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 Implementation of the NanoBRET Target Engagement Intracellular Kinase Assay to Reveal Differential Compound Engagement by SIK2/3 Isoforms

2019 ◽  
Vol 25 (2) ◽  
pp. 215-222
Author(s):  
Hyun Yong Jin ◽  
Yanyan Tudor ◽  
Kaylee Choi ◽  
Zhifei Shao ◽  
Brian A. Sparling ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Inflammatory Responses ◽  
Therapeutic Benefit ◽  
Kinase Assay ◽  
Target Engagement ◽  
Biologically Relevant ◽  
Safety Margins ◽  
Small Molecule Ligands

The real-time quantification of target engagement (TE) by small-molecule ligands in living cells remains technically challenging. Systematic quantification of such interactions in a high-throughput setting holds promise for identification of target-specific, potent small molecules within a pathophysiological and biologically relevant cellular context. The salt-inducible kinases (SIKs) belong to a subfamily of the AMP-activated protein kinase (AMPK) family and are composed of three isoforms in humans (SIK1, SIK2, and SIK3). They modulate the production of pro- and anti-inflammatory cytokines in immune cells. Although pan-SIK inhibitors are sufficient to reverse SIK-dependent inflammatory responses, the apparent toxicity associated with SIK3 inhibition suggests that isoform-specific inhibition is required to realize therapeutic benefit with acceptable safety margins. Here, we used the NanoBRET TE intracellular kinase assay, a sensitive energy transfer technique, to directly measure molecular proximity and quantify TE in HEK293T cells overexpressing SIK2 or SIK3. Our 384-well high-throughput screening of 530 compounds demonstrates that the NanoBRET TE intracellular kinase assay was sensitive and robust enough to reveal differential engagement of candidate compounds with the two SIK isoforms and further highlights the feasibility of high-throughput implementation of NanoBRET TE intracellular kinase assays for target-driven small-molecule screening.

scholarly journals Integration of an In Situ MALDI-Based High-Throughput Screening Process: A Case Study with Receptor Tyrosine Kinase c-MET

2017 ◽  
Vol 22 (10) ◽  
pp. 1203-1210 ◽  
Author(s):  
Katrin Beeman ◽  
Jens Baumgärtner ◽  
Manuel Laubenheimer ◽  
Karlheinz Hergesell ◽  
Martin Hoffmann ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Maldi Ms ◽  
Kinase Assay ◽  
Label Free ◽  
Screening Process ◽  
Label Free Detection ◽  
Ic50 Values

Mass spectrometry (MS) is known for its label-free detection of substrates and products from a variety of enzyme reactions. Recent hardware improvements have increased interest in the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for high-throughput drug discovery. Despite interest in this technology, several challenges remain and must be overcome before MALDI-MS can be integrated as an automated “in-line reader” for high-throughput drug discovery. Two such hurdles include in situ sample processing and deposition, as well as integration of MALDI-MS for enzymatic screening assays that usually contain high levels of MS-incompatible components. Here we adapt our c-MET kinase assay to optimize for MALDI-MS compatibility and test its feasibility for compound screening. The pros and cons of the Echo (Labcyte) as a transfer system for in situ MALDI-MS sample preparation are discussed. We demonstrate that this method generates robust data in a 1536-grid format. We use the MALDI-MS to directly measure the ratio of c-MET substrate and phosphorylated product to acquire IC50 curves and demonstrate that the pharmacology is unaffected. The resulting IC50 values correlate well between the common label-based capillary electrophoresis and the label-free MALDI-MS detection method. We predict that label-free MALDI-MS-based high-throughput screening will become increasingly important and more widely used for drug discovery.

scholarly journals Cross-Platform Bayesian Optimization System for Autonomous Biological Assay Development

2021 ◽  
Vol 26 (6) ◽  
pp. 579-590
Author(s):  
Sam Elder ◽  
Carleen Klumpp-Thomas ◽  
Adam Yasgar ◽  
Jameson Travers ◽  
Shayne Frebert ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Assay Development ◽  
Bayesian Optimization ◽  
Biological Assay ◽  
Proof Of Concept ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Cross Platform ◽  
Assay Conditions

Current high-throughput screening assay optimization is often a manual and time-consuming process, even when utilizing design-of-experiment approaches. A cross-platform, Cloud-based Bayesian optimization-based algorithm was developed as part of the National Center for Advancing Translational Sciences (NCATS) ASPIRE (A Specialized Platform for Innovative Research Exploration) Initiative to accelerate preclinical drug discovery. A cell-free assay for papain enzymatic activity was used as proof of concept for biological assay development and system operationalization. Compared with a brute-force approach that sequentially tested all 294 assay conditions to find the global optimum, the Bayesian optimization algorithm could find suitable conditions for optimal assay performance by testing 21 assay conditions on average, with up to 20 conditions being tested simultaneously, as confirmed by repeated simulation. The algorithm could achieve a sevenfold reduction in costs for lab supplies and high-throughput experimentation runtime, all while being controlled from a remote site through a secure connection. Based on this proof of concept, this technology is expected to be applied to more complex biological assays and automated chemistry reaction screening at NCATS, and should be transferable to other institutions. Graphical Abstract

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 Fluorescence Polarization Assays for High Throughput Screening of G Protein-Coupled Receptors

2000 ◽  
Vol 5 (3) ◽  
pp. 159-167 ◽  
Author(s):  
Peter Banks ◽  
Mylene Gosselin ◽  
Linda Prystay
Keyword(s):  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Rank Order ◽  
Drug Binding ◽  
G Protein Coupled Receptors ◽  
Microtiter Plates ◽  
High Throughput Drug Screening ◽  
G Protein Coupled ◽  
Speed Of Analysis

Fluorescence polarization assays in 384-well microtiter plates have been demonstrated. The performance is suitable for high throughput drug screening applications with respect to speed of analysis, displaceable signal, precision, and sensitivity to various reagents. Rank order of potency was maintained relative to ['251]-ligand filtration assays, and the effects of the highly colored compounds, tartrazine and Chicago Sky Blue, were insignificant on the polarization signal up to a concentration of 1 tiM. These attributes suggest that accurate assessment of drug binding can be obtained.

An improved zinc cocktail-mediated fluorescence polarization-based kinase assay for high-throughput screening of kinase inhibitors

2008 ◽  
Vol 380 (1) ◽  
pp. 143-145 ◽  
Author(s):  
Puneet Chopra ◽  
Kamna Nanda ◽  
Mou Chatterjee ◽  
Malini Bajpai ◽  
Sunanda G. Dastidar ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Kinase Assay

scholarly journals A High-Throughput Screening Assay for Small Molecules That Disrupt Yeast Cell Integrity

2008 ◽  
Vol 13 (7) ◽  
pp. 657-664 ◽  
Author(s):  
Damian J. Krysan ◽  
Louis Didone
Keyword(s):  
Yeast Cell ◽  
Small Molecules ◽  
High Throughput ◽  
Adenylate Kinase ◽  
High Throughput Screening ◽  
Cell Lysis ◽  
Kinase Assay ◽  
Screening Assay ◽  
Drug Concentrations ◽  
High Throughput Screening Assay

Lead compounds for antifungal drug development are urgently needed because invasive fungal infections are an important cause of morbidity and mortality in immunocompromised patients. Here, a high-throughput screening assay for small molecules that cause yeast cell lysis is described. The assay is based on the detection of the intracellular enzyme adenylate kinase in the culture medium as a reporter of yeast cell lysis. Features of the assay protocol include 1) the ability to detect cell lysis at drug concentrations that cause no apparent growth defect, 2) specificity for fungicidal molecules, 3) a simple 1-plate, add-and-read protocol using a commercially available adenylate kinase assay kit, 4) short, 5-h incubation time, and 5) low cost. The assay is applicable to the model yeast Saccharomyces cerevisiae and to Candida albicans, the most common human fungal pathogen. The adenylate kinase assay is validated in a pilot screen of 4505 compounds. Consistent with its specificity for fungicidal molecules, the largest class of molecules identified in 2 libraries of known bioactive molecules targeted the plasma membrane. Fungistatic compounds are not detected by the assay. Adenylate kinase—based screening appears to be a useful approach to the direct identification of small molecules that kill yeast cells. ( Journal of Biomolecular Screening 2008:657-664)

Miniaturization and Validation of a High-Throughput Serine Kinase Assay Using the Alpha Screen Platform

2004 ◽  
Vol 9 (8) ◽  
pp. 719-725 ◽  
Author(s):  
Achim Von Leoprechting ◽  
Renate Kumpf ◽  
Susanne Menzel ◽  
Dominique Reulle ◽  
Ralf Griebel ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Assay ◽  
Serine Kinase ◽  
Serine Threonine Kinase ◽  
Active Compounds ◽  
Threonine Kinase ◽  
Cost Efficient ◽  
Reducing Costs ◽  
Volume Range

Reducing costs while maintaining the highest readout quality is a precept of modern high-throughput screening. Given the trend toward nonradiometric screening platforms, this has been a big challenge for some kinase target classes. Common issues include lowsensitivity, susceptibility to nonspecific interference, or the need for costly reagents. In this study, the authors describe the feasibility ofminiaturization of a serine kinase assay using generic reagents in the Alpha Screen format. They have validated the robustness of this assay in the course of miniaturization from a 35-to 4.375-µ L final assay volume in 384-and 1536-well formats. Within this volume range, they consistently obtained Z• values above 0.5 and have investigated the suitability of these assay formats for measuring compound effects by testing a set of 25 previously identified active compounds. These active compounds were also reliably identified in the miniaturized assay formats. The results presented here show that the Alpha Screen technology permits robust and cost-efficient miniaturization of serine/threonine kinase assays.

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