scholarly journals Development of a Homogeneous Time-Resolved Fluorescence Assay for High Throughput Screening to Identify Lck Inhibitors: Comparison with Scintillation Proximity Assay and Streptavidin-Coated Plate Assay

2000 ◽  
Vol 5 (6) ◽  
pp. 463-470 ◽  
Author(s):  
Natsue Ohml ◽  
Jonathan M. Wingfield ◽  
Hidenori Yazawa ◽  
Osamu Inagaki
Keyword(s):  
High Throughput ◽  
Tyrosine Kinases ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Time Resolved Fluorescence ◽  
Screening Assay ◽  
Time Resolved ◽  
Atp Binding Site ◽  
High Throughput Screening Assay ◽  
Coated Plate

This study details the development of a homogeneous time-resolved fluorescence (HTRF) high throughput screening assay to identify inhibitors of Lck. HTRF was compared with scintillation proximity and streptavidin-coated plate assays. Because of the differences in the sensitivity of detection of phosphotyrosine among the three assays, different amounts of enzyme were used. However, the concentrations of the other assay components were standardized. When using similar assay conditions, the calculated IC50 values of inhibitory compounds were independent of assay format. Furthermore, filtration experiments revealed that phosphorylation of a biotinyl poly-Glu,Ala, Tyr peptide substrate was less than autophosphorylation of the Lck enzyme; this was due to the low Km value for biotinyl poly-Glu,Ala,Tyr. In the HTRF assay, small amounts of enzyme and high concentrations of ATP could be used, thereby minimizing the effects of autophosphorylation. Higher ATP concentration would also minimize the effect of ATP competitors. Using this technology, it may be possible to find novel kinase inhibitors that do not act at the ATP binding site of protein tyrosine kinases.

A Generic High-Throughput Screening Assay for Kinases: Protein Kinase A as an Example

2003 ◽  
Vol 8 (2) ◽  
pp. 198-204 ◽  
Author(s):  
Rommel Mallari ◽  
Elissa Swearingen ◽  
Wei Liu ◽  
Arnold Ow ◽  
Stephen W. Young ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Signal To Noise Ratio ◽  
Product Formation ◽  
Screening Assay ◽  
Chemical Libraries ◽  
Coulombic Interactions ◽  
High Throughput Screening Assay ◽  
Peptide Product

A generic high-throughput screening assay based on the scintillation proximity assay technology has been developed for protein kinases. In this assay, the biotinylated 33P-peptide product is captured onto polylysine Ysi bead via avidin. The scintillation signal measuring the product formation increases linearly with avidin concentration due to effective capture of the product on the bead surface via strong coulombic interactions. This novel assay has been optimized and validated in 384-well microplates. In a pilot screen, a signal-to-noise ratio of 5-to 9-fold and a Z′ factor ranging from 0.6 to 0.8 were observed, demonstrating the suitability of this assay for high-throughput screening of random chemical libraries for kinase inhibitors. ( Journal of Biomolecular Screening 2003:198-204)

scholarly journals TR-FRET-Based High-Throughput Screening Assay for Identification of UBC13 Inhibitors

2011 ◽  
Vol 17 (2) ◽  
pp. 163-176 ◽  
Author(s):  
Charitha Madiraju ◽  
Kate Welsh ◽  
Michael P. Cuddy ◽  
Paulo H. Godoi ◽  
Ian Pass ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Inflammatory Diseases ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Chemical Library ◽  
Screening Assay ◽  
Time Resolved ◽  
High Throughput Screening Assay

UBC13 is a noncanonical ubiquitin conjugating enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of lysine 63–linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair and thus a potential radiosensitizer and chemosensitizer target for oncology. The authors developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)–conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)–conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. The authors defined conditions for optimized performance of the TR-FRET assay in both 384- and 1536-well formats. Chemical library screens (total 456 865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z′ scores (typically >0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13.

scholarly journals High-Throughput Screening Assay for Sphingosine Kinase Inhibitors in Whole Blood Using RapidFire® Mass Spectrometry

2011 ◽  
Vol 16 (2) ◽  
pp. 272-277 ◽  
Author(s):  
Maureen K. Highkin ◽  
Matthew P. Yates ◽  
Olga V. Nemirovskiy ◽  
William A. Lamarr ◽  
Grace E. Munie ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Whole Blood ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Mass Spectrometry Technology ◽  
Sphingosine 1 Phosphate ◽  
Mass Spectrometry System

To facilitate discovery of compounds modulating sphingosine-1-phosphate (S1P) signaling, the authors used high-throughput mass spectrometry technology to measure S1P formation in human whole blood. Since blood contains endogenous sphingosine (SPH) and S1P, mass spectrometry was chosen to detect the conversion of an exogenously added 17-carbon-long variant of sphingosine, C17SPH, into C17S1P. The authors developed procedures to achieve homogeneous mixing of whole blood in 384-well plates and for a method requiring minimal manipulations to extract S1P from blood in 96- and 384-well plates prior to analyses using the RapidFire® mass spectrometry system.

scholarly journals Corrigendum

2014 ◽  
Vol 19 (10) ◽  
pp. 1418-1418
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Screening Assay ◽  
Atm Kinase ◽  
High Throughput Screening Assay ◽  
A Cell

Kexiao Guo, Anang A. Shelat, R. Kiplin Guy, and Michael B. Kastan. Development of a Cell-Based, High-Throughput Screening Assay for ATM Kinase Inhibitors J. Biomol. Screen. 2014, 19( 4)538-546.

scholarly journals Identification of Autophosphorylation Inhibitors of the Inositol-Requiring Enzyme 1 Alpha (IRE1α) by High-Throughput Screening Using a DELFIA Assay

2012 ◽  
Vol 18 (3) ◽  
pp. 298-308 ◽  
Author(s):  
Yvette Newbatt ◽  
Anthea Hardcastle ◽  
P. Craig McAndrew ◽  
Jade A. Strover ◽  
Amin Mirza ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protein Misfolding ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Screening Assay ◽  
Unfolded Protein ◽  
High Throughput Screening Assay ◽  
The Unfolded Protein Response

Inositol-requiring enzyme 1 alpha (IRE1α) is a transmembrane sensor protein with both kinase and ribonuclease activity, which plays a crucial role in the unfolded protein response (UPR). Protein misfolding in the endoplasmic reticulum (ER) lumen triggers dimerization and subsequent trans-autophosphorylation of IRE1α. This leads to the activation of its endoribonuclease (RNase) domain and splicing of the mRNA of the transcriptional activator XBP1, ultimately generating an active XBP1 (XBP1s) implicated in multiple myeloma survival. Previously, we have identified human IRE1α as a target for the development of kinase inhibitors that could modulate the UPR in human cells, which has particular relevance for multiple myeloma and other secretory malignancies. Here we describe the development and validation of a 384-well high-throughput screening assay using DELFIA technology that is specific for IRE1α autophosphorylation. Using this format, a focused library of 2312 potential kinase inhibitors was screened, and several novel IRE1α kinase inhibitor scaffolds were identified that could potentially be developed toward new therapies to treat multiple myeloma.

scholarly journals Development of a High-Throughput Screening Assay for Inhibitors of Aggrecan Cleavage Using Luminescent Oxygen Channeling (AlphaScreen™)

2003 ◽  
Vol 8 (2) ◽  
pp. 149-156 ◽  
Author(s):  
Jane Peppard ◽  
Fraser Glickman ◽  
Yang He ◽  
Shou-Ih Hu ◽  
John Doughty ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Cross Link ◽  
Screening Assay ◽  
Time Resolved ◽  
High Throughput Screening Assay ◽  
Donor And Acceptor ◽  
A Disintegrin And Metalloproteinase ◽  
Set Up

Aggrecan is one of the most important structural components of joint cartilage, and members of the metalloprotease (MMP) and ADAM (a disintegrin and metalloproteinase) protease families have been shown to degrade aggrecan in vivo. A robust assay for aggrecan-degrading activity suitable for high-throughput screening (HTS) was set up and measured using AlphaScreen™. In this technology, beads brought into proximity through cross-linking and stimulated with laser light generate a signal through luminescent oxygen tunneling, the outcome of which is a time-resolved fluorescent signal. Specific antibodies to the carbohydrate side chains of aggrecan were harnessed to create a scaffold whereby aggrecan could form a cross-link between donor and acceptor AlphaScreen detector beads. Digested aggrecan, which failed to form a cross-link, generated no signal, so that inhibitors of the digestion could be detected as a restoration of signal. The development of this assay and its validation for HTS are described in this report. ( Journal of Biomolecular Screening 2003:136-148)

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