High-Throughput Yeast-Based Reporter Assay to Identify Compounds with Anti-inflammatory Potential

2016 ◽  
pp. 441-452 ◽  
Author(s):  
G. Garcia ◽  
C. Nunes do Santos ◽  
R. Menezes
Keyword(s):  
High Throughput ◽  
Reporter Assay ◽  
Anti Inflammatory

scholarly journals Evaluation of Division-Arrested Cells for Cell-Based High-Throughput Screening and Profiling

2005 ◽  
Vol 10 (6) ◽  
pp. 615-623 ◽  
Author(s):  
Mary Ellen Digan ◽  
Chantevy Pou ◽  
Honglin Niu ◽  
Ji-Hu Zhang
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Rank Order ◽  
Kinase Assay ◽  
Just In Time ◽  
Reporter Assay ◽  
A431 Cells ◽  
Dividing Cells ◽  
Epidermal Growth ◽  
Assay Conditions

Just-in-time cell supply for cell-based high-throughput screening (HTS) is frequently problematic. In addition to scheduling and logistical issues, quality issues and variability due to passage effect, cell cycle, or confluency contribute to day-to-day signal variability in the course of cell-based HTS campaigns. Cell division-arrest and cryopreservation technologies permit the use of cells as assay-ready reagents for HTS and other cell-based profiling and structure-activity studies. In this report, the authors compare division-arrested and dividing cells in 2 assay types that are dependent on movement of proteins within or through cell membranes: a receptor tyrosine kinase assay involving A431 cells responsive to epidermal growth factor, and a secretion reporter assay, which measures secretion of a reporter gene, secreted alkaline phosphatase. In both assays, dividing and division-arrested cells yielded similar basal and maximal signals at a given cell density. Similar IC50s were obtained for reference inhibitors in each assay, type in both dividing and division-arrested cells. In addition, for the secretion reporter assay, when comparing IC50s obtained from 44 compounds randomly chosen from a primary screening hit list, the rank order of potency obtained from dividing cells and division-arrested cells was essentially identical. Furthermore, the results show that, under certain assay conditions, data generated using division-arrested cells are less variable than those generated using dividing cells. In summary, the results suggest that, in many cases, division-arrested cells can substitute for dividing cells and offer certain advantages for cell-based assays.

scholarly journals High-Throughput Screening for CEBPD-Modulating Compounds in THP-1-Derived Reporter Macrophages Identifies Anti-Inflammatory HDAC and BET Inhibitors

2021 ◽  
Vol 22 (6) ◽  
pp. 3022
Author(s):  
Tatjana Ullmann ◽  
Sonja Luckhardt ◽  
Markus Wolf ◽  
Michael J. Parnham ◽  
Eduard Resch
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
High Throughput ◽  
Transcription Initiation ◽  
High Throughput Screening ◽  
Inflammatory Responses ◽  
Hdac Inhibitors ◽  
Screening Assay ◽  
Bet Inhibitors ◽  
Anti Inflammatory

This study aimed to identify alternative anti-inflammatory compounds that modulate the activity of a relevant transcription factor, CCAAT/enhancer binding protein delta (C/EBPδ). C/EBPδ is a master regulator of inflammatory responses in macrophages (Mϕ) and is mainly regulated at the level of CEBPD gene transcription initiation. To screen for CEBPD-modulating compounds, we generated a THP-1-derived reporter cell line stably expressing secreted alkaline phosphatase (SEAP) under control of the defined CEBPD promoter (CEBPD::SEAP). A high-throughput screening of LOPAC®1280 and ENZO®774 libraries on LPS- and IFN-γ-activated THP-1 reporter Mϕ identified four epigenetically active hits: two bromodomain and extraterminal domain (BET) inhibitors, I-BET151 and Ro 11-1464, as well as two histone deacetylase (HDAC) inhibitors, SAHA and TSA. All four hits markedly and reproducibly upregulated SEAP secretion and CEBPD::SEAP mRNA expression, confirming screening assay reliability. Whereas BET inhibitors also upregulated the mRNA expression of the endogenous CEBPD, HDAC inhibitors completely abolished it. All hits displayed anti-inflammatory activity through the suppression of IL-6 and CCL2 gene expression. However, I-BET151 and HDAC inhibitors simultaneously upregulated the mRNA expression of pro-inflammatory IL-1ß. The modulation of CEBPD gene expression shown in this study contributes to our understanding of inflammatory responses in Mϕ and may offer an approach to therapy for inflammation-driven disorders.

Development of high-throughput multi-residue method for non-steroidal anti-inflammatory drugs monitoring in swine muscle by LC-MS/MS

2016 ◽  
Vol 33 (7) ◽  
pp. 1166-1174 ◽  
Author(s):  
Tamara S. Castilhos ◽  
Fabiano Barreto ◽  
Leonardo Meneghini ◽  
Ana Maria Bergold
Keyword(s):  
High Throughput ◽  
Inflammatory Drugs ◽  
Anti Inflammatory

Development of fluorescence-based genomic reporter assay for high-throughput screening of potential fetal haemoglobin inducers

2007 ◽  
Vol 38 (2) ◽  
pp. 184
Author(s):  
Preeyachan Lourthai ◽  
Hady Wardan ◽  
John Parisot ◽  
Ian Street ◽  
Suthat Fucharoen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Reporter Assay ◽  
Fetal Haemoglobin

Development of a β-Lactamase Reporter Gene Assay for Metabotropic Glutamate Receptor 1 by Using Coexpression of Glutamate Transporter

2010 ◽  
Vol 15 (2) ◽  
pp. 148-158 ◽  
Author(s):  
Gentaroh Suzuki ◽  
Hiroshi Kawamoto ◽  
Hisashi Ohta
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Metabotropic Glutamate Receptor ◽  
Chinese Hamster ◽  
High Sensitivity ◽  
Reporter Assay ◽  
Activated T Cells ◽  
Basal Activity ◽  
Gene Assay ◽  
Very High

mGluR1 antagonists have been postulated to be novel CNS drugs, including antipsychotics. Toward this end, the authors developed a β-lactamase reporter assay to identify mGluR1 antagonists. β-Lactamase has several interesting features for high-throughput screening, including very high sensitivity and less well-to-well variation than other reporter enzymes. mGluR1-expressing Chinese hamster ovary (CHO) cells with the β-lactamase gene under control of the nuclear factor of activated T cells (NFAT) promoter (CHO-NFAT-bla-hmGluR1b) exhibited very high basal activity, resulting in an inadequate signal-to-basal (S/B) ratio. Coexpression of glutamate/aspartate transporter (GLAST) with mGluR1 in the cell line (CHO-NFAT-bla-hmGluR1b-GLAST) dramatically decreased basal activity and improved the S/B ratio (from 2- to 20-fold). The contribution of GLAST to lowering basal activity and increasing the S/B ratio was validated by the expression level of GLAST mRNA and by a GLAST inhibitor. Antagonistic activities of known mGluR1 antagonists in the β-lactamase reporter assay were comparable with those in the conventional Ca2+ mobilization assay. The Z′ factor of the β-lactamase reporter assay was 0.89 under optimized conditions. Taken together, the β-lactamase reporter assay with CHO-NFAT-bla-hmGluR1b-GLAST could be a novel high-throughput assay for mGluR1 antagonist screening. This is the first description of a successful β-lactamase reporter assay among all mGluR subtypes.

scholarly journals SIRT1 Modulating Compounds from High-Throughput Screening as Anti-Inflammatory and Insulin-Sensitizing Agents

2006 ◽  
Vol 11 (8) ◽  
pp. 959-967 ◽  
Author(s):  
Vasantha M. Nayagam ◽  
Xukun Wang ◽  
Yong Cheng Tan ◽  
Anders Poulsen ◽  
Kee Chuan Goh ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Metabolic Diseases ◽  
Necrosis Factor Alpha ◽  
Insulin Resistant ◽  
Ppar Γ ◽  
Inhibitory Compound ◽  
Cytokine Tumor Necrosis Factor ◽  
Anti Inflammatory ◽  
Mouse Adipocytes

The nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase SIRT1 has been linked to fatty acid metabolism via suppression of peroxysome proliferator-activated receptor gamma (PPAR-γ) and to inflammatory processes by deacetylating the transcription factor NF-κB. First, modulation of SIRT1 activity affects lipid accumulation in adipocytes, which has an impact on the etiology of a variety of human metabolic diseases such as obesity and insulin-resistant diabetes. Second, activation of SIRT1 suppresses inflammation via regulation of cytokine expression. Using high-throughput screening, the authors identified compounds with SIRT1 activating and inhibiting potential. The biological activity of these SIRT1-modulating compounds was confirmed in cell-based assays using mouse adipocytes, as well as human THP-1 monocytes. SIRT1 activators were found to be potent lipolytic agents, reducing the overall lipid content of fully differentiated NIH L1 adipocytes. In addition, the same compounds have anti-inflammatory properties, as became evident by the reduction of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α). In contrast, a SIRT1 inhibitory compound showed a stimulatory activity on the differentiation of adipocytes, a feature often linked to insulin sensitization.

scholarly journals Bioactivity Profiling of Plant Biodiversity of Panama by High Throughput Screening

2019 ◽  
Vol 14 (1) ◽  
pp. 1934578X1901400 ◽  
Author(s):  
Anuradha Roy ◽  
Peter McDonald ◽  
Barbara N. Timmermann ◽  
Mahabir Gupta ◽  
Rathnam Chaguturu
Keyword(s):  
National Parks ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cancer Cell Line ◽  
Antioxidant Response ◽  
Lung Cancer Cell Line ◽  
High Throughput Analysis ◽  
Anticancer Properties ◽  
Methanolic Extracts ◽  
Anti Inflammatory

We report relative bioactivities of extracts prepared from a large collection of plants from three national parks in Panama. Over 181 plants were collected, taxonomically identified and their detannified dichloromethane (DCM)-methanolic extracts were used for profiling selected bioactivities. Assays were performed to evaluate the antioxidant activity of the extracts for Antioxidant Response Element (ARE) induction, total non-enzymatic antioxidant potential, anti-inflammatory and anticancer properties. The high throughput analysis of 280 extracts resulted in identification of 57.5% of the extracts that could induce ARE at one or more concentrations tested, 93.5% that harbored total antioxidant capacity, and 2.1% of the extracts that showed lung cancer cell line-specific cytotoxicity. Data from our profiling experiments indicate that a large number of extracts could be a source for further isolation and chemical identification of compounds that could serve as leads for discovery of antioxidant, anticancer and anti-inflammatory agents to prevent or treat complex diseases like cancer and neurodegenerative disorders.

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