scholarly journals Assay Concordance between SPA and TR-FRET in High-Throughput Screening

2006 ◽  
Vol 11 (6) ◽  
pp. 606-616 ◽  
Author(s):  
Oliver Von Ahsen ◽  
Anne Schmidt ◽  
Monika Klotz ◽  
Karsten Parczyk
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Statistical Significance ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Detection Technology ◽  
Significant Difference ◽  
Set Up ◽  
Detection Technologies

High-throughput screening (HTS) of large chemical libraries has become the main source of new lead compounds for drug development. Several specialized detection technologies have been developed to facilitate the cost- and time-efficient screening of millions of compounds. However, concerns have been raised, claiming that different HTS technologies may produce different hits, thus limiting trust in the reliability of HTS data. This study was aimed to investigate the reliability of the authors most frequently used assay techniques: scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET). To investigate the data concordance between these 2 detection technologies, the authors screened a large subset of the Schering compound library consisting of 300,000 compounds for inhibitors of a nonreceptor tyrosine kinase. They chose to set up this study in realistic HTS scale to ensure statistical significance of the results. The findings clearly demonstrate that the choice of detection technology has no significant impact on hit finding, provided that assays are biochemically equivalent. Data concordance is up to 90%. The little differences in hit findings are caused by threshold setting but not by systematic differences between the technologies. The most significant difference between the compared techniques is that in the SPA format, more false-positive primary hits were obtained.

scholarly journals Repurposing a Histamine Detection Platform for High-Throughput Screening of Histidine Decarboxylase

2018 ◽  
Vol 23 (9) ◽  
pp. 974-981
Author(s):  
Yu-Chi Juang ◽  
Xavier Fradera ◽  
Yongxin Han ◽  
Anthony William Partridge
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Histidine Decarboxylase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Gastric Ulcers ◽  
Statistical Parameters ◽  
Time Resolved ◽  
Neurological Response

Histidine decarboxylase (HDC) is the primary enzyme that catalyzes the conversion of histidine to histamine. HDC contributes to many physiological responses as histamine plays important roles in allergic reaction, neurological response, gastric acid secretion, and cell proliferation and differentiation. Small-molecule modulation of HDC represents a potential therapeutic strategy for a range of histamine-associated diseases, including inflammatory disease, neurological disorders, gastric ulcers, and select cancers. High-throughput screening (HTS) methods for measuring HDC activity are currently limited. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring HDC activity. The assay is based on competition between HDC-generated histamine and fluorophore-labeled histamine for binding to a Europium cryptate (EuK)-labeled anti-histamine antibody. We demonstrated that the assay is highly sensitive and simple to develop. Assay validation experiments were performed using low-volume 384-well plates and resulted in good statistical parameters. A pilot HTS screen gave a Z′ score > 0.5 and a hit rate of 1.1%, and led to the identification of a validated hit series. Overall, the presented assay should facilitate the discovery of therapeutic HDC inhibitors by acting as a novel tool suitable for large-scale HTS and subsequent interrogation of compound structure–activity relationships.

scholarly journals Thermally Denatured BSA, a Surrogate Additive to Replace BSA in Buffers for High-Throughput Screening

2010 ◽  
Vol 15 (10) ◽  
pp. 1281-1286 ◽  
Author(s):  
Imanol Peña ◽  
Juan Manuel Domínguez
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Negative Impact ◽  
Specific Binding ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Masking Effect ◽  
Binding Properties ◽  
Time Resolved ◽  
Beneficial Effects

The use of thermally denatured bovine serum albumin (tdBSA) as an additive in high-throughput screening (HTS) buffers has been studied with the aim of finding a surrogate to native albumin devoid of its inconveniences, in particular its compound masking effect. The presence of aggregates in the thermally denatured material did not have any negative impact on common readout technologies used in HTS such as fluorescence intensity (FLINT), fluorescence polarization, time-resolved fluorescence resonance energy transfer (TR-FRET) and luminescence. tdBSA rendered the same beneficial effects as native albumin in several assays or even improved its performance due to the lack of specific binding properties. Although tdBSA still binds compounds nonspecifically as any other protein does, it mitigates the compound masking effect observed with native albumin and can be postulated as a convenient surrogate to BSA for HTS purposes.

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

2016 ◽  
Vol 22 (4) ◽  
pp. 338-347 ◽  
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.

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

2019 ◽  
Vol 24 (6) ◽  
pp. 693-700 ◽  
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.

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

2005 ◽  
Vol 10 (6) ◽  
pp. 581-589 ◽  
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.

scholarly journals Homogeneous High-Throughput Screening Assays for HIV-1 Integrase 3β-Processing and Strand Transfer Activities

2005 ◽  
Vol 10 (5) ◽  
pp. 456-462 ◽  
Author(s):  
Yu Wang ◽  
Heath Klock ◽  
Hong Yin ◽  
Karen Wolff ◽  
Kimberly Bieza ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Antiviral Drug ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Biologically Active ◽  
Strand Transfer ◽  
Virus Propagation ◽  
Time Resolved ◽  
Hiv 1

HIV-1 integrase (HIV-IN) is a well-validated antiviral drug target catalyzing a multistep reaction to incorporate the HIV-1 provirus into the genome of the host cell. Smallmolecule inhibitors of HIV-1 integrase that specifically target the strand transfer step have demonstrated efficacy in the suppression of virus propagation. However, only fewspecific strand transfer inhibitors have been identified to date, and the need to screen for novel compound scaffolds persists. Here, the authors describe 2 homogeneous time-resolved fluorescent resonance energy transfer-based assays for the measurement of HIV-1 integrase 3'-processing and strand transfer activities. Both assayswere optimized for high-throughput screening formats, and a diverse library containingmore than 1million compoundswas screened in 1536-well plates for HIV-IN strand transfer inhibitors. As a result, compounds were found that selectively affect the enzymatic strand transfer reaction over 3β processing. Moreover, several bioactivemoleculeswere identified that inhibited HIV-1 reporter virus infection in cellularmodel systems. In conclusion, the assays presented herein have proven their utility for the identification ofmechanistically interesting and biologically active inhibitors of HIV-1 integrase that hold potential for further development into potent antiviral drugs.

scholarly journals Comparison of Miniaturized Time-Resolved Fluorescence Resonance Energy Transfer and Enzyme-Coupled Luciferase High-Throughput Screening Assays to Discover Inhibitors of Rho-Kinase II (ROCK-II)

2007 ◽  
Vol 13 (1) ◽  
pp. 17-28 ◽  
Author(s):  
Thomas Schröter ◽  
Dmitriy Minond ◽  
Amiee Weiser ◽  
Chinh Dao ◽  
Jeff Habel ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Rho Kinase ◽  
Resonance Energy ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Fluorescence Resonance

Kinases are important drug discovery targets for a wide variety of therapeutic indications; consequently, the measurement of kinase activity remains a common high-throughput screening (HTS) application. Recently, enzyme-coupled luciferase-kinase (LK) format assays have been introduced. This format measures luminescence resulting from metabolism of adenosine triphosphate (ATP) via a luciferin/luciferase-coupled reaction. In the research presented here, 1536-well format time-resolved fluorescence resonance energy transfer (TR-FRET) and LK assays were created to identify novel Rho-associated kinase II (ROCK-II) inhibitors. HTS campaigns for both assays were conducted in this miniaturized format. It was found that both assays were able to consistently reproduce the expected pharmacology of inhibitors known to be specific to ROCK-II (fasudil IC50: 283 ± 27 nM and 336 ± 54 nM for TR-FRET and LK assays, respectively; Y-27632 IC50: 133 ± 7.8 nM and 150 ± 22 nM for TR-FRET and LK assays, respectively). In addition, both assays proved robust for HTS efforts, demonstrating excellent plate Z′ values during the HTS campaign (0.84 ± 0.03; 0.72 ± 0.05 for LK and TR-FRET campaigns, respectively). Both formats identified scaffolds of known and novel ROCK-II inhibitors with similar sensitivity. A comparison of the performance of these 2 assay formats in an HTS campaign was enabled by the existence of a subset of 25,000 compounds found in both our institutional and the Molecular Library Screening Center Network screening files. Analysis of the HTS campaign results based on this subset of common compounds showed that both formats had comparable total hit rates, hit distributions, amount of hit clusters, and format-specific artifact. It can be concluded that both assay formats are suitable for the discovery of ROCK-II inhibitors, and the choice of assay format depends on reagents and/or screening technology available. ( Journal of Biomolecular Screening 2008:17-28)

scholarly journals A Widely Applicable, High-Throughput TR-FRET Assay for the Measurement of Kinase Autophosphorylation: VEGFR-2 as a Prototype

2003 ◽  
Vol 8 (4) ◽  
pp. 447-452 ◽  
Author(s):  
Deborah J. Moshinsky ◽  
Lany Ruslim ◽  
Robert A. Blake ◽  
Flora Tang
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Growth Factor Receptor ◽  
Resonance Energy ◽  
Antiangiogenic Agents ◽  
General Technique ◽  
Time Resolved ◽  
Exogenous Substrate ◽  
Dependent Inhibition

Homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assays represent a highly sensitive and robust high-throughput screening (HTS) method for the quantification of kinase activity. Traditional TR-FRET kinase assays detect the phosphorylation of an exogenous substrate. The authors describe the development and optimization of a TR-FRET technique that measures the autophosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) kinase and extend its applicability to a variety of other kinases. The VEGFR-2 assay demonstrated dose-dependent inhibition by compounds known to modulate the catalytic activity of this receptor. In addition, kinetic analysis of a previously characterized VEGFR-2 inhibitor was performed using the method, and results were consistent with those obtained using a different assay format. Because of the known involvement of VEGFR-2 in angiogenesis, this assay should facilitate HTS for antiangiogenic agents. In addition, this general technique should have utility for the screening for inhibitors of kinases as potential therapeutic agents for many other disease indications.

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