scholarly journals Development of a High-Throughput Cell-Based Assay for Superoxide Production in HL-60 Cells

2010 ◽  
Vol 15 (4) ◽  
pp. 388-397 ◽  
Author(s):  
Patricia M. Seitz ◽  
Rona Cooper ◽  
Gregory J. Gatto ◽  
Fernando Ramon ◽  
Thomas D. Sweitzer ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Superoxide Production ◽  
Test Set ◽  
Cellular Processes ◽  
Detection Assay ◽  
Z Value ◽  
Compound Concentration ◽  
Compound Test ◽  
Early Drug

Superoxide affects many normal and pathogenic cellular processes, and the detection of superoxide produced by cells is therefore of interest for potential therapeutic applications. To develop a high-throughput cell-based assay for the detection of extracellular superoxide production that could be run in a 384-well or 1536-well format, 2 luminescent reagents, Lucigenin and Diogenes, and one fluorescent reagent, Oxyburst Green BSA, were tested. HL-60 cells, which had been differentiated to a neutrophil-like phenotype with DMSO and frozen in large batches, were used in assays. All 3 superoxide detection reagents performed well statistically in terms of IC50 reproducibility and met a desired Z′ value requirement of >0.4. When tested against a 1408-compound test set at 5 or 10 µM compound concentration, a higher hit rate was obtained with the 2 luminescent reagents compared with that obtained with the fluorescent Oxyburst Green BSA reagent. The Oxyburst Green BSA assay was ultimately chosen for compound profiling and high-throughput screening activities. This 1536 superoxide detection assay using cryopreserved differentiated HL-60 cells represents a shifting paradigm toward the utilization of more therapeutically relevant cells in early drug development activities.

scholarly journals Development of a Fluorescent Intensity Assay Amenable for High-Throughput Screening for Determining 15-Lipoxygenase Activity

2010 ◽  
Vol 15 (6) ◽  
pp. 671-679 ◽  
Author(s):  
Märta Dahlström ◽  
Daniel Forsström ◽  
Malin Johannesson ◽  
Yasmin Huque-Andersson ◽  
Marie Björk ◽  
...  
Keyword(s):  
Fatty Acids ◽  
High Throughput ◽  
High Throughput Screening ◽  
Lipid Hydroperoxide ◽  
Lipoxygenase Activity ◽  
Lipid Hydroperoxides ◽  
Fluorescent Compound ◽  
Fluorescent Intensity ◽  
Cellular Assays ◽  
Z Value

15-Lipoxygenase-1 catalyzes the introduction of molecular oxygen into polyunsaturated fatty acids to form a lipid hydroperoxide. The authors have developed an assay for the detection of lipid hydroperoxides formed by human 15-lipoxygenase (15-LO) in enzyme or cellular assays using either a 96-well or a 384-well format. The assays described take advantage of the ability of lipid hydroperoxides to oxidize nonfluorescent diphenyl-1-pyrenylphosphine (DPPP) to a fluorescent phosphine oxide. Oxidation of DPPP yields a fluorescent compound, which is not sensitive to temperature and is stable for more than 2 h. The assay is sensitive toward inhibition and robust with a Z′ value of 0.79 and 0.4 in a 96- and 384-well format, respectively, and thus amenable for high-throughput screening. The utility of DPPP as a marker for 15-lipoxygenase activity was demonstrated with both enzyme- and cell-based assays for the identification of hits and to determine potency by IC50 determinations.

scholarly journals High-Throughput Screening of a 100,000-Compound Library for Inhibitors of Influenza A Virus (H3N2)

2008 ◽  
Vol 13 (9) ◽  
pp. 879-887 ◽  
Author(s):  
William E. Severson ◽  
Michael McDowell ◽  
Subramaniam Ananthan ◽  
Dong-Hoon Chung ◽  
Lynn Rasmussen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Influenza A ◽  
Moderate Activity ◽  
Compound Library ◽  
Highly Active ◽  
Virus Life Cycle ◽  
Influenza Activity ◽  
Compound Concentration ◽  
Viral Cytopathic Effect

Using a highly reproducible and robust cell-based high-throughput screening (HTS) assay, the authors screened a 100,000-compound library at 14- and 114-µM compound concentration against influenza strain A/Udorn/72 (H3N2). The “hit” rates (>50% inhibition of the viral cytopathic effect) from the 14- and 114-µM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen at the lower concentration yielded 3 compounds, which displayed moderate activity (SI50 = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI50 > 50. Time of addition experiments revealed 1 compound that inhibited early and 4 other compounds that inhibited late in the virus life cycle, suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides, and benzothiazinones, which have not been previously identified as having antiviral/anti-influenza activity. ( Journal of Biomolecular Screening 2008:879-887)

scholarly journals A Homogeneous, High-Throughput Fluorescence Anisotropy-Based DNA Supercoiling Assay

2010 ◽  
Vol 15 (9) ◽  
pp. 1088-1098 ◽  
Author(s):  
Adam Shapiro ◽  
Haris Jahic ◽  
Swati Prasad ◽  
David Ehmann ◽  
Jason Thresher ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Anisotropy ◽  
High Throughput Screening ◽  
Topoisomerase I ◽  
Dna Supercoiling ◽  
New Drugs ◽  
E Coli ◽  
Cellular Processes ◽  
The Difference ◽  
Cellular Replication

The degree of supercoiling of DNA is vital for cellular processes, such as replication and transcription. DNA topology is controlled by the action of DNA topoisomerase enzymes. Topoisomerases, because of their importance in cellular replication, are the targets of several anticancer and antibacterial drugs. In the search for new drugs targeting topoisomerases, a biochemical assay compatible with automated high-throughput screening (HTS) would be valuable. Gel electrophoresis is the standard method for measuring changes in the extent of supercoiling of plasmid DNA when acted upon by topoisomerases, but this is a low-throughput and laborious method. A medium-throughput method was described previously that quantitatively distinguishes relaxed and supercoiled plasmids by the difference in their abilities to form triplex structures with an immobilized oligonucleotide. In this article, the authors describe a homogeneous supercoiling assay based on triplex formation in which the oligonucleotide strand is labeled with a fluorescent dye and the readout is fluorescence anisotropy. The new assay requires no immobilization, filtration, or plate washing steps and is therefore well suited to HTS for inhibitors of topoisomerases. The utility of this assay is demonstrated with relaxation of supercoiled plasmid by Escherichia coli topoisomerase I, supercoiling of relaxed plasmid by E. coli DNA gyrase, and inhibition of gyrase by fluoroquinolones and nalidixic acid.

scholarly journals Antagonism screen for inhibitors of bacterial cell wall biogenesis uncovers an inhibitor of undecaprenyl diphosphate synthase

2015 ◽  
Vol 112 (35) ◽  
pp. 11048-11053 ◽  
Author(s):  
Maya A. Farha ◽  
Tomasz L. Czarny ◽  
Cullen L. Myers ◽  
Liam J. Worrall ◽  
Shawn French ◽  
...  
Keyword(s):  
Cell Wall ◽  
High Throughput ◽  
High Throughput Screening ◽  
Teichoic Acid ◽  
Antibacterial Drug ◽  
Wall Teichoic Acid ◽  
Chemical Screening ◽  
Cellular Processes ◽  
Cell Wall Biogenesis ◽  
Antagonistic Interactions

Drug combinations are valuable tools for studying biological systems. Although much attention has been given to synergistic interactions in revealing connections between cellular processes, antagonistic interactions can also have tremendous value in elucidating genetic networks and mechanisms of drug action. Here, we exploit the power of antagonism in a high-throughput screen for molecules that suppress the activity of targocil, an inhibitor of the wall teichoic acid (WTA) flippase in Staphylococcus aureus. Well-characterized antagonism within the WTA biosynthetic pathway indicated that early steps would be sensitive to this screen; however, broader interactions with cell wall biogenesis components suggested that it might capture additional targets. A chemical screening effort using this approach identified clomiphene, a widely used fertility drug, as one such compound. Mechanistic characterization revealed the target was the undecaprenyl diphosphate synthase, an enzyme that catalyzes the synthesis of a polyisoprenoid essential for both peptidoglycan and WTA synthesis. The work sheds light on mechanisms contributing to the observed suppressive interactions of clomiphene and in turn reveals aspects of the biology that underlie cell wall synthesis in S. aureus. Further, this effort highlights the utility of antagonistic interactions both in high-throughput screening and in compound mode of action studies. Importantly, clomiphene represents a lead for antibacterial drug discovery.

scholarly journals Development of a Fluorescence Based High Throughput Assay for Antagonists of the Human Chorionic Gonadotropin Receptor Extracellular Domain: Analysis of Peptide Inhibitors

2001 ◽  
Vol 6 (3) ◽  
pp. 151-158 ◽  
Author(s):  
Leslie I. Lobel ◽  
John P. Morseman ◽  
Xiangfei Zeng ◽  
Joyce W. Lustbader ◽  
Hao Chen ◽  
...  
Keyword(s):  
Chorionic Gonadotropin ◽  
High Throughput ◽  
Human Chorionic Gonadotropin ◽  
High Throughput Screening ◽  
Extracellular Domain ◽  
Fluorescent Detection ◽  
Recombinant Phage ◽  
Simple Method ◽  
Blue Green Algae ◽  
Detection Assay

A simple method for prompt fluorescent detection of inhibitors of human chorionic gonadotropin (hCG) binding to the extracellular domain of the human luteinizing hormone/chorionic gonadotropin (hLHICG) receptor was developed for high throughput screening (HTS). Construction and analysis of a recombinant phage that displays the extracellular binding domain of the hLH/CG receptor on its surface and specifically binds hCG was previously described. To facilitate the identification of molecules that disrupt the interaction of hCG with its receptor, a method for prompt fluorescent detection of these phage bound to hCG was developed. This technique is extremely sensitive and employs fluorescent labels (PBXL dyes) that are derived from red and blue-green algae. Antibodies labeled with PBXL dye were able to specifically detect phage that display the extracellular domain of the hLH/CG receptor when bound to hCG immobilized in 96-well microplates. Decreases in fluorescence correlate with the concentration of exogenous hCG or hCG antagonists in the assay. This prompt fluorescence detection assay was optimized in a 96-well format as a model system for HTS applications that target the receptors for the group of hormones known as the gonadotropins. Low-affinity molecules that disrupt binding of the phage-displayed receptor extracellular domain to hCG can be rapidly identified in this high throughput screen.

Development of a High-Throughput Assay to Identify Inhibitors of ENPP1

2021 ◽  
pp. 247255522098232
Author(s):  
Meera Kumar ◽  
Robert G. Lowery
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Type I Interferon ◽  
Direct Detection ◽  
Innate Immune ◽  
Assay Method ◽  
Type I ◽  
Immune Response To Cancer ◽  
Z Value ◽  
High Throughput Assay

The innate immune response to cancer is initiated by cytosolic DNA, where it binds to cGAS and triggers type I interferon (IFN) expression via the STING receptor, leading to activation of tumor-specific T cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as the primary enzyme responsible for degrading cGAMP, and therefore it is under intense investigation as a therapeutic target for cancer immunotherapy. ENPP1 hydrolyzes cGAMP to produce AMP and GMP, and hydrolyzes ATP and other nucleotides to monophosphates and pyrophosphate. We developed a robust, high-throughput screening (HTS)-compatible enzymatic assay method for ENPP1 using the Transcreener AMP2/GMP2 Assay, a competitive fluorescence polarization (FP) immunoassay that enables direct detection of AMP and GMP in a homogenous format. The monoclonal antibody used in the Transcreener AMP2/GMP2 Assay showed more than 104-fold selectivity for AMP and GMP versus cGAMP, and 3000-fold selectivity for AMP over ATP, indicating that the assay can be used for detection at initial velocity with either substrate. A working concentration of 100 pM ENPP1 was determined as optimal with a 60 min reaction period, enabling screening with very low quantities of enzyme. A Z′ value of 0.72 was determined using ATP as substrate, indicating a high-quality assay. Consistent with previous studies, we found that ENPP1 preferred ATP as a substrate when compared with other nucleotides like GTP, ADP, and GDP. ENPP1 showed a 20-fold selectivity for 2′3′cGAMP compared with 2′3′c-diGMP and showed no activity with 3′3′c-diAMP. The Transcreener AMP2/GMP2 Assay should prove to be a valuable tool for the discovery of ENPP1 lead molecules.

scholarly journals Development of a High-Throughput Screening Assay to Identify Inhibitors of the Lipid Kinase PIP5K1C

2014 ◽  
Vol 20 (5) ◽  
pp. 655-662 ◽  
Author(s):  
Brittany D. Wright ◽  
Catherine Simpson ◽  
Michael Stashko ◽  
Dmitri Kireev ◽  
Emily A. Hull-Ryde ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Mobility Shift ◽  
Screening Assay ◽  
Lipid Kinase ◽  
Cellular Processes ◽  
High Throughput Screening Assay ◽  
Lipid Kinases ◽  
Mobility Shift Assay ◽  
Phosphatidylinositol 4

Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) regulate a variety of cellular processes, including signaling through G protein-coupled receptors (GPCRs), endocytosis, exocytosis, and cell migration. These lipid kinases synthesize phosphatidylinositol 4,5-bisphosphate (PIP2) from phosphatidylinositol 4-phosphate [PI(4)P]. Because small-molecule inhibitors of these lipid kinases did not exist, molecular and genetic approaches were predominantly used to study PIP5K1 regulation of these cellular processes. Moreover, standard radioisotope-based lipid kinase assays cannot be easily adapted for high-throughput screening. Here, we report a novel, high-throughput, microfluidic mobility shift assay to identify inhibitors of PIP5K1C. This assay uses fluorescently labeled phosphatidylinositol 4-phosphate as the substrate and recombinant human PIP5K1C. Our assay exhibited high reproducibility, had a calculated adenosine triphosphate Michaelis constant (Km) of 15 µM, performed with z’ values >0.7, and was used to screen a kinase-focused library of ~4700 compounds. From this screen, we identified several potent inhibitors of PIP5K1C, including UNC3230, a compound that we recently found can reduce nociceptive sensitization in animal models of chronic pain. This novel assay will allow continued drug discovery efforts for PIP5K1C and can be adapted easily to screen additional lipid kinases.

scholarly journals Functional annotation of lncRNA in high-throughput screening

2021 ◽  
Author(s):  
Chi Wai Yip ◽  
Divya M. Sivaraman ◽  
Anika V. Prabhu ◽  
Jay W. Shin
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Functional Annotation ◽  
Cell Types ◽  
Specific Expression ◽  
Functional Roles ◽  
Cellular Processes ◽  
Cell Type Specific Expression ◽  
Cell Type Specific ◽  
Different Cell Types

Abstract Recent efforts on the characterization of long non-coding RNAs (lncRNAs) revealed their functional roles in modulating diverse cellular processes. These include pluripotency maintenance, lineage commitment, carcinogenesis, and pathogenesis of various diseases. By interacting with DNA, RNA and protein, lncRNAs mediate multifaceted mechanisms to regulate transcription, RNA processing, RNA interference and translation. Of more than 173000 discovered lncRNAs, the majority remain functionally unknown. The cell type-specific expression and localization of the lncRNA also suggest potential distinct functions of lncRNAs across different cell types. This highlights the niche of identifying functional lncRNAs in different biological processes and diseases through high-throughput (HTP) screening. This review summarizes the current work performed and perspectives on HTP screening of functional lncRNAs where different technologies, platforms, cellular responses and the downstream analyses are discussed. We hope to provide a better picture in applying different technologies to facilitate functional annotation of lncRNA efficiently.

scholarly journals Compartmentalization of enhanced biomolecular interactions for high-throughput drug screening in test tubes

2020 ◽  
Author(s):  
Min Zhou ◽  
Weiping Li ◽  
Jian Li ◽  
Leiming Xie ◽  
Rongbo Wu ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
Biomedical Research ◽  
High Throughput Screening ◽  
Biomolecular Interactions ◽  
Compound Library ◽  
Cellular Processes ◽  
High Throughput Drug Screening ◽  
Binding Partners ◽  
Discovery Method

AbstractModification-dependent and -independent biomolecular interactions (BIs), including protein-protein, protein-DNA/RNA and protein-lipid, play crucial roles in all cellular processes. Dysregulation of BIs or malfunction of the associated enzymes results in various diseases, thus they are attractive targets for therapies. High-throughput screening (HTS) can greatly facilitate the discovery of drugs for these targets. Here we describe a HTS drug discovery method, called compartmentalization of enhanced biomolecular interactions in test tubes (CEBIT). CEBIT uses selective recruitment of biomolecules into phase separated compartments harboring their cognate binding partners as readouts. CEBIT were tailored to detect various BIs and associated modifying enzymes. Using CEBIT-based HTS assays, we successfully identified known inhibitors of the p53/MDM2 interaction and of SUV39H1 from a compound library. CEBIT is simple and versatile, and is likely to become a powerful tool for drug discovery and basic biomedical research.

scholarly journals Differentiated SH-SY5Y Cells for High-Throughput Screening in a 96-Well Plate Format: Protocol Optimisation and Application for Neurite Outgrowth Assays

2021 ◽  
Author(s):  
Anusha Dravid ◽  
Brad Raos ◽  
Darren Svirskis ◽  
Simon J O’Carroll
Keyword(s):  
Neurite Outgrowth ◽  
High Throughput ◽  
High Throughput Screening ◽  
Initial Density ◽  
Neuronal Model ◽  
Neuronal Markers ◽  
Cellular Processes ◽  
Undifferentiated State ◽  
Mature Neurons ◽  
High Throughput Assay

Abstract Neuronal models are a crucial tool in neuroscientific research, helping to elucidate the molecular and cellular processes involved in disorders of the nervous system. Adapting these models to a high-throughput format enables simultaneous screening of multiple agents within a single assay. SH-SY5Y cells have been widely used as a neuronal model, yet commonly in an undifferentiated state that is not representative of mature neurons. Differentiation of the SH-SY5Y cells is a necessary step to obtain cells that express mature neuronal markers. Despite this understanding, the absence of a standardised protocol has limited the use of differentiated SH-SY5Y cells in high-throughput assay formats. Here, we describe a protocol to differentiate and re-plate SH-SY5Y cells within a 96-well plate for high-throughput screening. SH-SY5Y cells seeded at an initial density of 2,500 cells/well in a 96-well plate provide sufficient space for neurites to extend, without impacting cell viability. Room temperature pre-incubation for 1 hour improved the plating homogeneity within the well and the ability to analyse neurites. We then demonstrated the efficacy of this protocol by optimising it further for neurite outgrowth analysis. The presented protocol achieves homogenously distributed differentiated SH-SY5Y cells, useful for researchers using these cells in high-throughput screening assays.

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