Application of Micro Arrayed Compound Screening (pcARCS) to Identify Inhibitors of Caspase-3

Micro Arrayed Compound Screening (pARCS) is a miniaturized ultra-high-throughput screening platform developed at Abbott Laboratories. In this format, 8640 discrete compounds are spotted and dried onto a polystyrene sheet, which has the same footprint as a 96-well plate. A homogeneous time-resolved fluorescence assay format (LANCE) was applied to identify the inhibitors of caspase-3 using a peptide substrate labeled with a fluorescent europium chelate and a dabcyl quencher. The caspase-3 enzyme was cast into a thin agarose gel, which was placed on a sheet containing test compounds. A second gel containing caspase substrate was then laid above the enzyme gel to initiate the reaction. Caspase-3 cleaves the substrate and separates the europium from the quencher, giving rise to a time-resolved fluorescent signal, which was detected using a ViewLux charge-coupled device imaging system. Potential inhibitors of caspase-3 appeared as dark spots on a bright fluorescent background. Results from the pARCS assay format were compared to those from a conventional 96-well plate-screening format.

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Time-Resolved Luminescent High-Throughput Screening Platform for Lysosomotropic Compounds in Living Cells

ACS Sensors ◽

10.1021/acssensors.0c02046 ◽

2020 ◽

Author(s):

Ke-Jia Wu ◽

Chun Wu ◽

Feng Chen ◽

Sha-Sha Cheng ◽

Dik-Lung Ma ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Living Cells ◽

Time Resolved ◽

Screening Platform

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High-Throughput Screening to Identify Small Molecules That Selectively Inhibit APOL1 Protein Level in Podocytes

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/24725552211026245 ◽

2021 ◽

pp. 247255522110262

Author(s):

Jonathan Choy ◽

Yanqing Kan ◽

Steve Cifelli ◽

Josephine Johnson ◽

Michelle Chen ◽

...

Keyword(s):

Small Molecule ◽

High Throughput ◽

High Throughput Screening ◽

Screening Strategy ◽

Time Resolved ◽

Protein Levels ◽

Increased Risk ◽

Compound Screening ◽

High Throughput Screens ◽

Screening Library

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

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Assay Establishment and Validation of a High-Throughput Screening Platform for Three-Dimensional Patient-Derived Colon Cancer Organoid Cultures

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057116650965 ◽

2016 ◽

Vol 21 (9) ◽

pp. 931-941 ◽

Cited By ~ 48

Author(s):

Karsten Boehnke ◽

Philip W. Iversen ◽

Dirk Schumacher ◽

María José Lallena ◽

Rubén Haro ◽

...

Keyword(s):

Colon Cancer ◽

Drug Discovery ◽

High Throughput ◽

High Throughput Screening ◽

Single Cells ◽

Three Dimensional ◽

Compound Screening ◽

Organoid Cultures ◽

Screening Platform ◽

Disease Specific

The application of patient-derived three-dimensional culture systems as disease-specific drug sensitivity models has enormous potential to connect compound screening and clinical trials. However, the implementation of complex cell-based assay systems in drug discovery requires reliable and robust screening platforms. Here we describe the establishment of an automated platform in 384-well format for three-dimensional organoid cultures derived from colon cancer patients. Single cells were embedded in an extracellular matrix by an automated workflow and subsequently self-organized into organoid structures within 4 days of culture before being exposed to compound treatment. We performed validation of assay robustness and reproducibility via plate uniformity and replicate-experiment studies. After assay optimization, the patient-derived organoid platform passed all relevant validation criteria. In addition, we introduced a streamlined plate uniformity study to evaluate patient-derived colon cancer samples from different donors. Our results demonstrate the feasibility of using patient-derived tumor samples for high-throughput assays and their integration as disease-specific models in drug discovery.

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Potential inhibitors for blocking the interaction of the coronavirus SARS-CoV-2 spike protein and its host cell receptor ACE2

10.1101/2021.12.14.472545 ◽

2021 ◽

Author(s):

Changzhi Li ◽

Hongjuan Zhou ◽

Lingling Guo ◽

Dehuan Xie ◽

Huiping He ◽

...

Keyword(s):

Cell Receptor ◽

Economic Stability ◽

Time Resolved ◽

S Protein ◽

Host Cell Receptor ◽

Compound Libraries ◽

Screening Platform ◽

Potential Inhibitors ◽

Validation Experiments

The outbreak of SARS-CoV-2 continues to pose a serious threat to human health and social and economic stability. In this study, we established an anti-coronavirus drug screening platform based on the Homogeneous Time Resolved Fluorescence (HTRF) technology and the interaction between the coronavirus S protein and its host receptor ACE2. This platform is a rapid, sensitive, specific, and high throughput system. With this platform, we screened two compound libraries of 2,864 molecules and identified three potential anti-coronavirus compounds: tannic acid (TA), TS-1276 (anthraquinone), and TS-984 (9-Methoxycanthin-6-one). Our in vitro validation experiments indicated that TS-984 strongly inhibits the interaction of the coronavirus S-protein and the human cell ACE2 receptor. This data suggests that TS-984 is a potent blocker of the interaction between the S-protein and ACE2, which might have the potential to be developed into an effective anti-coronavirus drug.

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Identification of New ATG4B Inhibitors Based on a Novel High-Throughput Screening Platform

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/1087057116639202 ◽

2016 ◽

Vol 22 (4) ◽

pp. 338-347 ◽

Cited By ~ 12

Author(s):

Danqing Xu ◽

Zhiheng Xu ◽

Li Han ◽

Cheng Liu ◽

Zheng Zhou ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Covalent Bond ◽

Fold Increase ◽

Dependent Manner ◽

Time Resolved ◽

Liquid Chromatography Tandem Mass ◽

Screening Platform

Autophagy is an evolutionarily conserved homeostasis process through which aggregated proteins or damaged organelles are enveloped in a double-membrane structure called an autophagosome and then digested in a lysosome-dependent manner. Growing evidence suggests that malfunction of autophagy contributes to the pathogenesis of a variety of diseases, including cancer, viral infection, and neurodegeneration. However, autophagy is a complicated process, and understanding of the relevance of autophagy to disease is limited by lack of specific and potent autophagy modulators. ATG4B, a Cys-protease that cleaves ATG8 family proteins, such as LC3B, is a key protein in autophagosome formation and maturation process. A novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay measuring protease activity of ATG4B was developed, validated, and adapted into a high-throughput screening (HTS) format. HTS was then conducted with a Roche focus library of 57,000 compounds. After hit confirmation and a counterscreen to filter out fluorescence interference compounds, 267 hits were confirmed, constituting a hit rate of 0.49%. Furthermore, among 65 hits with an IC50 < 50 µM, one compound mimics the LC3 peptide substrate (-TFG-). Chemistry modification based on this particular hit gave preliminary structure activity relationship (SAR) resulting in a compound with a 10-fold increase in potency. This compound forms a stable covalent bond with Cys74 of ATG4B in a 1:1 ratio as demonstrated by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Furthermore, this compound displayed cellular ATG4B inhibition activity. Overall, the novel TR-FRET ATG4B protease assay plus counterscreen assay provides a robust platform to identify ATG4B inhibitors, which would help to elucidate the mechanism of the autophagy pathway and offer opportunities for drug discovery.

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A General TR-FRET Assay Platform for High-Throughput Screening and Characterizing Inhibitors of Methyl-Lysine Reader Proteins

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555219844569 ◽

2019 ◽

Vol 24 (6) ◽

pp. 693-700 ◽

Cited By ~ 1

Author(s):

Justin M. Rectenwald ◽

P. Brian Hardy ◽

Jacqueline L. Norris-Drouin ◽

Stephanie H. Cholensky ◽

Lindsey I. James ◽

...

Keyword(s):

High Throughput ◽

Posttranslational Modifications ◽

High Throughput Screening ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Time Resolved ◽

Disease States ◽

Downstream Effects ◽

Compound Screening ◽

Inhibitor Selectivity

Chromatin regulatory complexes localize to specific sites via recognition of posttranslational modifications (PTMs) on N-terminal tails of histone proteins (e.g., methylation, acetylation, and phosphorylation). Molecular recognition of modified histones is mediated by “reader” protein subunits. The recruited complexes govern processes such as gene transcription, DNA replication, and chromatin remodeling. Dysregulation of histone modifications and consequent downstream effects have been associated with a variety of disease states, leading to an interest in developing small-molecule inhibitors of reader proteins. Herein, we describe a generalized time-resolved fluorescence resonance energy transfer (TR-FRET) assay for a panel of methyl-lysine (Kme) reader proteins. These assays are facile, robust, and reproducible. Importantly, this plug-and-play assay can be used for high-throughput screening (HTS) campaigns, generation of structure–activity relationships (SARs), and evaluation of inhibitor selectivity. Successful demonstration of this assay format for compound screening is highlighted with a pilot screen of a focused compound set with CBX2. This assay platform enables the discovery and characterization of chemical probes that can potently and selectively inhibit Kme reader proteins to ultimately accelerate studies of chromatin reader proteins in normal biology and disease states.

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Further Comparison of Primary Hit Identification by Different Assay Technologies and Effects of Assay Measurement Variability

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057105275628 ◽

2005 ◽

Vol 10 (6) ◽

pp. 581-589 ◽

Cited By ~ 29

Author(s):

Xiang Wu ◽

Matthew A. Sills ◽

Ji-Hu Zhang

Keyword(s):

High Throughput Screening ◽

Single Point ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Critical Factor ◽

Screening Process ◽

Time Resolved ◽

Measurement Variability ◽

Assay Format ◽

Detection Technologies

High-throughput screening (HTS) has grown rapidly in the past decade, with many advances in new assay formats, detection technologies, and laboratory automation. Recently, several studies have shown that the choice of assay technology used for the screening process is particularly important and can yield quite different primary screening outcomes. However, because the screening assays in these previous studies were performed in a single-point determination, it is not clear to what extent the difference observed in the screening results between different assay technologies is attributable to inherent assay variability and day-to-day measurement variation. To address this question, a nuclear receptor coactivator recruitment assay was carried out in 2 different assay formats, namely, AlphaScreen™ and time-resolved fluorescence resonance energy transfer, which probed the same biochemical binding events but with different detection technologies. For each assay format, 4 independent screening runs in a typical HTS setting were completed to evaluate the run-to-run screening variability. These multiple tests with 2 assay formats allow an unambiguous comparison between the discrepancies of different assay formats and the effects of the variability of assay and screening measurements on the screening outcomes. The results provide further support that the choice of assay format or technology is a critical factor in HTS assay development.

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Development of a Fluorescence-Based, Ultra High-Throughput Screening Platform for Nanoliter-Scale Cytochrome P450 Microarrays

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109336592 ◽

2009 ◽

Vol 14 (6) ◽

pp. 668-678 ◽

Cited By ~ 24

Author(s):

Sumitra M. Sukumaran ◽

Benjamin Potsaid ◽

Moo-Yeal Lee ◽

Douglas S. Clark ◽

Jonathan S. Dordick

Keyword(s):

Enzyme Activity ◽

Cytochrome P450 ◽

High Throughput ◽

High Throughput Screening ◽

Imaging System ◽

Early Stage ◽

Wide Field ◽

Cytochrome P450 Enzyme ◽

Assay Sensitivity ◽

Screening Platform

Cytochrome P450 enzyme (CYP450s) assays are critical enzymes in early-stage lead discovery and optimization in drug development. Currently available fluorescence-based reaction assays provide a rapid and reliable method for monitoring CYP450 enzyme activity but are confined to medium-throughput well-plate systems. The authors present a high-throughput, integrated screening platform for CYP450 assays combining enzyme encapsulation techniques, microarraying methods, and wide-field imaging. Alginate-containing microarrays consisting of up to 1134 CYP450 reaction elements were fabricated on functionalized glass slides (reaction volumes 20 to 80 nL, total enzyme content in pg) and imaged to yield endpoint activity, stability, and kinetic data. A charge-coupled device imager acquired quantitative, high-resolution images of a 20 × 20 mm area/snapshot using custom-built wide-field optics with telecentric lenses and easily interchangeable filter sets. The imaging system offered a broad dynamic intensity range (linear over 3 orders of magnitude) and sensitivity down to fluorochrome quantities of <5 fmols, with read accuracy similar to a laser scanner or a fluorescence plate reader but with higher throughput. Rapid image acquisition enabled analysis of CYP450 kinetics. Fluorogenic assays with CYP3A4, CYP2C9, and CYP2D6 on the alginate microarrays exhibited Z′ factors ranging from 0.75 to 0.85, sensitive detection of inhibitory compounds, and reactivity comparable to that in solution, thereby demonstrating the reliability and accuracy of the microarray platform. This system enables for the first time a significant miniaturization of CYP enzyme assays with significant conservation of assay reagents, greatly increased throughput, and no apparent loss of enzyme activity or assay sensitivity. ( Journal of Biomolecular Screening 2009:668-678)

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Homogeneous and Nonradioactive High-Throughput Screening Platform for the Characterization of Kinase Inhibitors in Cell Lysates

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057106294697 ◽

2006 ◽

Vol 11 (8) ◽

pp. 1015-1026 ◽

Cited By ~ 17

Author(s):

Sylvie Guenat ◽

Nathalie Rouleau ◽

Christelle Bielmann ◽

Julie Bedard ◽

Fabienne Maurer ◽

...

Keyword(s):

High Throughput Screening ◽

Kinase Inhibitors ◽

Kinase Assay ◽

Binding Studies ◽

Cell Lysates ◽

Therapeutic Drugs ◽

Jnk Inhibitors ◽

Screening Platform ◽

Potential Inhibitors

Protein kinases are directly implicated in many human diseases; therefore, kinase inhibitors show great promises as new therapeutic drugs. In an effort to facilitate the screening and the characterization of kinase inhibitors, a novel application of the AlphaScreen technology was developed to monitor JNK activity from (1) purified kinase preparations and (2) endogenous kinase from cell lysates preactivated with different cytokines. The authors confirmed that both adenosine triphosphate (ATP) competitive as well as peptide-based JNK inhibitors were able to block the activity of both recombinant and HepG2 endogenous JNK activity. Using the same luminescence technique adapted for binding studies, the authors characterized peptide inhibitor mechanisms by measuring the binding affinity of the inhibitors for JNK. Because of the versatility of the technology, this cell-based JNK kinase assay could be adapted to other kinases and would represent a powerful tool to evaluate endogenous kinase activity and test a large number of potential inhibitors in a more physiologically relevant environment.

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Homogeneous Time-Resolved Fluorescence Quenching Assay (LANCE) for Caspase-3

CrossRef Listing of Deleted DOIs ◽

10.1177/108705710200700306 ◽

2002 ◽

Vol 7 (3) ◽

pp. 223-231 ◽

Cited By ~ 41

Author(s):

Jarkko Karvinen ◽

Pertti Hurskainen ◽

Sujatha Gopalakrishnan ◽

David Burns ◽

Usha Warrior ◽

...

Keyword(s):

Fluorescence Quenching ◽

High Throughput ◽

High Throughput Screening ◽

Caspase 3 ◽

New Drugs ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Viral Proteases ◽

Modern Drug ◽

Quenching Assay

In addition to kinases and G protein—coupled receptors, proteases are one of the main targets in modern drug discovery. Caspases and viral proteases, for instance, are potential targets for new drugs. To satisfy the current need for fast and sensitive high-throughput screening for inhibitors, new homogeneous protease assays are needed. We used a caspase-3 assay as a model to develop a homogeneous time-resolved fluorescence quenching assay technology. The assay utilizes a peptide labeled with both a luminescent europium chelate and a quencher. Cleavage of the peptide by caspase-3 separates the quencher from the chelate and thus recovers europium fluorescence. The sensitivity of the assay was 1 pg/μl for active caspase-3 and 200 pM for the substrate. We evaluated the assay for high-throughput usage by screening 9600 small-molecule compounds. We also evaluated this format for absorption/distribution/metabolism/excretion assays with cell lysates. Additionally, the assay was compared to a commercial fluorescence caspase-3 assay.

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