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 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 High-Throughput Turbidometric Assay for Screening Inhibitors of Protein Disulfide Isomerase Activity

2004 ◽  
Vol 9 (7) ◽  
pp. 614-620 ◽  
Author(s):  
Anthony M. Smith ◽  
John Chan ◽  
Donna Oksenberg ◽  
Roman Urfer ◽  
David S. Wexler ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Protein Disulfide Isomerase ◽  
Secretory Proteins ◽  
Screening Assay ◽  
Kinetic Assay ◽  
Chemical Libraries ◽  
Disulfide Isomerase ◽  
High Throughput Screening Assay ◽  
Protein Disulfide

Protein disulfide isomerase (PDI) plays a key role in protein folding by catalyzing rearrangements of disulfide bonds in substrate proteins following their synthesis in eukaryotic cells. Besides its major role in the processing and maturation of secretory proteins in the endoplasmic reticulum, this enzyme and its homologs have been implicated in multiple important cellular processes; however, they have not served as targets for the development of therapeutic agents. The authors developed a high-throughput screening assay for PDI and its homologous enzymes in 384-well microplates. The method is based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol and measures the aggregation of reduced insulin chains at 650 nm. This kinetic assay was converted to an end-point assay by using hydrogen peroxide as a stop reagent. The feasibility of this high-throughput assay for screening chemical libraries was demonstrated in a pilot screen. The authors show that this homogenous turbidometric assay is robust and cost-effective and can be applied to identify PDI inhibitors from chemical libraries, opening this class of enzymes for therapeutic exploration.

Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry

2020 ◽  
Vol 25 (9) ◽  
pp. 1064-1071
Author(s):  
Maikel Izquierdo ◽  
De Lin ◽  
Sandra O’Neill ◽  
Martin Zoltner ◽  
Lauren Webster ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Trypanosoma Cruzi ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Signal To Noise Ratio ◽  
Screening Method ◽  
Cellular Functions ◽  
Screening Assay ◽  
High Throughput Screening Assay

Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin–based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)–based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 μM) and arphamenine A (IC50 = 15.75 μM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.

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 Development of High-Throughput Screening Assay for Antihantaviral Therapeutics

2017 ◽  
Vol 22 (6) ◽  
pp. 767-774
Author(s):  
Anuradha Roy ◽  
Mohammad A. Mir
Keyword(s):  
High Throughput ◽  
Therapeutic Intervention ◽  
Nucleocapsid Protein ◽  
High Throughput Screening ◽  
Critical Role ◽  
Hantavirus Infection ◽  
Screening Assay ◽  
Chemical Libraries ◽  
Conserved Sequence ◽  
High Throughput Screening Assay

Humans acquire hantavirus infection by the inhalation of aerosolized excreta of infected rodent hosts. There is no treatment for hantavirus diseases at present. Therapeutic intervention during early stages of viral infection can improve the outcome of this zoonotic viral illness. The interaction between an evolutionary conserved sequence at the 5′ terminus of hantaviral genomic RNA and hantavirus nucleocapsid protein plays a critical role in the hantavirus replication cycle. This unique interaction is a novel target for therapeutic intervention of hantavirus disease. We developed a very sensitive, tractable, and cost-effective fluorescence-based assay to monitor the interaction between the nucleocapsid protein and the target RNA sequence. The assay was optimized for high-throughput screening of chemical libraries to identify molecules that interrupt this RNA–protein interaction. The assay was validated using a library of 6880 chemical compounds. This validation screen demonstrated the reproducibility and validity of required statistical criteria for high-throughput screening. The assay is ready to use for high-throughput screening of large chemical libraries to identify antihantaviral therapeutic molecules and can be amenable to similar targets in other viruses.

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.

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