scholarly journals Optimizing the Expression of Recombinant αβγ GABAA Receptors in HEK293 Cells for High-Throughput Screening

2008 ◽  
Vol 14 (1) ◽  
pp. 86-91 ◽  
Author(s):  
Daniel Gilbert ◽  
Abolghasem Esmaeili ◽  
Joseph W. Lynch
Keyword(s):  
High Throughput ◽  
Gabaa Receptors ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Effective Means ◽  
Hek293 Cells ◽  
Gaba A ◽  
Protein Assay ◽  
Lead Compounds

Despite being important clinical targets, it is not straightforward to reliably express recombinant trimeric αβγ GABA-A receptors (GABAARs) for high-throughput screening. This study therefore sought to devise a simple and reliable means of transiently expressing α1β1γ1 and α1β1γ2 GABAARs in HEK293 cells. Expression efficiencies resulting from 5 different transfection strategies were assessed by flow cytometry and pharmacological analysis using an anion-sensitive yellow fluorescent protein-based assay. PolyFect™ and Effectene™, employed according to the manufacturers' instructions, conferred the strongest and most reliable expression of trimeric αβγ GABAARs. Functional analysis via the yellow fluorescent protein assay revealed dramatic differences in the pharmacological properties of γ1- and γ2-containing receptors, consistent with previous electrophysiological characterizations. The authors conclude that this method of expressing and screening recombinant GABAARs provides an effective means of discovering novel GABAAR modulators for use as therapeutic lead compounds and pharmacological probes. ( Journal of Biomolecular Screening 2009:86-91)

scholarly journals Development and utilization of human decidualization reporter cell line uncovers new modulators of female fertility

2019 ◽  
Vol 116 (39) ◽  
pp. 19541-19551
Author(s):  
Meade Haller ◽  
Yan Yin ◽  
Liang Ma
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Patient Specific ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
A Genome ◽  
Decidual Cells

Failure of embryo implantation accounts for a significant percentage of female infertility. Exquisitely coordinated molecular programs govern the interaction between the competent blastocyst and the receptive uterus. Decidualization, the rapid proliferation and differentiation of endometrial stromal cells into decidual cells, is required for implantation. Decidualization defects can cause poor placentation, intrauterine growth restriction, and early parturition leading to preterm birth. Decidualization has not yet been systematically studied at the genetic level due to the lack of a suitable high-throughput screening tool. Herein we describe the generation of an immortalized human endometrial stromal cell line that uses yellow fluorescent protein under the control of the prolactin promoter as a quantifiable visual readout of the decidualization response (hESC-PRLY cells). Using this cell line, we performed a genome-wide siRNA library screen, as well as a screen of 910 small molecules, to identify more than 4,000 previously unrecognized genetic and chemical modulators of decidualization. Ontology analysis revealed several groups of decidualization modulators, including many previously unappreciated transcription factors, sensory receptors, growth factors, and kinases. Expression studies of hits revealed that the majority of decidualization modulators are acutely sensitive to ovarian hormone exposure. Gradient treatment of exogenous factors was used to identify EC50 values of small-molecule hits, as well as verify several growth factor hits identified by the siRNA screen. The high-throughput decidualization reporter cell line and the findings described herein will aid in the development of patient-specific treatments for decidualization-based recurrent pregnancy loss, subfertility, and infertility.

A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABAA receptor chloride channels

2005 ◽  
Vol 380 (3) ◽  
pp. 340-345 ◽  
Author(s):  
Wade Kruger ◽  
Daniel Gilbert ◽  
Rebecca Hawthorne ◽  
Deanne H. Hryciw ◽  
Stephan Frings ◽  
...  
Keyword(s):  
Gabaa Receptor ◽  
High Throughput ◽  
High Throughput Screening ◽  
Chloride Channels ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein

scholarly journals New mechanisms of radioiodide uptake revealed via a novel high throughput drug screening approach in thyroid cancer

2020 ◽  
Author(s):  
Martin L. Read ◽  
Katie Brookes ◽  
Caitlin E.M. Thornton ◽  
Alice Fletcher ◽  
Mohammed Alshahrani ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Cancer Genes ◽  
Iodide Uptake ◽  
Approved Drugs ◽  
The Impact ◽  
Fda Approved Drugs

ABSTRACTNew combinatorial drug strategies are urgently needed to improve radioiodide (RAI) uptake and efficiently ablate thyroid cancer cells, thereby addressing recurrent and metastatic disease. Cellular iodide uptake is accomplished solely by the sodium iodide symporter (NIS), but the complexity of NIS functional regulation and a lack of amenable high-throughput screening assays has impeded progress. We utilised mutated yellow fluorescent protein (YFP) as a surrogate biosensor of intracellular iodide for ∼1200 FDA-approved drugs, allowing us to appraise the impact of 73 leading compounds at 10 doses on 125I uptake in thyroid cancer cell lines. Subsequent mechanistic analysis suggests three predominant modes of drug action: Firstly, a number of drugs inhibited specific regulation of NIS function by the protein VCP. Secondly, some drugs enhanced transcriptional or post-transcriptional regulation of NIS expression. Thirdly, several drugs strongly implicated proteasomal degradation and the unfolded protein response in the cellular processing of NIS. Exploiting these mechanistic insights, multiple compounds gave striking increases in radioiodide uptake when combined with the drug SAHA. Importantly, our new drug combination strategies were also effective in human primary thyrocytes, suggesting they target endogenous NIS physiology. In patients with papillary thyroid cancer, genes involved in proteostasis were remarkably altered and predicted significantly worse outcome, but only in those patients who received RAI therapy. Collectively, we therefore propose a new model of intracellular NIS processing, and identify key nodes which may now be druggable in patients with aggressive thyroid cancer.SUMMARYOur data identify FDA-approved drugs that enhance radioiodide uptake outside of the canonical pathways of NIS processing, leading to a new mechanistic understanding of endogenous NIS function which is subverted in cancer.

scholarly journals A High-throughput Screening Method to Identify Proteins Involved in Unfolded Protein Response Signaling in Plants

2019 ◽  
Author(s):  
André Alcântara ◽  
Denise Seitner ◽  
Fernando Navarrete ◽  
Armin Djamei
Keyword(s):  
Unfolded Protein Response ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Screening Method ◽  
Screening Assay ◽  
Unfolded Protein ◽  
High Throughput Screening Assay ◽  
Protein Response

AbstractBackgroundThe unfolded protein response (UPR) is a highly conserved process in eukaryotic organisms that plays a crucial role in adaptation and development. While the most ubiquitous components of this pathway have been characterized, current efforts are focused on identifying and characterizing other UPR factors that play a role in specific conditions, such as developmental changes, abiotic cues, and biotic interactions. Considering the central role of protein secretion in plant pathogen interactions, there has also been a recent focus on understanding how pathogens manipulate their host’s UPR to facilitate infection.ResultsWe developed a high-throughput screening assay to identify proteins that interfere with UPR signalingin planta. A set of 35 genes from a library of secreted proteins from the maize pathogenUstilago maydiswere transiently co-expressed with a reporter construct that upregulates enhanced yellow fluorescent protein (eYFP) expression upon UPR stress inNicotiana benthamianaplants. After UPR stress induction, leaf discs were placed in 96 well plates and eYFP expression was measured. This allowed us to identify a previously undescribed fungal protein that inhibits plant UPR signaling, which was then confirmed using the classical but more laborious qRT-PCR method.ConclusionsWe have established a rapid and reliable fluorescence-based method to identify heterologously expressed proteins involved in UPR stress in plants. This system can be used for initial screens with libraries of proteins and potentially other molecules to identify candidates for further validation and characterization.

scholarly journals Diethylstilbestrol, a Novel ANO1 Inhibitor, Exerts an Anticancer Effect on Non-Small Cell Lung Cancer via Inhibition of ANO1

2021 ◽  
Vol 22 (13) ◽  
pp. 7100
Author(s):  
Yohan Seo ◽  
Sung Baek Jeong ◽  
Joo Han Woo ◽  
Oh-Bin Kwon ◽  
Sion Lee ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Small Cell ◽  
Channel Activity ◽  
Small Cell Lung ◽  
Protein Levels

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca2+-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca2+ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent.

scholarly journals Beeporter: a high-throughput tool for non-invasive analysis of honey bee virus infection

2021 ◽  
Author(s):  
Jay D. Evans ◽  
Olubukola Banmeke ◽  
Evan C. Palmer-Young ◽  
Yanping Chen ◽  
Eugene V. Ryabov
Keyword(s):  
Virus Infection ◽  
Honey Bee ◽  
High Throughput ◽  
Honey Bees ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Imaging System ◽  
Uv Light ◽  
Light Sources ◽  
Deformed Wing Virus

ABSTRACTHoney bees face numerous pests and pathogens but arguably none are as devastating as Deformed wing virus (DWV). Development of antiviral therapeutics and virus-resistant honey bee lines to control DWV in honey bees is slowed by the lack of a cost-effective high-throughput screening of DWV infection. Currently, analysis of virus infection and screening for antiviral treatments in bees and their colonies is tedious, requiring a well-equipped molecular biology laboratory and the use of hazardous chemicals. Here we utilize a cDNA clone of DWV tagged with green fluorescent protein (GFP) to develop the Beeporter assay, a method for detection and quantification of DWV infection in live honey bees. The assay involves infection of honey bee pupae by injecting a standardized DWV-GFP inoculum, followed by incubation for up to 44 hours. GFP fluorescence is recorded at intervals via commonly available long-wave UV light sources and a smartphone camera or a standard ultraviolet transilluminator gel imaging system. Nonlethal DWV monitoring allows high-throughput screening of antiviral candidates and a direct breeding tool for identifying honey bee parents with increased antivirus resistance. For even more rapid drug screening, we also describe a method for screening bees using 96-well trays and a spectrophotometer.

scholarly journals Therapeutic Drugs Targeting 2019-nCoV Main Protease by High-Throughput Screening

2020 ◽  
Author(s):  
Yan Li ◽  
Jinyong Zhang ◽  
Ning Wang ◽  
Haibo Li ◽  
Yun Shi ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Binding Capacity ◽  
Effective Means ◽  
Clinical Applications ◽  
Therapeutic Drugs ◽  
Main Protease ◽  
Novel Coronavirus ◽  
Clinical Drug ◽  
Specific Inhibitors

Abstract2019 Novel Coronavirus (2019-nCoV) is a virus identified as the cause of the outbreak of pneumonia first detected in Wuhan, China. Investigations on the transmissibility, severity, and other features associated with this virus are ongoing. Currently, there is no vaccine or therapeutic antibody to prevent the infection, and more time is required to develop an effective immune strategy against the pathogen. In contrast, specific inhibitors targeting the key protease involved in replication and proliferation of the virus are the most effective means to alleviate the epidemic. The main protease of SARS-CoV is essential for the life cycle of the virus, which showed 96.1% of similarity with the main proteaseof 2019-nCoV, is considered to be an attractive target for drug development. In this study, we have identified 4 small molecular drugs with high binding capacity with SARS-CoV main protease by high-throughput screening based on the 8,000 clinical drug libraries, all these drugs have been widely used in clinical applications with guaranteed safety, which may serve as promising candidates to treat the infection of 2019-nCoV.

scholarly journals Identification of Compounds That Inhibit Estrogen-Related Receptor Alpha Signaling Using High-Throughput Screening Assays

Molecules ◽  
2019 ◽  
Vol 24 (5) ◽  
pp. 841 ◽  
Author(s):  
Caitlin Lynch ◽  
Jinghua Zhao ◽  
Srilatha Sakamuru ◽  
Li Zhang ◽  
Ruili Huang ◽  
...  
Keyword(s):  
Cancer Progression ◽  
High Throughput ◽  
High Throughput Screening ◽  
Biological Properties ◽  
Environmental Chemicals ◽  
Hek293 Cells ◽  
Peroxisome Proliferator ◽  
Receptor Alpha ◽  
Screening Assays ◽  
Estrogen Related Receptor

The nuclear receptor, estrogen-related receptor alpha (ERRα; NR3B1), plays a pivotal role in energy homeostasis. Its expression fluctuates with the demands of energy production in various tissues. When paired with the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), the PGC/ERR pathway regulates a host of genes that participate in metabolic signaling networks and in mitochondrial oxidative respiration. Unregulated overexpression of ERRα is found in many cancer cells, implicating a role in cancer progression and other metabolism-related diseases. Using high throughput screening assays, we screened the Tox21 10K compound library in stably transfected HEK293 cells containing either the ERRα-reporter or the reporter plus PGC-1α expression plasmid. We identified two groups of antagonists that were potent inhibitors of ERRα activity and/or the PGC/ERR pathway: nine antineoplastic agents and thirteen pesticides. Results were confirmed using gene expression studies. These findings suggest a novel mechanism of action on bioenergetics for five of the nine antineoplastic drugs. Nine of the thirteen pesticides, which have not been investigated previously for ERRα disrupting activity, were classified as such. In conclusion, we demonstrated that high-throughput screening assays can be used to reveal new biological properties of therapeutic and environmental chemicals, broadening our understanding of their modes of action.

scholarly journals The Receptor for Parathyroid Hormone and Parathyroid Hormone-Related Peptide Is Hydrolyzed and Its Signaling Properties Are Altered by Directly Binding the Calpain Small Subunit

Endocrinology ◽  
2005 ◽  
Vol 146 (5) ◽  
pp. 2336-2344 ◽  
Author(s):  
Masako Shimada ◽  
Matthew J. Mahon ◽  
Peter A. Greer ◽  
Gino V. Segre
Keyword(s):  
Parathyroid Hormone ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Small Subunit ◽  
Hek293 Cells ◽  
Calpain Inhibitor ◽  
Dependent Manner ◽  
Intact Cells ◽  
Calcium Dependent

Abstract We show calcium-dependent, direct binding between the N-terminal portion of the PTH/PTHrP receptor (PTH1R) C-terminal intracellular tail and the calpain small subunit. Binding requires, but may not be limited to, amino acids W474, S475, and W477. The wild-type, full-length rat (r) PTH1R, but not rPTH1R with W474A/W477A substitutions, copurifies with the endogenous calpain small subunit in HEK293 cells. Calpain hydrolyzes ΔNt-rPTH1R, a receptor with a 156-amino acid N-terminal deletion, in a calcium-dependent manner in vitro and in intact cells. Most importantly, PTH stimulation increases the cleavage of ΔNt-rPTH1R and rPTH1R-yellow fluorescent protein in HEK293 cells, and of talin in HEK293 cells expressing rPTH1R-yellow fluorescent protein and in ROS17/2.8 osteoblast-like cells that express rPTH1R endogenously. The absence of calpain in Capn4-null embryonic fibroblasts and the lowered calpain activity in MC3T3-E1 osteoblastic cells due to stable expression of the calpain inhibitor, calpastatin, reduce PTH-stimulated cAMP accumulation. The calpain small subunit is the second protein, in addition to the sodium-hydrogen exchanger regulatory factor, and the first enzyme that binds the PTH1R; PTH1R bound to both of these proteins results in altered PTH signaling.

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