does not lead to a decrease in IC(50. Molecular modeling of structures of complexes of the ligands with the enzyme, as well as QSAR analysis, pointed to a balance of hydrophobic and electrostatic interactions as a discriminator of inhibitory activity. The role of halogen bonding remains debatable, as originally noted for the crystal structure of TBBt with CK2α (pdb1j91. Finally we direct attention to the promising applicability of our series of well-defined halogenated benzotriazoles to studies on inhibition of kinases other than CK2.

Identification and Characterization of Roseltide, a Knottin-type Neutrophil Elastase Inhibitor Derived from Hibiscus sabdariffa

Science.gov (United States)

Loo, Shining; Kam, Antony; Xiao, Tianshu; Nguyen, Giang K. T.; Liu, Chuan Fa; Tam, James P.

2016-01-01

Plant knottins are of therapeutic interest due to their high metabolic stability and inhibitory activity against proteinases involved in human diseases. The only knottin-type proteinase inhibitor against porcine pancreatic elastase was first identified from the squash family in 1989. Here, we report the identification and characterization of a knottin-type human neutrophil elastase inhibitor from Hibiscus sabdariffa of the Malvaceae family. Combining proteomic and transcriptomic methods, we identified a panel of novel cysteine-rich peptides, roseltides (rT1-rT8), which range from 27 to 39 residues with six conserved cysteine residues. The 27-residue roseltide rT1 contains a cysteine spacing and amino acid sequence that is different from the squash knottin-type elastase inhibitor. NMR analysis demonstrated that roseltide rT1 adopts a cystine-knot fold. Transcriptome analyses suggested that roseltides are bioprocessed by asparagine endopeptidases from a three-domain precursor. The cystine-knot structure of roseltide rT1 confers its high resistance against degradation by endopeptidases, 0.2 N HCl, and human serum. Roseltide rT1 was shown to inhibit human neutrophil elastase using enzymatic and pull-down assays. Additionally, roseltide rT1 ameliorates neutrophil elastase-stimulated cAMP accumulation in vitro. Taken together, our findings demonstrate that roseltide rT1 is a novel knottin-type neutrophil elastase inhibitor with therapeutic potential for neutrophil elastase associated diseases. PMID:27991569

The human complement inhibitor Sushi Domain-Containing Protein 4 (SUSD4) expression in tumor cells and infiltrating T cells is associated with better prognosis of breast cancer patients

OpenAIRE

Englund, Emelie; Reitsma, Bart; King, Ben C.; Escudero-Esparza, Astrid; Owen, Sioned; Orimo, Akira; Okroj, Marcin; Anagnostaki, Lola; Jiang, Wen G.; Jirström, Karin; Blom, Anna M.

2015-01-01

Background: The human Sushi Domain-Containing Protein 4 (SUSD4) was recently shown to function as a novel inhibitor of the complement system, but its role in tumor progression is unknown. \\ud \\ud Methods: Using immunohistochemistry and quantitative PCR, we investigated SUSD4 expression in breast cancer tissue samples from two cohorts. The effect of SUSD4 expression on cell migration and invasion was studied in vitro using two human breast cancer cell lines overexpressing SUSD4. \\ud \\ud Result...

Thermodynamics parameters for binding of halogenated benzotriazole inhibitors of human protein kinase CK2α.

Science.gov (United States)

Winiewska, Maria; Kucińska, Katarzyna; Makowska, Małgorzata; Poznański, Jarosław; Shugar, David

2015-10-01

The interaction of human CK2α (hCK2α) with nine halogenated benzotriazoles, TBBt and its analogues representing all possible patterns of halogenation on the benzene ring of benzotriazole, was studied by biophysical methods. Thermal stability of protein-ligand complexes, monitored by calorimetric (DSC) and optical (DSF) methods, showed that the increase in the mid-point temperature for unfolding of protein-ligand complexes (i.e. potency of ligand binding to hCK2α) follow the inhibitory activities determined by biochemical assays. The dissociation constant for the ATP-hCK2α complex was estimated with the aid of microscale thermophoresis (MST) as 4.3±1.8 μM, and MST-derived dissociation constants determined for halogenated benzotriazoles, when converted according to known ATP concentrations, perfectly reconstruct IC50 values determined by the biochemical assays. Ligand-dependent quenching of tyrosine fluorescence, together with molecular modeling and DSC-derived heats of unfolding, support the hypothesis that halogenated benzotriazoles bind in at least two alternative orientations, and those that are efficient hCK2α inhibitors bind in the orientation which TBBt adopts in its complex with maize CK2α. DSC-derived apparent heat for ligand binding (ΔΔHbind) is driven by intermolecular electrostatic interactions between Lys68 and the triazole ring of the ligand, as indicated by a good correlation between ΔΔHbind and ligand pKa. Overall results, additionally supported by molecular modeling, confirm that a balance of hydrophobic and electrostatic interactions contribute predominantly (~40 kJ/mol), relative to possible intermolecular halogen/hydrogen bonding (less than 10 kJ/mol), in binding of halogenated benzotriazoles to the ATP-binding site of hCK2α. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. Copyright © 2015 Elsevier B.V. All rights reserved.

Structural and biophysical analysis of interactions between cod and human uracil-DNA N-glycosylase (UNG) and UNG inhibitor (Ugi)

Energy Technology Data Exchange (ETDEWEB)

Assefa, Netsanet Gizaw [UiT The Arctic University of Norway, 9037 Tromsø (Norway); Niiranen, Laila [UiT The Arctic University of Norway, 9037 Tromsø (Norway); University of Turku, FIN-20014 Turku (Finland); Johnson, Kenneth A.; Leiros, Hanna-Kirsti Schrøder; Smalås, Arne Oskar; Willassen, Nils Peder [UiT The Arctic University of Norway, 9037 Tromsø (Norway); Moe, Elin, E-mail: elin.moe@uit.no [UiT The Arctic University of Norway, 9037 Tromsø (Norway); Universidade Nova de Lisboa, Avenida da Republica (EAN), 2780-157 Oeiras (Portugal)

2014-08-01

A structural and biophysical study of the interactions between cod and human uracil-DNA N-glycosylase (UNG) and their inhibitor Ugi is presented. The stronger interaction between cod UNG and Ugi can be explained by a greater positive electrostatic surface potential. Uracil-DNA N-glycosylase from Atlantic cod (cUNG) shows cold-adapted features such as high catalytic efficiency, a low temperature optimum for activity and reduced thermal stability compared with its mesophilic homologue human UNG (hUNG). In order to understand the role of the enzyme–substrate interaction related to the cold-adapted properties, the structure of cUNG in complex with a bacteriophage encoded natural UNG inhibitor (Ugi) has been determined. The interaction has also been analyzed by isothermal titration calorimetry (ITC). The crystal structure of cUNG–Ugi was determined to a resolution of 1.9 Å with eight complexes in the asymmetric unit related through noncrystallographic symmetry. A comparison of the cUNG–Ugi complex with previously determined structures of UNG–Ugi shows that they are very similar, and confirmed the nucleotide-mimicking properties of Ugi. Biophysically, the interaction between cUNG and Ugi is very strong and shows a binding constant (K{sub b}) which is one order of magnitude larger than that for hUNG–Ugi. The binding of both cUNG and hUNG to Ugi was shown to be favoured by both enthalpic and entropic forces; however, the binding of cUNG to Ugi is mainly dominated by enthalpy, while the entropic term is dominant for hUNG. The observed differences in the binding properties may be explained by an overall greater positive electrostatic surface potential in the protein–Ugi interface of cUNG and the slightly more hydrophobic surface of hUNG.

Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

DEFF Research Database (Denmark)

Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter Durand

2003-01-01

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosyla...

Two structurally distinct inhibitors of glycogen synthase kinase 3 induced centromere positive micronuclei in human lymphoblastoid TK6 cells.

Science.gov (United States)

Mishima, Masayuki; Tanaka, Kenji; Takeiri, Akira; Harada, Asako; Kubo, Chiyomi; Sone, Sachiko; Nishimura, Yoshikazu; Tachibana, Yukako; Okazaki, Makoto

2008-08-25

Glycogen synthase kinase 3 (GSK3) is an attractive novel pharmacological target. Inhibition of GSK3 is recently regarded as one of the viable approaches to therapy for Alzheimer's disease, cancer, diabetes mellitus, osteoporosis, and bipolar mood disorder. Here, we have investigated the aneugenic potential of two potent and highly specific inhibitors of GSK3 by using an in vitro micronucleus test with human lymphoblastoid TK6 cells. One inhibitor was a newly synthesized maleimide derivative and the other was a previously known aminopyrimidine derivative. Both compounds elicited statistically significant and concentration-dependent increases in micronucleated cells. One hundred micronuclei (MN) of each were analyzed using centromeric DNA staining with fluorescence in situ hybridization. Both the two structurally distinct compounds induced centromere-positive micronuclei (CMN). Calculated from the frequency of MN cells and the percentage of CMN, CMN cell incidence after treatment with the maleimide compound at 1.2 microM, 2.4 microM, and 4.8 microM was 11.6, 27.7, and 56.3 per 1000 cells, respectively; the negative control was 4.5. CMN cell incidence after the treatment with the aminopyrimidine compound at 1.8 microM, 3.6 microM, and 5.4 microM was 6.7, 9.8 and 17.2 per 1000 cells, respectively. Both compounds exhibited concentration-dependent increase in the number of mitotic cells. The frequency of CMN cells correlated well with mitotic cell incidence after treatment with either compound. Furthermore, both inhibitors induced abnormal mitotic cells with asymmetric mitotic spindles and lagging anaphase chromosomes. These results lend further support to the hypothesis that the inhibition of GSK3 activity affects microtubule function and exhibits an aneugenic mode of action.

Chemical structure and properties of low-molecular furin inhibitors

Directory of Open Access Journals (Sweden)

T. V. Osadchuk

2016-12-01

Full Text Available The review is devoted to the analysis of the relationship between a chemical structure and properties of low-molecular weight inhibitors of furin, the most studied proprotein convertase, which is involved in the development of some pathologies, such as oncologic diseases, viral and bacterial infections, etc. The latest data concerning the influence of peptides, pseudo-peptides, aromatic and heterocyclic compounds, some natural ones such as flavonoids, coumarins, and others on enzyme inactivation are considered. The power of furin inhibition is shown to rise with the increasing number of positively charged groups in the structure of these compounds. Peptidomimetics (Ki = 5-8 pM are shown to be the most effective furin inhibitors. The synthesized substances, however, have not been used in practical application yet. Nowadays it is very important to find more selective inhibitors, improve their stability, bioavailability and safety for the human organism.

Vorinostat, an HDAC inhibitor attenuates epidermoid squamous cell carcinoma growth by dampening mTOR signaling pathway in a human xenograft murine model

Energy Technology Data Exchange (ETDEWEB)

Kurundkar, Deepali; Srivastava, Ritesh K.; Chaudhary, Sandeep C. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States); Ballestas, Mary E. [Department of Pediatrics Infectious Disease, Children' s of Alabama, School of Medicine, University of Alabama at Birmingham, AL (United States); Kopelovich, Levy [Division of Cancer Prevention, National Cancer Institute, 6130 Executive Blvd., Suite 2114, Bethesda, MD 20892 (United States); Elmets, Craig A. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States)

2013-01-15

Histone deacetylase (HDAC) inhibitors are potent anticancer agents and show efficacy against various human neoplasms. Vorinostat is a potent HDAC inhibitor and has shown potential to inhibit growth of human xenograft tumors. However, its effect on the growth of skin neoplasm remains undefined. In this study, we show that vorinostat (2 μM) reduced expression of HDAC1, 2, 3, and 7 in epidermoid carcinoma A431 cells. Consistently, it increased acetylation of histone H3 and p53. Vorinostat (100 mg/kg body weight, IP) treatment reduced human xenograft tumor growth in highly immunosuppressed nu/nu mice. Histologically, the vorinostat-treated tumor showed features of well-differentiation with large necrotic areas. Based on proliferating cell nuclear antigen (PCNA) staining and expression of cyclins D1, D2, E, and A, vorinostat seems to impair proliferation by down-regulating the expression of these proteins. However, it also induced apoptosis. The mechanism by which vorinostat blocks proliferation and makes tumor cells prone to apoptosis, involved inhibition of mTOR signaling which was accompanied by reduction in cell survival AKT and extracellular-signal regulated kinase (ERK) signaling pathways. Our data provide a novel mechanism-based therapeutic intervention for cutaneous squamous cell carcinoma (SCC). Vorinostat may be utilized to cure skin neoplasms in organ transplant recipient (OTR). These patients have high morbidity and surgical removal of these lesions which frequently develop in these patients, is difficult. -- Highlights: ► Vorinostat reduces SCC growth in a xenograft murine model. ► Vorinostat dampens proliferation and induces apoptosis in tumor cells. ► Diminution in mTOR, Akt and ERK signaling underlies inhibition in proliferation. ► Vorinostat by inhibiting HDACs inhibits epithelial–mesenchymal transition.

Vorinostat, an HDAC inhibitor attenuates epidermoid squamous cell carcinoma growth by dampening mTOR signaling pathway in a human xenograft murine model

International Nuclear Information System (INIS)

Kurundkar, Deepali; Srivastava, Ritesh K.; Chaudhary, Sandeep C.; Ballestas, Mary E.; Kopelovich, Levy; Elmets, Craig A.; Athar, Mohammad

2013-01-01

Histone deacetylase (HDAC) inhibitors are potent anticancer agents and show efficacy against various human neoplasms. Vorinostat is a potent HDAC inhibitor and has shown potential to inhibit growth of human xenograft tumors. However, its effect on the growth of skin neoplasm remains undefined. In this study, we show that vorinostat (2 μM) reduced expression of HDAC1, 2, 3, and 7 in epidermoid carcinoma A431 cells. Consistently, it increased acetylation of histone H3 and p53. Vorinostat (100 mg/kg body weight, IP) treatment reduced human xenograft tumor growth in highly immunosuppressed nu/nu mice. Histologically, the vorinostat-treated tumor showed features of well-differentiation with large necrotic areas. Based on proliferating cell nuclear antigen (PCNA) staining and expression of cyclins D1, D2, E, and A, vorinostat seems to impair proliferation by down-regulating the expression of these proteins. However, it also induced apoptosis. The mechanism by which vorinostat blocks proliferation and makes tumor cells prone to apoptosis, involved inhibition of mTOR signaling which was accompanied by reduction in cell survival AKT and extracellular-signal regulated kinase (ERK) signaling pathways. Our data provide a novel mechanism-based therapeutic intervention for cutaneous squamous cell carcinoma (SCC). Vorinostat may be utilized to cure skin neoplasms in organ transplant recipient (OTR). These patients have high morbidity and surgical removal of these lesions which frequently develop in these patients, is difficult. -- Highlights: ► Vorinostat reduces SCC growth in a xenograft murine model. ► Vorinostat dampens proliferation and induces apoptosis in tumor cells. ► Diminution in mTOR, Akt and ERK signaling underlies inhibition in proliferation. ► Vorinostat by inhibiting HDACs inhibits epithelial–mesenchymal transition.

Live-cell microscopy reveals small molecule inhibitor effects on MAPK pathway dynamics.

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Daniel J Anderson

Full Text Available Oncogenic mutations in the mitogen activated protein kinase (MAPK pathway are prevalent in human tumors, making this pathway a target of drug development efforts. Recently, ATP-competitive Raf inhibitors were shown to cause MAPK pathway activation via Raf kinase priming in wild-type BRaf cells and tumors, highlighting the need for a thorough understanding of signaling in the context of small molecule kinase inhibitors. Here, we present critical improvements in cell-line engineering and image analysis coupled with automated image acquisition that allow for the simultaneous identification of cellular localization of multiple MAPK pathway components (KRas, CRaf, Mek1 and Erk2. We use these assays in a systematic study of the effect of small molecule inhibitors across the MAPK cascade either as single agents or in combination. Both Raf inhibitor priming as well as the release from negative feedback induced by Mek and Erk inhibitors cause translocation of CRaf to the plasma membrane via mechanisms that are additive in pathway activation. Analysis of Erk activation and sub-cellular localization upon inhibitor treatments reveals differential inhibition and activation with the Raf inhibitors AZD628 and GDC0879 respectively. Since both single agent and combination studies of Raf and Mek inhibitors are currently in the clinic, our assays provide valuable insight into their effects on MAPK signaling in live cells.

Blockade of the ERK pathway markedly sensitizes tumor cells to HDAC inhibitor-induced cell death

International Nuclear Information System (INIS)

Ozaki, Kei-ichi; Minoda, Ai; Kishikawa, Futaba; Kohno, Michiaki

2006-01-01

Constitutive activation of the extracellular signal-regulated kinase (ERK) pathway is associated with the neoplastic phenotype of a large number of human tumor cells. Although specific blockade of the ERK pathway by treating such tumor cells with potent mitogen-activated protein kinase/ERK kinase (MEK) inhibitors completely suppresses their proliferation, it by itself shows only a modest effect on the induction of apoptotic cell death. However, these MEK inhibitors markedly enhance the efficacy of histone deacetylase (HDAC) inhibitors to induce apoptotic cell death: such an enhanced cell death is observed only in tumor cells in which the ERK pathway is constitutively activated. Co-administration of MEK inhibitor markedly sensitizes tumor cells to HDAC inhibitor-induced generation of reactive oxygen species, which appears to mediate the enhanced cell death induced by the combination of these agents. These results suggest that the combination of MEK inhibitors and HDAC inhibitors provides an efficient chemotherapeutic strategy for the treatment of tumor cells in which the ERK pathway is constitutively activated

The Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Cellular Senescence of Cultivated Human Corneal Endothelial Cells.

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Hongo, Akane; Okumura, Naoki; Nakahara, Makiko; Kay, EunDuck P; Koizumi, Noriko

2017-07-01

We have begun a clinical trial of a cell-based therapy for corneal endothelial dysfunction in Japan. The purpose of this study was to investigate the usefulness of a p38 MAPK inhibitor for prevention cellular senescence in cultivated human corneal endothelial cells (HCECs). HCECs of 10 donor corneas were divided and cultured with or without SB203580 (a p38 MAPK inhibitor). Cell density and morphology were evaluated by phase-contrast microscopy. Expression of function-related proteins was examined by immunofluorescent staining. Cellular senescence was evaluated by SA-β-gal staining and Western blotting for p16 and p21. Senescence-associated factors were evaluated by membrane blotting array, quantitative PCR, and ELISA. Phase-contrast microscopy showed a significantly higher cell density for HCECs cultured with SB203580 than without SB203580 (2623 ± 657 cells/mm2 and 1752 ± 628 cells/mm2, respectively). The HCECs cultured with SB203580 maintained a hexagonal morphology and expressed ZO-1, N-cadherin, and Na+/K+-ATPase in the plasma membrane, whereas the control HCECs showed an altered staining pattern for these marker proteins. HCECs cultured without SB203580 showed high positive SA-β-gal staining, a low nuclear/cytoplasm ratio, and expression of p16 and p21. IL-6, IL-8, CCL2, and CXCL1 were observed at high levels in low cell density HCECs cultured without SB203580. Activation of p38 MAPK signaling due to culture stress might be a causative factor that induces cellular senescence; therefore, the use of p38 MAPK inhibitor to counteract senescence may achieve sufficient numbers of HCECs for tissue engineering therapy for corneal endothelial dysfunction.

EGFR inhibitor C225 increases the radiosensitivity of human lung squamous cancer cells

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Yang Ruijie

2010-10-01

Full Text Available Abstract Background The purpose of the present study is to investigate the direct biological effects of the epidermal growth factor receptor (EGFR inhibitor C225 on the radiosensitivity of human lung squamous cancer cell-H520. H520 cells were treated with different dosage of 60Co γ ray irradiation (1.953 Gy/min in the presence or absence of C225. The cellular proliferation, colony forming capacity, apoptosis, the cell cycle distribution as well as caspase-3 were analyzed in vitro. Results We found that C225 treatment significantly increased radiosensitivity of H-520 cells to irradiation, and led to cell cycle arrest in G1 phase, whereas 60Co γ ray irradiation mainly caused G2 phase arrest. H-520 cells thus displayed both the G1 and G2 phase arrest upon treatment with C225 in combination with 60Co γ ray irradiation. Moreover, C225 treatment significantly increased the apoptosis percentage of H-520 cells (13.91% ± 1.88% compared with the control group (5.75% ± 0.64%, P Conclusion In this regard, C225 treatment may make H-520 cells more sensitive to irradiation through the enhancement of caspase-3 mediated tumor cell apoptosis and cell cycle arrest.

Genetics Home Reference: complete plasminogen activator inhibitor 1 deficiency

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... well studied in a large family belonging to the Old Order Amish population of eastern and southern Indiana. Additional cases in North ... Human plasminogen activator inhibitor-1 (PAI-1) deficiency: characterization of a large kindred with a null mutation in the PAI-1 gene. Blood. 1997 Jul 1;90( ...

Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies.

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Duong, Minh Ngoc; Matera, Eva-Laure; Mathé, Doriane; Evesque, Anne; Valsesia-Wittmann, Sandrine; Clémenceau, Béatrice; Dumontet, Charles

2015-01-01

Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated.

The structure of the human interferon alpha/beta receptor gene.

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Lutfalla, G; Gardiner, K; Proudhon, D; Vielh, E; Uzé, G

1992-02-05

Using the cDNA coding for the human interferon alpha/beta receptor (IFNAR), the IFNAR gene has been physically mapped relative to the other loci of the chromosome 21q22.1 region. 32,906 base pairs covering the IFNAR gene have been cloned and sequenced. Primer extension and solution hybridization-ribonuclease protection have been used to determine that the transcription of the gene is initiated in a broad region of 20 base pairs. Some aspects of the polymorphism of the gene, including noncoding sequences, have been analyzed; some are allelic differences in the coding sequence that induce amino acid variations in the resulting protein. The exon structure of the IFNAR gene and of that of the available genes for the receptors of the cytokine/growth hormone/prolactin/interferon receptor family have been compared with the predictions for the secondary structure of those receptors. From this analysis, we postulate a common origin and propose an hypothesis for the divergence from the immunoglobulin superfamily.

Perinatal administration of aromatase inhibitors in rodents as animal models of human male homosexuality: similarities and differences.

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Olvera-Hernández, Sandra; Fernández-Guasti, Alonso

2015-01-01

In this chapter we briefly review the evidence supporting the existence of biological influences on sexual orientation. We focus on basic research studies that have affected the estrogen synthesis during the critical periods of brain sexual differentiation in male rat offspring with the use of aromatase inhibitors, such as 1,4,6-androstatriene-3,17 (ATD) and letrozole. The results after prenatal and/or postnatal treatment with ATD reveal that these animals, when adults, show female sexual responses, such as lordosis or proceptive behaviors, but retain their ability to display male sexual activity with a receptive female. Interestingly, the preference and sexual behavior of these rats vary depending upon the circadian rhythm.Recently, we have established that the treatment with low doses of letrozole during the second half of pregnancy produces male rat offspring, that when adults spend more time in the company of a sexually active male than with a receptive female in a preference test. In addition, they display female sexual behavior when forced to interact with a sexually experienced male and some typical male sexual behavior when faced with a sexually receptive female. Interestingly, these males displayed both sexual behavior patterns spontaneously, i.e., in absence of exogenous steroid hormone treatment. Most of these features correspond with those found in human male homosexuals; however, the "bisexual" behavior shown by the letrozole-treated rats may be related to a particular human population. All these data, taken together, permit to propose letrozole prenatal treatment as a suitable animal model to study human male homosexuality and reinforce the hypothesis that human sexual orientation is underlied by changes in the endocrine milieu during early development.

Dehydroandrographolide, an iNOS inhibitor, extracted from Andrographis paniculata (Burm.f.) Nees, induces autophagy in human oral cancer cells.

Science.gov (United States)

Hsieh, Ming-Ju; Lin, Chiao-Wen; Chiou, Hui-Ling; Yang, Shun-Fa; Chen, Mu-Kuan

2015-10-13

Autophagy, which is constitutively executed at the basal level in all cells, promotes cellular homeostasis by regulating the turnover of organelles and proteins. Andrographolide and dehydroandrographolide (DA) are the two principle components of Andrographis paniculata (Burm.f.) Nees. and are the main contributors to its therapeutic properties. However, the pharmacological activities of dehydroandrographolide (DA) remain unclear. In this study, DA induces oral cancer cell death by activating autophagy. Treatment with autophagy inhibitors inhibited DA-induced human oral cancer cell death. In addition, DA increased LC3-II expression and reduced p53 expression in a time- and concentration-dependent manner. Furthermore, DA induced autophagy and decreased cell viability through modulation of p53 expression. DA-induced autophagy was triggered by an activation of JNK1/2 and an inhibition of Akt and p38. In conclusion, this study demonstrated that DA induced autophagy in human oral cancer cells by modulating p53 expression, activating JNK1/2, and inhibiting Akt and p38. Finally, an administration of DA effectively suppressed the tumor formation in the oral carcinoma xenograft model in vivo. This is the first study to reveal the novel function of DA in activating autophagy, suggesting that DA could serve as a new and potential chemopreventive agent for treating human oral cancer.

Protective effects of an aptamer inhibitor of neutrophil elastase in lung inflammatory injury

DEFF Research Database (Denmark)

Bless, N M; Smith, D; Charlton, J

1997-01-01

Neutrophils play an important part in the development of acute inflammatory injury. Human neutrophils contain high levels of the serine protease elastase, which is stored in azurophilic granules and is secreted in response to inflammatory stimuli. Elastase is capable of degrading many components...... of extracellular matrix [1-4] and has cytotoxic effects on endothelial cells [5-7] and airway epithelial cells. Three types of endogenous protease inhibitors control the activity of neutrophil elastase, including alpha-1 protease inhibitor (alpha-1PI), alpha-2 macroglobulin and secreted leukoproteinase inhibitor...... (SLPI) [8-10]. A disturbed balance between neutrophil elastase and these inhibitors has been found in various acute clinical conditions (such as adult respiratory syndrome and ischemia-reperfusion injury) and in chronic diseases. We investigated the effect of NX21909, a selected oligonucleotide (aptamer...

Novel 11β-hydroxysteroid dehydrogenase 1 inhibitors reduce cortisol levels in keratinocytes and improve dermal collagen content in human ex vivo skin after exposure to cortisone and UV.

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Stéphanie M Boudon

Full Text Available Activity and selectivity assessment of new bi-aryl amide 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1 inhibitors, prepared in a modular manner via Suzuki cross-coupling, are described. Several compounds inhibiting 11β-HSD1 at nanomolar concentrations were identified. Compounds 2b, 3e, 7b and 12e were shown to selectively inhibit 11β-HSD1 over 11β-HSD2, 17β-HSD1 and 17β-HSD2. These inhibitors also potently inhibited 11β-HSD1 activity in intact HEK-293 cells expressing the recombinant enzyme and in intact primary human keratinocytes expressing endogenous 11β-HSD1. Moreover, compounds 2b, 3e and 12e were tested for their activity in human skin biopsies. They were able to prevent, at least in part, both the cortisone- and the UV-mediated decreases in collagen content. Thus, inhibition of 11β-HSD1 by these compounds can be further investigated to delay or prevent UV-mediated skin damage and skin aging.

Human serum albumin binding assay based on displacement of a non selective fluorescent inhibitor.

Science.gov (United States)

Thorarensen, Atli; Sarver, Ronald W; Tian, Fang; Ho, Andrea; Romero, Donna L; Marotti, Keith R

2007-08-15

In this paper, we describe a fluorescent antibacterial analog, 6, with utility as a competition probe to determine affinities of other antibacterial analogs for human serum albumin (HSA). Analog 6 bound to HSA with an affinity of 400+/-100 nM and the fluorescence was environmentally sensitive. With 370 nm excitation, environmental sensitivity was indicated by a quenching of the 530 nm emission when the probe bound to HSA. Displacement of dansylsarcosine from HSA by 6 indicated it competed with compounds that bound at site II (ibuprofen binding site) on HSA. Analog 6 also shifted the NMR peaks of an HSA bound oleic acid molecule that itself was affected by compounds that bound at site II. In addition to binding at site II, 6 interacted at site I (warfarin binding site) as indicated by displacement of dansylamide and the shifting of NMR peaks of an HSA bound oleic acid molecule affected by warfarin site binding. Additional evidence for multiple site interaction was discovered when a percentage of 6 could be displaced by either ibuprofen or phenylbutazone. A competition assay was established using 6 to determine relative affinities of other antibacterial inhibitors for HSA.

The Structural Basis of Cryptosporidium-Specific IMP Dehydrogenase Inhibitor Selectivity

Energy Technology Data Exchange (ETDEWEB)

MacPherson, Iain S.; Kirubakaran, Sivapriya; Gorla, Suresh Kumar; Riera, Thomas V.; D’Aquino, J. Alejandro; Zhang, Minjia; Cuny, Gregory D.; Hedstrom, Lizbeth (BWH); (Brandeis)

2010-03-29

Cryptosporidium parvum is a potential biowarfare agent, an important AIDS pathogen, and a major cause of diarrhea and malnutrition. No vaccines or effective drug treatment exist to combat Cryptosporidium infection. This parasite relies on inosine 5{prime}-monophosphate dehydrogenase (IMPDH) to obtain guanine nucleotides, and inhibition of this enzyme blocks parasite proliferation. Here, we report the first crystal structures of CpIMPDH. These structures reveal the structural basis of inhibitor selectivity and suggest a strategy for further optimization. Using this information, we have synthesized low-nanomolar inhibitors that display 10{sup 3} selectivity for the parasite enzyme over human IMPDH2.

Pharmacological modulation of human platelet leukotriene C4-synthase.

Science.gov (United States)

Sala, A; Folco, G; Henson, P M; Murphy, R C

1997-03-21

The aim of this study was to test if human platelet leukotriene C4-synthase (LTC4-S) is pharmacologically different from cloned and expressed LTC4-S and, in light of the significant homologies between 5-lipoxygenase activating protein (FLAP) and LTC4-S, if different potencies of leukotriene synthesis inhibitors acting through binding with FLAP (FLAP inhibitors) reflect in different potencies as LTC4-S inhibitors. Leukotriene C4 (LTC4) synthesis by washed human platelets supplemented with synthetic leukotriene A4 (LTA4) was studied in the absence and presence of two different, structurally unrelated FLAP inhibitors (MK-886 and BAY-X1005) as well as a direct 5-lipoxygenase inhibitor (zileuton). LTC4 production was analyzed by RP-HPLC coupled to diode array detection. We report that human platelet LTC4-S was inhibited by MK-886 and BAY-X1005 (IC50 of 4.7 microM and 91.2 microM, respectively), but not by zileuton (inactive up to 300 microM); all 3 compounds were able to inhibit 5-lipoxygenase metabolite biosynthesis in intact human polymorphonuclear leukocytes (IC50 of 0.044 microM, 0.85 microM, and 1.5 microM, respectively). Platelet LTC4-S does not appear pharmacologically different from expression cloned LTC4-S. LTC4-S inhibition by FLAP inhibitors is in agreement with the significant homology reported for expression-cloned LTC4-S with FLAP, Furthermore, functional homology of the binding sites for inhibitors on LTC4-S and FLAP is suggested by the conservation of the relative potencies of MK-886 and BAY-X1005 vs FLAP-dependent 5-lipoxygenase activity and LTC4-S inhibition: MK-886 was 19.3-fold more potent than BAY-X1005 as FLAP inhibitor and 19.6-fold more potent than BAY-X1005 as LTC4-S inhibitor.

CoMFA and CoMSIA 3D-QSAR studies on S(6)-(4-nitrobenzyl)mercaptopurine riboside (NBMPR) analogs as inhibitors of human equilibrative nucleoside transporter 1 (hENT1).

Science.gov (United States)

Gupte, Amol; Buolamwini, John K

2009-01-15

3D-QSAR (CoMFA and CoMSIA) studies were performed on human equlibrative nucleoside transporter (hENT1) inhibitors displaying K(i) values ranging from 10,000 to 0.7nM. Both CoMFA and CoMSIA analysis gave reliable models with q2 values >0.50 and r2 values >0.92. The models have been validated for their stability and robustness using group validation and bootstrapping techniques and for their predictive abilities using an external test set of nine compounds. The high predictive r2 values of the test set (0.72 for CoMFA model and 0.74 for CoMSIA model) reveals that the models can prove to be a useful tool for activity prediction of newly designed nucleoside transporter inhibitors. The CoMFA and CoMSIA contour maps identify features important for exhibiting good binding affinities at the transporter, and can thus serve as a useful guide for the design of potential equilibrative nucleoside transporter inhibitors.

Clinical trials for BET inhibitors run ahead of the science.

Science.gov (United States)

Andrieu, Guillaume; Belkina, Anna C; Denis, Gerald V

2016-03-01

Several cancer clinical trials for small molecule inhibitors of BET bromodomain proteins have been initiated. There is enthusiasm for the anti-proliferative effect of inhibiting BRD4, one of the targets of these inhibitors, which is thought to cooperate with MYC, a long-desired target for cancer therapeutics. However, no current inhibitor is selective for BRD4 among the three somatic BET proteins, which include BRD2 and BRD3; their respective functions are partially overlapping and none are functionally redundant with BRD4. Each BET protein controls distinct transcriptional pathways that are important for functions beyond cancer cell proliferation, including insulin production, cytokine gene transcription, T cell differentiation, adipogenesis and most seriously, active repression of dangerous latent viruses like HIV. BET inhibitors have been shown to reactivate HIV in human cells. Failure to appreciate that at concentrations used, no available BET inhibitor is member-selective, or to develop a sound biological basis to understand the diverse functions of BET proteins before undertaking for these clinical trials is reckless and likely to lead to adverse events. More mechanistic information from new basic science studies should enable proper focus on the most relevant cancers and define the expected side effect profiles. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparative Amino Acid Decomposition Analysis of Potent Type I P38α Inhibitors

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Ahmad Ebadi

2013-05-01

Full Text Available Background and purpose of the study:p38α is a member of mitogen-activated protein kinases (MAPK considered as a prominent target in development of anti-inflammatory agents. Any abnormality in the phosphorylation process leads to the different human diseases such as cancer, diabetes and inflammatory diseases. Several small molecule p38α inhibitors have been developed up to now. In this regard, structural elucidation of p38 inhibitors needs to be done enabling us in rational lead development strategies.Methods:Various interactions of three potent inhibitors with p38α active site have been evaluated in terms of binding energies and bond lengths via density function theory and MD simulations.Results:Our comparative study showed that both ab initio and MD simulation led to the relatively similar results in pharmacophore discrimination of p38α inhibitors.Conclusion:The results of the present study may find their usefulness in pharmacophore based modification of p38α inhibitors.

Canine osteosarcoma cells exhibit resistance to aurora kinase inhibitors.

Science.gov (United States)

Cannon, C M; Pozniak, J; Scott, M C; Ito, D; Gorden, B H; Graef, A J; Modiano, J F

2015-03-01

We evaluated the effect of Aurora kinase inhibitors AZD1152 and VX680 on canine osteosarcoma cells. Cytotoxicity was seen in all four cell lines; however, half-maximal inhibitory concentrations were significantly higher than in human leukaemia and canine lymphoma cells. AZD1152 reduced Aurora kinase B phosphorylation, indicating resistance was not because of failure of target recognition. Efflux mediated by ABCB1 and ABCG2 transporters is one known mechanism of resistance against these drugs and verapamil enhanced AZD1152-induced apoptosis; however, these transporters were only expressed by a small percentage of cells in each line and the effects of verapamil were modest, suggesting other mechanisms contribute to resistance. Our results indicate that canine osteosarcoma cells are resistant to Aurora kinase inhibitors and suggest that these compounds are unlikely to be useful as single agents for this disease. Further investigation of these resistance mechanisms and the potential utility of Aurora kinase inhibitors in multi-agent protocols is warranted. © 2013 Blackwell Publishing Ltd.

Comparison of GLUT1, GLUT3, and GLUT4 mRNA and the subcellular distribution of their proteins in normal human muscle

Science.gov (United States)

Stuart, C. A.; Wen, G.; Gustafson, W. C.; Thompson, E. A.

2000-01-01

Basal, "insulin-independent" glucose uptake into skeletal muscle is provided by glucose transporters positioned at the plasma membrane. The relative amount of the three glucose transporters expressed in muscle has not been previously quantified. Using a combination of qualitative and quantitative ribonuclease protection assay (RPA) methods, we found in normal human muscle that GLUT1, GLUT3, and GLUT4 mRNA were expressed at 90 +/- 10, 46 +/- 4, and 156 +/- 12 copies/ng RNA, respectively. Muscle was fractionated by DNase digestion and differential sedimentation into membrane fractions enriched in plasma membranes (PM) or low-density microsomes (LDM). GLUT1 and GLUT4 proteins were distributed 57% to 67% in LDM, whereas GLUT3 protein was at least 88% in the PM-enriched fractions. These data suggest that basal glucose uptake into resting human muscle could be provided in part by each of these three isoforms.

A bioavailable cathepsin S nitrile inhibitor abrogates tumor development.

Science.gov (United States)

Wilkinson, Richard D A; Young, Andrew; Burden, Roberta E; Williams, Rich; Scott, Christopher J

2016-04-21

Cathepsin S has been implicated in a variety of malignancies with genetic ablation studies demonstrating a key role in tumor invasion and neo-angiogenesis. Thus, the application of cathepsin S inhibitors may have clinical utility in the treatment of cancer. In this investigation, we applied a cell-permeable dipeptidyl nitrile inhibitor of cathepsin S, originally developed to target cathepsin S in inflammatory diseases, in both in vitro and in vivo tumor models. Validation of cathepsin S selectivity was carried out by assaying fluorogenic substrate turnover using recombinant cathepsin protease. Complete kinetic analysis was carried out and true K i values calculated. Abrogation of tumour invasion using murine MC38 and human MCF7 cell lines were carried out in vitro using a transwell migration assay. Effect on endothelial tube formation was evaluated using primary HUVEC cells. The effect of inhibitor in vivo on MC38 and MCF7 tumor progression was evaluated using cells propagated in C57BL/6 and BALB/c mice respectively. Subsequent immunohistochemical staining of proliferation (Ki67) and apoptosis (TUNEL) was carried out on MCF7 tumors. We confirmed that this inhibitor was able to selectively target cathepsin S over family members K, V, L and B. The inhibitor also significantly reduced MC38 and MCF7 cell invasion and furthermore, significantly reduced HUVEC endothelial tubule formation in vitro. In vivo analysis revealed that the compound could significantly reduce tumor volume in murine MC38 syngeneic and MCF7 xenograft models. Immunohistochemical analysis of MCF7 tumors revealed cathepsin S inhibitor treatment significantly reduced proliferation and increased apoptosis. In summary, these results highlight the characterisation of this nitrile cathepsin S inhibitor using in vitro and in vivo tumor models, presenting a compound which may be used to further dissect the role of cathepsin S in cancer progression and may hold therapeutic potential.

Development of a monoclonal antibody that specifically detects tissue inhibitor of metalloproteinase-4 (TIMP-4) in formalin-fixed, paraffin-embedded human tissues.

Science.gov (United States)

Donover, P Scott; Wojciechowski, Brian S; Thirumaran, Rajesh; Zemba-Palko, Vlasta; Prendergast, George C; Wallon, U Margaretha

2010-08-01

Overexpression of the extracellular metalloproteinase inhibitor TIMP-4 in estrogen receptor-negative breast cancers was found recently to be associated with a poor prognosis for survival. To pursue exploration of the theranostic applications of TIMP-4, specific antibodies with favorable properties for immunohistochemical use and other clinical assays are needed. Here we report the characterization of a monoclonal antibody (clone 9:4-7) specific for full-length human TIMP-4 with suitable qualities. The antibody was determined to be an IgG(2b) immunoglobulin. In enzyme-linked immunosorbent assay (ELISA) and immunoblotting assays, it did not exhibit any detectable crossreactivity with recombinant forms of the other human TIMPs 1, 2, and 3. In contrast, the antibody displayed high specificity and sensitivity for TIMP-4 including in formalin-fixed and paraffin-embedded specimens of human breast specimens. An analysis of tissue microarrays of human cancer and corresponding normal tissues revealed specific staining patterns with excellent signal-to-noise ratios. This study documents TIMP-4 monoclonal antibody clone 9:4-7 as an effective tool for preclinical and clinical investigations. Published 2010 Wiley-Liss, Inc.

Molecular design and structure--activity relationships leading to the potent, selective, and orally active thrombin active site inhibitor BMS-189664.

Science.gov (United States)

Das, Jagabandhu; Kimball, S David; Hall, Steven E; Han, Wen Ching; Iwanowicz, Edwin; Lin, James; Moquin, Robert V; Reid, Joyce A; Sack, John S; Malley, Mary F; Chang, Chiehying Y; Chong, Saeho; Wang-Iverson, David B; Roberts, Daniel G M; Seiler, Steven M; Schumacher, William A; Ogletree, Martin L

2002-01-07

A series of structurally novel small molecule inhibitors of human alpha-thrombin was prepared to elucidate their structure-activity relationships (SARs), selectivity and activity in vivo. BMS-189664 (3) is identified as a potent, selective, and orally active reversible inhibitor of human alpha-thrombin which is efficacious in vivo in a mouse lethality model, and at inhibiting both arterial and venous thrombosis in cynomolgus monkey models.

Raltegravir: first in class HIV integrase inhibitor

Directory of Open Access Journals (Sweden)

Zelalem Temesgen

2008-06-01

Full Text Available Zelalem Temesgen1, Dawd S Siraj21Mayo Clinic, Rochester, MN, USA; 2East Carolina University Greenville, NC, USAAbstract: On October 16, 2007, the US Food and Drug Administration (FDA approved raltegravir for treatment of human immunodeficiency virus (HIV-1 infection in combination with other antiretroviral agents in treatment-experienced adult patients who have evidence of viral replication and HIV-1 strains resistant to multiple antiretroviral agents. Raltegravir is first in a novel class of antiretroviral drugs known as integrase inhibitors. It has demonstrated potent anti HIV activity in both antiretroviral treatment-naïve and experienced patients. The most common adverse events reported with raltegravir during phase 2 and 3 clinical trials were diarrhea, nausea, and headache. Laboratory abnormalities include mild elevations in liver transaminases and creatine phosphokinase.Keywords: raltegravir, HIV, antiretroviral agents, integrase inhibitors

Potent peptidic fusion inhibitors of influenza virus

Energy Technology Data Exchange (ETDEWEB)

Kadam, Rameshwar U.; Juraszek, Jarek; Brandenburg, Boerries; Buyck, Christophe; Schepens, Wim B. G.; Kesteleyn, Bart; Stoops, Bart; Vreeken, Rob J.; Vermond, Jan; Goutier, Wouter; Tang, Chan; Vogels, Ronald; Friesen, Robert H. E.; Goudsmit, Jaap; van Dongen, Maria J. P.; Wilson, Ian A.

2017-09-28

Influenza therapeutics with new targets and mechanisms of action are urgently needed to combat potential pandemics, emerging viruses, and constantly mutating strains in circulation. We report here on the design and structural characterization of potent peptidic inhibitors of influenza hemagglutinin. The peptide design was based on complementarity-determining region loops of human broadly neutralizing antibodies against the hemagglutinin (FI6v3 and CR9114). The optimized peptides exhibit nanomolar affinity and neutralization against influenza A group 1 viruses, including the 2009 H1N1 pandemic and avian H5N1 strains. The peptide inhibitors bind to the highly conserved stem epitope and block the low pH–induced conformational rearrangements associated with membrane fusion. These peptidic compounds and their advantageous biological properties should accelerate the development of new small molecule– and peptide-based therapeutics against influenza virus.

ETS1 mediates MEK1/2-dependent overexpression of cancerous inhibitor of protein phosphatase 2A (CIP2A in human cancer cells.

Directory of Open Access Journals (Sweden)

Anchit Khanna

2011-03-01

Full Text Available EGFR-MEK-ERK signaling pathway has an established role in promoting malignant growth and disease progression in human cancers. Therefore identification of transcriptional targets mediating the oncogenic effects of the EGFR-MEK-ERK pathway would be highly relevant. Cancerous inhibitor of protein phosphatase 2A (CIP2A is a recently characterized human oncoprotein. CIP2A promotes malignant cell growth and is over expressed at high frequency (40-80% in most of the human cancer types. However, the mechanisms inducing its expression in cancer still remain largely unexplored. Here we present systematic analysis of contribution of potential gene regulatory mechanisms for high CIP2A expression in cancer. Our data shows that evolutionary conserved CpG islands at the proximal CIP2A promoter are not methylated both in normal and cancer cells. Furthermore, sequencing of the active CIP2A promoter region from altogether seven normal and malignant cell types did not reveal any sequence alterations that would increase CIP2A expression specifically in cancer cells. However, treatment of cancer cells with various signaling pathway inhibitors revealed that CIP2A mRNA expression was sensitive to inhibition of EGFR activity as well as inhibition or activation of MEK-ERK pathway. Moreover, MEK1/2-specific siRNAs decreased CIP2A protein expression. Series of CIP2A promoter-luciferase constructs were created to identify proximal -27 to -107 promoter region responsible for MEK-dependent stimulation of CIP2A expression. Additional mutagenesis and chromatin immunoprecipitation experiments revealed ETS1 as the transcription factor mediating stimulation of CIP2A expression through EGFR-MEK pathway. Thus, ETS1 is probably mediating high CIP2A expression in human cancers with increased EGFR-MEK1/2-ERK pathway activity. These results also suggest that in addition to its established role in invasion and angiogenesis, ETS1 may support malignant cellular growth via regulation of

Proton pump inhibitor chemosensitization in human osteosarcoma: from the bench to the patients' bed.

Science.gov (United States)

Ferrari, Stefano; Perut, Francesca; Fagioli, Franca; Brach Del Prever, Adalberto; Meazza, Cristina; Parafioriti, Antonina; Picci, Piero; Gambarotti, Marco; Avnet, Sofia; Baldini, Nicola; Fais, Stefano

2013-10-24

Major goals in translational oncology are to reduce systemic toxicity of current anticancer strategies and improve effectiveness. An extremely efficient cancer cell mechanism to avoid and/or reduce the effects of highly cytotoxic drugs is the establishment of an acidic microenvironment, an hallmark of all malignant tumors. The H +-rich milieu that anticancer drugs meet once they get inside the tumor leads to their protonation and neutralization, therefore hindering their access into tumor cells. We have previously shown that proton pump inhibitors (PPI) may efficiently counterattack this tumor advantage leading to a consistent chemosensitization of tumors. In this study, we investigated the effects of PPI in chemosensitizing osteosarcoma. MG-63 and Saos-2 cell lines were used as human osteosarcoma models. Cell proliferation after pretreatment with PPI and subsequent treatment with cisplatin was evaluated by using erythrosin B dye vital staining. Tumour growth was evaluated in xenograft treated with cisplatin after PPI pretreatment. Subsequently, a multi-centre historically controlled trial, was performed to evaluate the activity of a pre-treatment administration of PPIs as chemosensitizers during neoadjuvant chemotherapy based on methotrexate, cisplatin, and adriamycin. Preclinical experiments showed that PPI sensitize both human osteosarcoma cell lines and xenografts to cisplatin. A clinical study subsequently showed that pretreatment with PPI drug esomeprazole leads to an increase in the local effect of chemotherapy, as expressed by percentage of tumor necrosis. This was particularly evident in chondroblastic osteosarcoma, an histological subtype that normally shows a poor histological response. Notably, no significant increase in toxicity was recorded in PPI treated patients. This study provides the first evidence that PPI may be beneficially added to standard regimens in combination to conventional chemotherapy.

Recognition and stabilization of geranylgeranylated human Rab5 by the GDP Dissociation Inhibitor (GDI).

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Edler, Eileen; Stein, Matthias

2017-10-25

The small GTPase Rab5 is the key regulator of early endosomal fusion. It is post-translationally modified by covalent attachment of two geranylgeranyl (GG) chains to adjacent cysteine residues of the C-terminal hypervariable region (HVR). The GDP dissociation inhibitor (GDI) recognizes membrane-associated Rab5(GDP) and serves to release it into the cytoplasm where it is kept in a soluble state. A detailed new structural and dynamic model for human Rab5(GDP) recognition and binding with human GDI at the early endosome membrane and in its dissociated state is presented. In the cytoplasm, the GDI protein accommodates the GG chains in a transient hydrophobic binding pocket. In solution, two different binding modes of the isoprenoid chains inserted into the hydrophobic pocket of the Rab5(GDP):GDI complex can be identified. This equilibrium between the two states helps to stabilize the protein-protein complex in solution. Interprotein contacts between the Rab5 switch regions and characteristic patches of GDI residues from the Rab binding platform (RBP) and the C-terminus coordinating region (CCR) reveal insight on the formation of such a stable complex. GDI binding to membrane-anchored Rab5(GDP) is initially mediated by the solvent accessible switch regions of the Rab-specific RBP. Formation of the membrane-associated Rab5(GDP):GDI complex induces a GDI reorientation to establish additional interactions with the Rab5 HVR. These results allow to devise a detailed structural model for the process of extraction of GG-Rab5(GDP) by GDI from the membrane and the dissociation from targeting factors and effector proteins prior to GDI binding.

Trichostatin A, a histone deacetylase inhibitor, potentiated cytotoxic effect of lionizing radiation in human head and neck cancer cell lines

International Nuclear Information System (INIS)

Kim, Jin Ho; Shin, Jin Hee; Chie, Eui Kyu; Wu, Hong Gyun; Kim, Jae Sung; Kim, Il Han; Ha, Sung Whan; Park, Charn Il; Kang, Wee Saing

2004-01-01

We have previously reported that human glioblastoma cells are sensitized to radiation-induced death after their exposure to trichostatin A (TSA), a histone deacetylase inhibitor (HDAC-I), prior to the irradiation. We aimed to measure the magnitude of the radiosensitizing effect of TSA in human head and neck cancer cell lines. human head and neck cancer cell lines, HN-3 and HN-9, were exposed to 0, 50, 100, and 200 nM TSA for 18 hr prior to irradiation. Then, the TSA-treated cells were irradiated with 0, 2, 4, 6, and 8 Gy, and cell survival was measured by clonogenic assay. Pre-irradiation exposure to TSA was found to radiosensitize HN-3 and HN-9 cell lines. In HN-9 cells, the fraction surviving after 2 Gy (SF2) was significantly reduced by treatment of TSA at concentration as low as 50 nM. However, a treatment with 200 nM TSA was required to significantly decrease SF2 in the HN-3 cell line. SER of pre-irradiation treatment with 200 nM TSA was 1.84 in HN-3 and 7.24 in HN-9, respectively. Our results clearly showed that human head and neck cancer cell lines can be sensitized to ionizing radiation by pre-irradiation inhibition of histone deacetylase (HDAC) using TSA, and that this potentiation might well be a general phenomenon

Gene expression response of A253 human salivary cell line to radiation, Cis-Pt, and EGF

International Nuclear Information System (INIS)

Woloschak, G.; Paunesku, T.; Mittal, B.; Dyck, P.; Pauloski, B.; Rademaker, A.; Logemann, J.; Quigg, R.

2003-01-01

We are interested in long and short term effects of head and neck cancers treatment, and prior to the studies of patient samples, experiments were designed to observe treatment effects in cultured cells, and examine gene expression profiles from A253 human salivary cells (derived from a head and neck tumor) following exposure to gamma-rays, cisplatin (cis-Pt), and a combination of either with epidermal growth factor (EGF) treatment. A253 cells were treated by: 2 Gy or 10 Gy of γ-rays (Cs137 source, 77 cGy/min), Cis-Pt at 50 μ/mL, and EGF at 40 ng/mL. RNAs were processed and hybridized with Affymetrix Hu95A arrays according to the manufacturer's instructions. Data were scanned and analyzed and we found significant differences in the expression patterns of numerous genes were observed. Some of the more interesting genes are: Requeim [a protein required for apoptosis]; Cyclin D1 (prad1/bcl1) [a cyclin that can function as an oncogene]; FK506 Binding Protein [which may be competing with TGF-beta type I receptor for binding with FK506 thus acting against this powerful immunosuppressant]; Thioredoxin (TXN) [an oxidoreductase with multiple in vitro substrates, including ribonuclease, choriogonadotropins, coagulation factors, glucocorticoid receptor, and insulin]; Glutathione Peroxidase (GPX) [whose role in protection against oxidative stress was long ago well documented]; Aquaporin 3 (AQP3) [protein with a water-channel function that was confirmed by functional expression in Xenopus oocytes]; Eukaryotic Initiation Factor 1A (EIF1A) [a translation factor, proposed as a candidate gene for Turner syndrome]; and finally Insulin-like Growth Factor-Binding Protein 6 (IGFBP6) [an autocrine growth inhibitor shown to inhibit growth of HaCat cells and other keratinocyte cell lines

Differentiation stage-specific regulation of primitive human hematopoietic progenitor cycling by exogenous and endogenous inhibitors in an in vivo model.

Science.gov (United States)

Cashman, J D; Clark-Lewis, I; Eaves, A C; Eaves, C J

1999-12-01

Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice transplanted with human cord blood or adult marrow cells and injected 6 weeks posttransplant with 2 daily doses of transforming growth factor-beta(1) (TGF-beta(1)), monocyte chemoattractant protein-1 (MCP-1), or a nonaggregating form of macrophage inflammatory protein-1alpha (MIP-1alpha) showed unique patterns of inhibition of human progenitor proliferation 1 day later. TGF-beta(1) was active on long-term culture initiating cells (LTC-IC) and on primitive erythroid and granulopoietic colony-forming cells (HPP-CFC), but had no effect on mature CFC. MCP-1 inhibited the cycling of both types of HPP-CFC but not LTC-IC. MIP-1alpha did not inhibit either LTC-IC or granulopoietic HPP-CFC but was active on erythroid HPP-CFC and mature granulopoietic CFC. All of these responses were independent of the source of human cells transplanted. LTC-IC of either human cord blood or adult marrow origin continue to proliferate in NOD/SCID mice for many weeks, although the turnover of all types of human CFC in mice transplanted with adult human marrow (but not cord blood) is downregulated after 6 weeks. Interestingly, administration of either MIP-1beta, an antagonist of both MIP-1alpha and MCP-1 or MCP-1(9-76), an antagonist of MCP-1 (and MCP-2 and MCP-3), into mice in which human marrow-derived CFC had become quiescent, caused the rapid reactivation of these progenitors in vivo. These results provide the first definition of stage-specific inhibitors of human hematopoietic progenitor cell cycling in vivo. In addition they show that endogenous chemokines can contribute to late graft failure, which can be reversed by the administration of specific antagonists.

Zinc oxide nanoparticles as novel alpha-amylase inhibitors

Science.gov (United States)

Dhobale, Sandip; Thite, Trupti; Laware, S. L.; Rode, C. V.; Koppikar, Soumya J.; Ghanekar, Ruchika-Kaul; Kale, S. N.

2008-11-01

Amylase inhibitors, also known as starch blockers, contain substances that prevent dietary starches from being absorbed by the body via inhibiting breakdown of complex sugars to simpler ones. In this sense, these materials are projected as having potential applications in diabetes control. In this context, we report on zinc oxide nanoparticles as possible alpha-amylase inhibitors. Zinc oxide nanoparticles have been synthesized using soft-chemistry approach and 1-thioglycerol was used as a surfactant to yield polycrystalline nanoparticles of size ˜18 nm, stabilized in wurtzite structure. Conjugation study and structural characterization have been done using x-ray diffraction technique, Fourier transform infrared spectroscopy, UV-visible spectroscopy, and transmission electron microscopy. Cytotoxicity studies on human fibrosarcoma (HT-1080) and skin carcinoma (A-431) cell lines as well as mouse primary fibroblast cells demonstrate that up to a dose of 20 μg/ml, ZnO nanoparticles are nontoxic to the cells. We report for the first time the alpha-amylase inhibitory activity of ZnO nanoparticles wherein an optimum dose of 20 μg/ml was sufficient to exhibit 49% glucose inhibition at neutral pH and 35 °C temperature. This inhibitory activity was similar to that obtained with acarbose (a standard alpha-amylase inhibitor), thereby projecting ZnO nanoparticles as novel alpha-amylase inhibitors.

Lysine Deacetylase Inhibitors in Parasites: Past, Present, and Future Perspectives.

Science.gov (United States)

Hailu, Gebremedhin S; Robaa, Dina; Forgione, Mariantonietta; Sippl, Wolfgang; Rotili, Dante; Mai, Antonello

2017-06-22

Current therapies for human parasite infections rely on a few drugs, most of which have severe side effects, and their helpfulness is being seriously compromised by the drug resistance problem. Globally, this is pushing discovery research of antiparasitic drugs toward new agents endowed with new mechanisms of action. By using a "drug repurposing" strategy, histone deacetylase inhibitors (HDACi), which are presently clinically approved for cancer use, are now under investigation for various parasite infections. Because parasitic Zn 2+ - and NAD + -dependent HDACs play crucial roles in the modulation of parasite gene expression and many of them are pro-survival for several parasites under various conditions, they are now emerging as novel potential antiparasitic targets. This Perspective summarizes the state of knowledge of HDACi (both class I/II HDACi and sirtuin inhibitors) targeted to the main human parasitic diseases (schistosomiasis, malaria, trypanosomiasis, leishmaniasis, and toxoplasmosis) and provides visions into the main issues that challenge their development as antiparasitic agents.

Biological and Molecular Effects of Small Molecule Kinase Inhibitors on Low-Passage Human Colorectal Cancer Cell Lines

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Falko Lange

2014-01-01

Full Text Available Low-passage cancer cell lines are versatile tools to study tumor cell biology. Here, we have employed four such cell lines, established from primary tumors of colorectal cancer (CRC patients, to evaluate effects of the small molecule kinase inhibitors (SMI vemurafenib, trametinib, perifosine, and regorafenib in an in vitro setting. The mutant BRAF (V600E/V600K inhibitor vemurafenib, but also the MEK1/2 inhibitor trametinib efficiently inhibited DNA synthesis, signaling through ERK1/2 and expression of genes downstream of ERK1/2 in BRAF mutant cells only. In case of the AKT inhibitor perifosine, three cell lines showed a high or intermediate responsiveness to the drug while one cell line was resistant. The multikinase inhibitor regorafenib inhibited proliferation of all CRC lines with similar efficiency and independent of the presence or absence of KRAS, BRAF, PIK3CA, and TP53 mutations. Regorafenib action was associated with broad-range inhibitory effects at the level of gene expression but not with a general inhibition of AKT or MEK/ERK signaling. In vemurafenib-sensitive cells, the antiproliferative effect of vemurafenib was enhanced by the other SMI. Together, our results provide insights into the determinants of SMI efficiencies in CRC cells and encourage the further use of low-passage CRC cell lines as preclinical models.

Hydroxybenzoic Acid Derivatives as Dual-Target Ligands: Mitochondriotropic Antioxidants and Cholinesterase Inhibitors.

Science.gov (United States)

Oliveira, Catarina; Cagide, Fernando; Teixeira, José; Amorim, Ricardo; Sequeira, Lisa; Mesiti, Francesco; Silva, Tiago; Garrido, Jorge; Remião, Fernando; Vilar, Santiago; Uriarte, Eugenio; Oliveira, Paulo J; Borges, Fernanda

2018-01-01

Alzheimer's disease (AD) is a multifactorial age-related disease associated with oxidative stress (OS) and impaired cholinergic transmission. Accordingly, targeting mitochondrial OS and restoring cholinergic transmission can be an effective therapeutic strategy toward AD. Herein, we report for the first time dual-target hydroxybenzoic acid (HBAc) derivatives acting as mitochondriotropic antioxidants and cholinesterase (ChE) inhibitors. The studies were performed with two mitochondriotropic antioxidants AntiOxBEN 1 (catechol derivative), and AntiOxBEN 2 (pyrogallol derivative) and compounds 15-18 , which have longer spacers. Compounds AntiOxBEN 1 and 15 , with a shorter carbon chain spacer (six- and eight-carbon) were shown to be potent antioxidants and BChE inhibitors (IC 50 = 85 ± 5 and 106 ± 5 nM, respectively), while compounds 17 and 18 with a 10-carbon chain were more effective AChE inhibitors (IC 50 = 7.7 ± 0.4 and 7.2 ± 0.5 μM, respectively). Interestingly, molecular modeling data pointed toward bifunctional ChEs inhibitors. The most promising ChE inhibitors acted by a non-competitive mechanism. In general, with exception of compounds 15 and 17 , no cytotoxic effects were observed in differentiated human neuroblastoma (SH-SY5Y) and human hepatocarcinoma (HepG2) cells, while Aβ-induced cytotoxicity was significantly prevented by the new dual-target HBAc derivatives. Overall, due to its BChE selectivity, favorable toxicological profile, neuroprotective activity and drug-like properties, which suggested blood-brain barrier (BBB) permeability, the mitochondriotropic antioxidant AntiOxBEN 1 is considered a valid lead candidate for the development of dual acting drugs for AD and other mitochondrial OS-related diseases.

Hydroxybenzoic Acid Derivatives as Dual-Target Ligands: Mitochondriotropic Antioxidants and Cholinesterase Inhibitors

Directory of Open Access Journals (Sweden)

Catarina Oliveira

2018-04-01

Full Text Available Alzheimer's disease (AD is a multifactorial age-related disease associated with oxidative stress (OS and impaired cholinergic transmission. Accordingly, targeting mitochondrial OS and restoring cholinergic transmission can be an effective therapeutic strategy toward AD. Herein, we report for the first time dual-target hydroxybenzoic acid (HBAc derivatives acting as mitochondriotropic antioxidants and cholinesterase (ChE inhibitors. The studies were performed with two mitochondriotropic antioxidants AntiOxBEN1 (catechol derivative, and AntiOxBEN2 (pyrogallol derivative and compounds 15–18, which have longer spacers. Compounds AntiOxBEN1 and 15, with a shorter carbon chain spacer (six- and eight-carbon were shown to be potent antioxidants and BChE inhibitors (IC50 = 85 ± 5 and 106 ± 5 nM, respectively, while compounds 17 and 18 with a 10-carbon chain were more effective AChE inhibitors (IC50 = 7.7 ± 0.4 and 7.2 ± 0.5 μM, respectively. Interestingly, molecular modeling data pointed toward bifunctional ChEs inhibitors. The most promising ChE inhibitors acted by a non-competitive mechanism. In general, with exception of compounds 15 and 17, no cytotoxic effects were observed in differentiated human neuroblastoma (SH-SY5Y and human hepatocarcinoma (HepG2 cells, while Aβ-induced cytotoxicity was significantly prevented by the new dual-target HBAc derivatives. Overall, due to its BChE selectivity, favorable toxicological profile, neuroprotective activity and drug-like properties, which suggested blood-brain barrier (BBB permeability, the mitochondriotropic antioxidant AntiOxBEN1 is considered a valid lead candidate for the development of dual acting drugs for AD and other mitochondrial OS-related diseases.

Hydroxybenzoic acid derivatives as dual-target ligands: mitochondriotropic antioxidants and cholinesterase inhibitors

Science.gov (United States)

Oliveira, Catarina; Cagide, Fernando; Teixeira, José; Amorim, Ricardo; Sequeira, Lisa; Mesiti, Francesco; Silva, Tiago; Garrido, Jorge; Remião, Fernando; Vilar, Santiago; Uriarte, Eugenio; Oliveira, Paulo J.; Borges, Fernanda

2018-04-01

Alzheimer’s disease (AD) is a multifactorial age-related disease associated with oxidative stress (OS) and impaired cholinergic transmission. Accordingly, targeting mitochondrial OS and restoring cholinergic transmission can be an effective therapeutic strategy towards AD. Herein, we report for the first time dual-target hydroxybenzoic acid (HBAc) derivatives acting as mitochondriotropic antioxidants and cholinesterase (ChE) inhibitors. The studies were performed with two mitochondriotropic antioxidants AntiOxBEN1 (catechol derivative), and AntiOxBEN2 (pyrogallol derivative) and compounds 15-18, which have longer spacers. Compounds AntiOxBEN1 and 15, with a shorter carbon chain spacer (six- and eight-carbon) were shown to be potent antioxidants and BChE inhibitors (IC50 = 85 ± 5 and 106 ± 5 nM, respectively), while compounds 17 and 18 with a ten-carbon chain were more effective AChE inhibitors (IC50 = 7.7 ± 0.4 and 7.2 ± 0.5 nM, respectively). Interestingly, molecular modelling data pointed towards bifunctional ChEs inhibitors. The most promising ChE inhibitors acted by a non-competitive mechanism. In general, with exception of compounds 15 and 17, no cytotoxic effects were observed in differentiated human neuroblastoma (SH-SY5Y) and human hepatocarcinoma (HepG2) cells, while Αβ-induced cytotoxicity was significantly prevented by the new dual-target HBAc derivatives. Overall, due to its BChE selectivity, favourable toxicological profile, neuroprotective activity and drug-like properties, which suggested blood-brain barrier (BBB) permeability, the mitochondriotropic antioxidant AntiOxBEN1 is considered a valid lead candidate for the development of dual acting drugs for AD and other mitochondrial OS-related disease

A comprehensive review of the pharmacodynamics of the SGLT2 inhibitor empagliflozin in animals and humans

Science.gov (United States)

Michel, Martin C.

2018-01-01

Empagliflozin (formerly known as BI 10773) is a potent, competitive, and selective inhibitor of the sodium glucose transporter SGLT2, which mediates glucose reabsorption in the early proximal tubule and most of the glucose reabsorption by the kidney, overall. Accordingly, empagliflozin treatment increased urinary glucose excretion. This has been observed across multiple species including humans and was reported under euglycemic conditions, in obesity and, most importantly, in type 2 diabetic patients and multiple animal models of type 2 diabetes and of type 1 diabetes. This led to a reduction in blood glucose, smaller blood glucose excursions during oral glucose tolerance tests, and, upon chronic treatment, a reduction in HbA1c in animal models and patients. In rodents, such effects were observed in early and late phases of experimental diabetes and were associated with preservation of pancreatic β-cell function. Combination studies in animals demonstrated that beneficial metabolic effects of empagliflozin may also manifest when added to other types of anti-hyperglycemic treatments including linagliptin and pioglitazone. While some anti-hyperglycemic drugs lead to weight gain, empagliflozin treatment was associated with reduced body weight in normoglycemic obese and non-obese animals despite an increased food intake, largely due to a loss of adipose tissue; on the other hand, empagliflozin preserved body weight in models of type 1 diabetes. Empagliflozin improved endothelial dysfunction in diabetic rats and arterial stiffness, reduced blood pressure in diabetic patients, and attenuated early signs of nephropathy in diabetic animal models. Taken together, the SGLT2 inhibitor empagliflozin improves glucose metabolism by enhancing urinary glucose excretion; upon chronic administration, at least in animal models, the reductions in blood glucose levels are associated with beneficial effects on cardiovascular and renal complications of diabetes. PMID:26108304

Rapid, Automated, and Specific Immunoassay to Directly Measure Matrix Metalloproteinase-9–Tissue Inhibitor of Metalloproteinase-1 Interactions in Human Plasma Using AlphaLISA Technology: A New Alternative to Classical ELISA

Directory of Open Access Journals (Sweden)

Helena Pulido-Olmo

2017-07-01

Full Text Available The protocol describes a novel, rapid, and no-wash one-step immunoassay for highly sensitive and direct detection of the complexes between matrix metalloproteinases (MMPs and their tissue inhibitor of metalloproteinases (TIMPs based on AlphaLISA® technology. We describe two procedures: (i one approach is used to analyze MMP-9–TIMP-1 interactions using recombinant human MMP-9 with its corresponding recombinant human TIMP-1 inhibitor and (ii the second approach is used to analyze native or endogenous MMP-9–TIMP-1 protein interactions in samples of human plasma. Evaluating native MMP-9–TIMP-1 complexes using this approach avoids the use of indirect calculations of the MMP-9/TIMP-1 ratio for which independent MMP-9 and TIMP-1 quantifications by two conventional ELISAs are needed. The MMP-9–TIMP-1 AlphaLISA® assay is quick, highly simplified, and cost-effective and can be completed in less than 3 h. Moreover, the assay has great potential for use in basic and preclinical research as it allows direct determination of native MMP-9–TIMP-1 complexes in circulating blood as biofluid.

Small molecule inhibitor screening identifified HSP90 inhibitor 17-AAG as potential therapeutic agent for gallbladder cancer.

Science.gov (United States)

Weber, Helga; Valbuena, José R; Barbhuiya, Mustafa A; Stein, Stefan; Kunkel, Hana; García, Patricia; Bizama, Carolina; Riquelme, Ismael; Espinoza, Jaime A; Kurtz, Stephen E; Tyner, Jeffrey W; Calderon, Juan Francisco; Corvalán, Alejandro H; Grez, Manuel; Pandey, Akhilesh; Leal-Rojas, Pamela; Roa, Juan C

2017-04-18

Gallbladder cancer (GBC) is a lethal cancer with poor prognosis associated with high invasiveness and poor response to chemotherapy and radiotherapy. New therapeutic approaches are urgently needed in order to improve survival and response rates of GBC patients. We screened 130 small molecule inhibitors on a panel of seven GBC cell lines and identified the HSP90 inhibitor 17-AAG as one of the most potent inhibitory drugs across the different lines. We tested the antitumor efficacy of 17-AAG and geldanamycin (GA) in vitro and in a subcutaneous preclinical tumor model NOD-SCID mice. We also evaluated the expression of HSP90 by immunohistochemistry in human GBC tumors.In vitro assays showed that 17-AAG and GA significantly reduced the expression of HSP90 target proteins, including EGFR, AKT, phospho-AKT, Cyclin B1, phospho-ERK and Cyclin D1. These molecular changes were consistent with reduced cell viability and cell migration and promotion of G2/M cell cycle arrest and apoptosis observed in our in vitro studies.In vivo, 17-AAG showed efficacy in reducing subcutaneous tumors size, exhibiting a 69.6% reduction in tumor size in the treatment group compared to control mice (p < 0.05).The HSP90 immunohistochemical staining was seen in 182/209 cases of GBC (87%) and it was strongly expressed in 70 cases (33%), moderately in 58 cases (28%), and weakly in 54 cases (26%).Our pre-clinical observations strongly suggest that the inhibition of HSP90 function by HSP90 inhibitors is a promising therapeutic strategy for gallbladder cancer that may benefit from new HSP90 inhibitors currently in development.

Breast Cancer Prevention by Hormonally Induced Mammary Gland Differentiation: The Role of a Novel Mammary Growth Inhibitor and Differentiation Factor MRG

National Research Council Canada - National Science Library

Shi, Y

2000-01-01

We have previously identified and characterized a novel tumor growth inhibitor and a fatty acid binding protein in human mammary gland and named it as Mammary derived growth inhibitor Related Gene MRG...

Omega-3 Fatty Acids and a Novel Mammary Derived Growth Inhibitor Fatty Acid Binding Protein MRG in Suppression of Mammary Tumor

National Research Council Canada - National Science Library

Liu, Yiliang

2001-01-01

We have previously identified and characterized a novel tumor growth inhibitor and a fatty acid binding protein in human mammary gland and named it as Mammary derived growth inhibitor Related Gene MRG...

RNase MC2: a new Momordica charantia ribonuclease that induces apoptosis in breast cancer cells associated with activation of MAPKs and induction of caspase pathways.

Science.gov (United States)

Fang, Evandro Fei; Zhang, Chris Zhi Yi; Fong, Wing Ping; Ng, Tzi Bun

2012-04-01

Ribonucleases (RNases) are ubiquitously distributed nucleases that cleave RNA into smaller pieces. They are promising drugs for different cancers based on their concrete antitumor activities in vitro and in vivo. Here we report for the first time purification and characterization of a 14-kDa RNase, designated as RNase MC2, in the seeds of bitter gourd (Momordica charantia). RNase MC2 manifested potent RNA-cleavage activity toward baker's yeast tRNA, tumor cell rRNA, and an absolute specificity for uridine. RNase MC2 demonstrated both cytostatic and cytotoxic activities against MCF-7 breast cancer cells. Treatment of MCF-7 cells with RNase MC2 caused nuclear damage (karyorrhexis, chromatin condensation, and DNA fragmentation), ultimately resulting in early/late apoptosis. Further molecular studies unveiled that RNase MC2 induced differential activation of MAPKs (p38, JNK and ERK) and Akt. On the other hand, RNase MC2 exposure activated caspase-8, caspase-9, caspase-7, increased the production of Bak and cleaved PARP, which in turn contributed to the apoptotic response. In conclusion, RNase MC2 is a potential agent which can be exploited in the worldwide fight against breast cancer.

Smad signaling pathway is a pivotal component of tissue inhibitor of metalloproteinases-3 regulation by transforming growth factor beta in human chondrocytes.

Science.gov (United States)

Qureshi, Hamid Yaqoob; Ricci, Gemma; Zafarullah, Muhammad

2008-09-01

Transforming growth factor beta (TGF-beta1) promotes cartilage matrix synthesis and induces tissue inhibitor of metalloproteinases-3 (TIMP-3), which inhibits matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme implicated in articular cartilage degradation and joint inflammation. TGF-beta1 activates Akt, ERK and Smad2 pathways in chondrocytes. Here we investigated previously unexplored roles of specific Smads in TGF-beta1 induction of TIMP-3 gene by pharmacological and genetic knockdown approaches. TGF-beta1-induced Smad2 phosphorylation and TIMP-3 protein expression could be inhibited by the Smad2/3 phosphorylation inhibitors, PD169316 and SB203580 and by Smad2-specific siRNA. Specific inhibitor of Smad3 (SIS3) and Smad3 siRNA abolished TGF-beta induction of TIMP-3. Smad2/3 siRNAs also down regulated TIMP-3 promoter-driven luciferase activities, suggesting transcriptional regulation. SiRNA-driven co-Smad4 knockdown abrogated TIMP-3 augmentation by TGF-beta. TIMP-3 promoter deletion analysis revealed that -828 deletion retains the original promoter activity while -333 and -167 deletions display somewhat reduced activity suggesting that most of the TGF-beta-responsive, cis-acting elements are found in the -333 fragment. Chromatin Immunoprecipitation (ChIP) analysis confirmed binding of Smad2 and Smad4 with the -940 and -333 promoter sequences. These results suggest that receptor-activated Smad2 and Smad3 and co-Smad4 critically mediate TGF-beta-stimulated TIMP-3 expression in human chondrocytes and TIMP-3 gene is a target of Smad signaling pathway.

Prevotella intermedia stimulates tissue-type plasminogen activator and plasminogen activator inhibitor-2 expression via multiple signaling pathways in human periodontal ligament cells.

Science.gov (United States)

Guan, Su-Min; He, Jian-Jun; Zhang, Ming; Shu, Lei

2011-06-01

Prevotella intermedia is an important periodontal pathogen that induces various inflammatory and immune responses. In this study, we investigated the effects of P. intermedia on the plasminogen system in human periodontal ligament (hPDL) cells and explored the signaling pathways involved. Using semi-quantitative reverse transcription (RT)-PCR and quantitative real-time RT-qPCR, we demonstrated that P. intermedia challenge increased tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI)-2 expression in a concentration- and time-dependent manner, but exerted no influence on urokinase-type plasminogen activator and PAI-1mRNA expression in hPDL cells. Prevotella intermedia stimulation also enhanced tPA protein secretion as confirmed by enzyme-linked immunosorbent assay. Western blot results revealed that P. intermedia treatment increased phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 kinase (p38). ERK, JNK and protein kinase C inhibitors significantly attenuated the P. intermedia-induced tPA and PAI-2 expression. Furthermore, p38 and phosphatidylinositol 3-kinase inhibitors markedly decreased PAI-2 expression, whereas they showed no or little inhibition on tPA expression. In contrast, inhibition of protein kinase A greatly enhanced the upregulatory effect of P. intermedia on tPA and PAI-2 expression. Our results suggest that P. intermedia may contribute to periodontal tissue destruction by upregulating tPA and PAI-2 expression in hPDL cells via multiple signaling pathways. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Serine proteinases and their inhibitors in fertilization

Czech Academy of Sciences Publication Activity Database

Jonáková, Věra; Jelínková-Slavíčková, Petra

2004-01-01

Roč. 8, 3,4 (2004), s. 108-110 ISSN 1211-8869. [Central European Conference on Human Tumor Markers /5./. Praha, 01.10.2004-03.10.2004] R&D Projects: GA ČR GA303/02/0433; GA ČR GP303/02/P069; GA ČR GP303/04/P070; GA MZd NJ7463 Institutional research plan: CEZ:AV0Z5052915 Keywords : serine proteinase * proteinase inhibitors * fertilization Subject RIV: CE - Biochemistry

Use of selective serotonin reuptake inhibitors reduces fertility in men

DEFF Research Database (Denmark)

Nørr, L; Bennedsen, Birgit; Fedder, Jens

2016-01-01

Clinical review of the present data on the effects of selective serotonin reuptake inhibitors (SSRIs) on male fertility was the objective of the study. PubMed and Scopus were searched for publications in English or Danish and reviewed. Human trials, animal studies and in vitro studies were included...