A robust and quantitative assay platform for multiplexed, high throughput screening of protein kinase inhibitors

2016 ◽  
Vol 52 (81) ◽  
pp. 12112-12115 ◽  
Author(s):  
Jieon Lee ◽  
Il-Soo Park ◽  
Ginam Park ◽  
Kyukwang Cho ◽  
Hee-Sung Park ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Protein Kinase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Protein Kinase Activity ◽  
Activity Assay ◽  
Kinase Activity Assay

We present a new platform for multiplexed protein kinase activity assay using TiO2decorated graphene oxide (GO), which is applicable to high throughput inhibitor screening.

Homogeneous single-label tyrosine kinase activity assay for high throughput screening

2015 ◽  
Vol 897 ◽  
pp. 96-101 ◽  
Author(s):  
Natalia Tong-Ochoa ◽  
Kari Kopra ◽  
Markku Syrjänpää ◽  
Nicolas Legrand ◽  
Harri Härmä
Keyword(s):  
Tyrosine Kinase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Activity Assay ◽  
Tyrosine Kinase Activity ◽  
Kinase Activity Assay

scholarly journals A Scintillating Microplate Assay for the Assessment of Protein Kinase Activity

1998 ◽  
Vol 3 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Grace R. Nakayama ◽  
Michael P. Nova ◽  
Zahra Parandoosh
Keyword(s):  
Protein Kinase ◽  
Tyrosine Kinases ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Kinase Assay ◽  
Protein Kinase Activity ◽  
Discovery Research ◽  
Cell Extracts ◽  
Drug Discovery Research

Protein kinases, a class of enzymes that phosphorylate certain tyrosine, serine, and threonine residues, play an important role in cellular functions and are important targets in drug discovery research. Thus, it is of interest to develop a simple assay that can be used to measure protein kinase activity toward specific substrates and is suitable for the high throughput screening (HTS) of potential kinase inhibitors. The scintillation proximity concept has been successfully applied for measuring specific kinase activity using surfaces passively coated with a peptide substrate. In this study, we evaluated kinase assay performance on three ScintiStrip platforms: unmodified surface, streptavidin-coated surface, and streptavidin covalently attached to surface. The high affinity of streptavidin toward biotin-linked peptide substrates makes it a unique platform for measuring specific incorporation of radiolabeled phosphate into selected substrates of specific enzymes in the presence of others. Therefore, this assay may be used with cell extracts containing impure kinases as well as with purified enzymes. The scope of this assay was demonstrated with purified tyrosine kinases (e.g., p60c-src kinase) and A431 cell extracts. This scintillation proximity assay is universal, simple, rapid, accurate, and can be adapted for use with robotics for HTS.

Fluorescent biosensors for high throughput screening of protein kinase inhibitors

2013 ◽  
Vol 9 (2) ◽  
pp. 253-265 ◽  
Author(s):  
Camille Prével ◽  
Morgan Pellerano ◽  
Thi Nhu Ngoc Van ◽  
May C. Morris
Keyword(s):  
Protein Kinase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Fluorescent Biosensors

scholarly journals Novel Chemical Class of pUL97 Protein Kinase-Specific Inhibitors with Strong Anticytomegaloviral Activity

2004 ◽  
Vol 48 (11) ◽  
pp. 4154-4162 ◽  
Author(s):  
Thomas Herget ◽  
Martina Freitag ◽  
Monika Morbitzer ◽  
Regina Kupfer ◽  
Thomas Stamminger ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Human Fibroblasts ◽  
Growth Factor Receptor ◽  
Protein Kinase Activity ◽  
Hcmv Replication

ABSTRACT Human cytomegalovirus (HCMV) is a major human pathogen frequently associated with life-threatening disease in immunosuppressed patients and newborns. The HCMV UL97-encoded protein kinase (pUL97) represents an important determinant of viral replication. Recent studies demonstrated that pUL97-specific kinase inhibitors are powerful tools for the control of HCMV replication. We present evidence that three related quinazoline compounds are potent inhibitors of the pUL97 kinase activity and block in vitro substrate phosphorylation, with 50% inhibitory concentrations (IC50s) between 30 and 170 nM. Replication of HCMV in primary human fibroblasts was suppressed with a high efficiency. The IC50s of these three quinazoline compounds (2.4 ± 0.4, 3.4 ± 0.6, and 3.9 ± 1.1 μM, respectively) were in the range of the IC50 of ganciclovir (1.2 ± 0.2 μM), as determined by the HCMV green fluorescent protein-based antiviral assay. Importantly, the quinazolines were demonstrated to have strong inhibitory effects against clinical HCMV isolates, including ganciclovir- and cidofovir-resistant virus variants. Moreover, in contrast to ganciclovir, the formation of resistance to the quinazolines was not observed. The mechanisms of action of these compounds were confirmed by kinetic analyses with infected cells. Quinazolines specifically inhibited viral early-late protein synthesis but had no effects at other stages of the replication cycle, such as viral entry, consistent with a blockage of the pUL97 function. In contrast to epithelial growth factor receptor inhibitors, quinazolines affected HCMV replication even when they were added hours after virus adsorption. Thus, our findings indicate that quinazolines are highly efficient inhibitors of HCMV replication in vitro by targeting pUL97 protein kinase activity.

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