scholarly journals High-Throughput Fluorescence Assay for Small-Molecule Inhibitors of Autophagins/Atg4

2011 ◽  
Vol 16 (2) ◽  
pp. 174-182 ◽  
Author(s):  
Chih-Wen Shu ◽  
Charitha Madiraju ◽  
Dayong Zhai ◽  
Kate Welsh ◽  
Paul Diaz ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Caspase 3 ◽  
Bioactive Molecules ◽  
Dependent Manner ◽  
Chemical Libraries ◽  
Evolutionarily Conserved ◽  
Identification And Characterization

Autophagy is an evolutionarily conserved process for catabolizing damaged proteins and organelles in a lysosome-dependent manner. Dysregulation of autophagy may cause various diseases, such as cancer and neurodegeneration. However, the relevance of autophagy to diseases remains controversial because of the limited availability of chemical modulators. Herein, the authors developed a fluorescence-based assay for measuring activity of the autophagy protease, autophagin-1(Atg4B). The assay employs a novel reporter substrate of Atg4B composed of a natural substrate (LC3B) fused to an assayable enzyme (PLA2) that becomes active upon cleavage by this cysteine protease. A high-throughput screening (HTS) assay was validated with excellent Z′ factor (>0.7), remaining robust for more than 5 h and suitable for screening of large chemical libraries. The HTS assay was validated by performing pilot screens with 2 small collections of compounds enriched in bioactive molecules ( n = 1280 for Lopac™ and 2000 for Spectrum™ library), yielding confirmed hit rates of 0.23% and 0.70%, respectively. As counterscreens, PLA2 and caspase-3 assays were employed to eliminate nonspecific inhibitors. In conclusion, the LC3B-PLA2 reporter assay provides a platform for compound library screening for identification and characterization of Atg4B-specific inhibitors that may be useful as tools for interrogating the role of autophagy in disease models.

scholarly journals High-Throughput Identification of Inhibitors of Human Mitochondrial Peptide Deformylase

2007 ◽  
Vol 12 (4) ◽  
pp. 521-535 ◽  
Author(s):  
Christophe Antczak ◽  
David Shum ◽  
Sindy Escobar ◽  
Bhramdeo Bassit ◽  
Earl Kim ◽  
...  
Keyword(s):  
High Throughput ◽  
Antiproliferative Activity ◽  
High Throughput Screening ◽  
Peptide Deformylase ◽  
Chemical Libraries ◽  
New Class ◽  
Cytotoxicity Studies ◽  
Antiproliferative Agent ◽  
And Performance

The human mitochondrial peptide deformylase (HsPDF) provides a potential new target for broadly acting antiproliferative agents. To identify novel nonpeptidomimetic and nonhydroxamic acid—based inhibitors of HsPDF, the authors have developed a high-throughput screening (HTS) strategy using a fluorescence polarization (FP)—based binding assay as the primary assay for screening chemical libraries, followed by an enzymatic-based assay to confirm hits, prior to characterization of their antiproliferative activity against established tumor cell lines. The authors present the results and performance of the established strategy tested in a pilot screen of 2880 compounds and the identification of the 1st inhibitors. Two common scaffolds were identified within the hits. Furthermore, cytotoxicity studies revealed that most of the confirmed hits have antiproliferative activity. These findings demonstrate that the designed strategy can identify novel functional inhibitors and provide a powerful alternative to the use of functional assays in HTS and support the hypothesis that HsPDF inhibitors may constitute a new class of antiproliferative agent. ( Journal of Biomolecular Screening 2007:521-535)

scholarly journals A Thallium-Sensitive, Fluorescence-Based Assay for Detecting and Characterizing Potassium Channel Modulators in Mammalian Cells

2004 ◽  
Vol 9 (8) ◽  
pp. 671-677 ◽  
Author(s):  
C. David Weaver ◽  
David Harden ◽  
Steven I. Dworetzky ◽  
Barbara Robertson ◽  
Ronald J. Knox
Keyword(s):  
Potassium Channels ◽  
High Throughput ◽  
High Throughput Screening ◽  
Mammalian Cells ◽  
Ease Of Use ◽  
Functional Assay ◽  
Chemical Libraries ◽  
Potassium Channel Modulators ◽  
Small Molecule Modulators

Potassium channels have been identified as targets for a large number of therapeutic indications. The ability to use a high-throughput functional assay for the detection and characterization of small-molecule modulators of potassium channels is very desirable. However, present techniques capable of screening very large chemical libraries are limited in terms of data quality, temporal resolution, ease of use, and requirements for specialized instrumentation. To address these issues, the authors have developed a fluorescence-based thalliumflux assay. This assay is capable of detectingmodulators of both voltageand ligand-gated potassium channels expressed inmammalian cells. The thalliumflux assay can use instruments standard to most high-throughput screening laboratories, and using such equipment has been successfully employed to screen large chemical libraries consisting of hundreds of thousands of compounds.

scholarly journals Scotin, a novel p53-inducible proapoptotic protein located in the ER and the nuclear membrane

2002 ◽  
Vol 158 (2) ◽  
pp. 235-246 ◽  
Author(s):  
J.-C. Bourdon ◽  
J. Renzing ◽  
P.L. Robertson ◽  
K.N. Fernandes ◽  
D.P. Lane
Keyword(s):  
Nuclear Membrane ◽  
Growth Arrest ◽  
Dependent Manner ◽  
Γ Irradiation ◽  
Expression Of Genes ◽  
Proapoptotic Protein ◽  
Identification And Characterization ◽  
Human And Mouse

p53 is a transcription factor that induces growth arrest or apoptosis in response to cellular stress. To identify new p53-inducible proapoptotic genes, we compared, by differential display, the expression of genes in spleen or thymus of normal and p53 nullizygote mice after γ-irradiation of whole animals. We report the identification and characterization of human and mouse Scotin homologues, a novel gene directly transactivated by p53. The Scotin protein is localized to the ER and the nuclear membrane. Scotin can induce apoptosis in a caspase-dependent manner. Inhibition of endogenous Scotin expression increases resistance to p53-dependent apoptosis induced by DNA damage, suggesting that Scotin plays a role in p53-dependent apoptosis. The discovery of Scotin brings to light a role of the ER in p53-dependent apoptosis.

High-Throughput Screening and Characterization of a High-Density Soybean Mutant Library Elucidate the Biosynthesis Pathway of Triterpenoid Saponins

2019 ◽  
Vol 60 (5) ◽  
pp. 1082-1097 ◽  
Author(s):  
Panneerselvam Krishnamurthy ◽  
Yukiko Fujisawa ◽  
Yuya Takahashi ◽  
Hanako Abe ◽  
Kentaro Yamane ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
High Density ◽  
Biosynthesis Pathway ◽  
Mutant Library ◽  
Triterpenoid Saponins

scholarly journals Molecular and Functional Characterization of a ToxR-Regulated Lipoprotein from a Clinical Isolate of Aeromonas hydrophila

2006 ◽  
Vol 74 (7) ◽  
pp. 3742-3755 ◽  
Author(s):  
Lakshmi Pillai ◽  
Jian Sha ◽  
Tatiana E. Erova ◽  
Amin A. Fadl ◽  
Bijay K. Khajanchi ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Aeromonas Hydrophila ◽  
Functional Characterization ◽  
Affinity Column ◽  
Serum Resistance ◽  
Classical Pathway ◽  
Dependent Manner ◽  
T7 Promoter

ABSTRACT Human diseases caused by species of Aeromonas have been classified into two major groups: septicemia and gastroenteritis. In this study, we reported the molecular and functional characterization of a new virulence factor, ToxR-regulated lipoprotein, or TagA, from a diarrheal isolate, SSU, of Aeromonas hydrophila. The tagA gene of A. hydrophila exhibited 60% identity with that of a recently identified stcE gene from Escherichia coli O157:H7, which encoded a protein (StcE) that provided serum resistance to the bacterium and prevented erythrocyte lysis by controlling classical pathway of complement activation by cleaving the complement C1-esterase inhibitor (C1-INH). We purified A. hydrophila TagA as a histidine-tagged fusion protein (rTagA) from E. coli DE3 strain using a T7 promoter-based pET30 expression vector and nickel affinity column chromatography. rTagA cleaved C1-INH in a time-dependent manner. The tagA isogenic mutant of A. hydrophila, unlike its corresponding wild-type (WT) or the complemented strain, was unable to cleave C1-INH, which is required to potentiate the C1-INH-mediated lysis of host and bacterial cells. We indeed demonstrated colocalization of C1-INH and TagA on the bacterial surface by confocal fluorescence microscopy, which ultimately resulted in increased serum resistance of the WT bacterium. Likewise, we delineated the role of TagA in contributing to the enhanced ability of C1-INH to inhibit the classical complement-mediated lysis of erythrocytes. Importantly, we provided evidence that the tagA mutant was significantly less virulent in a mouse model of infection (60%) than the WT bacterium at two 50% lethal doses, which resulted in 100% mortality within 48 h. Taken together, our data provided new information on the role of TagA as a virulence factor in bacterial pathogenesis. This is the first report of TagA characterization from any species of Aeromonas.

scholarly journals Differential role of SIRT1/MAPK pathway during cerebral ischemia in rats and humans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sireesh Kumar Teertam ◽  
Phanithi Prakash Babu
Keyword(s):  
Cerebral Ischemia ◽  
Caspase 3 ◽  
Mapk Pathway ◽  
Protective Role ◽  
Akt Signaling ◽  
Sirtuin 1 ◽  
Neurological Dysfunction ◽  
Dependent Manner ◽  
Erk Mapk

AbstractCerebral ischemia (CI) is a severe cause of neurological dysfunction and mortality. Sirtuin-1 (Silent information regulator family protein 1, SIRT1), an oxidized nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase, plays an important role in protection against several neurodegenerative disorders. The present study aims to investigate the protective role of SIRT1 after CI in experimental young and aged rats and humans. Also, the study examines the possible regulatory mechanisms of neuronal death in CI settings. Immunoblotting and immunohistochemistry were used to evaluate changes in the expression of SIRT1, JNK/ERK/MAPK/AKT signaling, and pro-apoptotic caspase-3 in experimental rats and CI patients. The study findings demonstrated that, in aged experimental rats, SIRT1 activation positively influenced JNK and ERK phosphorylation and modulated neuronal survival in AKT-dependent manner. Further, the protection conferred by SIRT1 was effectively reversed by JNK inhibition and increased pro-apoptotic caspase-3 expression. In young experimental rats, SIRT1 activation decreased the phosphorylation of stress-induced JNK, ERK, caspase-3, and increased the phosphorylation of AKT after CI. Inhibition of SIRT1 reversed the protective effect of resveratrol. More importantly, in human patients, SIRT1 expression, phosphorylation of JNK/ERK/MAPK/AKT signaling and caspase-3 were up-regulated. In conclusion, SIRT1 could possibly be involved in the modulation of JNK/ERK/MAPK/AKT signaling pathway in experimental rats and humans after CI.

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