Open AccessDevelopment 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
Author(s) -
Natsue Ohml,
Jonathan Wingfield,
Hidenori Yazawa,
Osamu Inagaki
Publication year - 2000
Publication title -
slas discovery
Language(s) - English
Resource type - Journals
eISSN - 2472-5560
pISSN - 2472-5552
DOI - 10.1177/108705710000500609
Subject(s) - autophosphorylation , biotinylation , streptavidin , high throughput screening , chemistry , enzyme , ligand binding assay , kinase , biochemistry , phosphorylation , microtiter plate , substrate (aquarium) , peptide , tyrosine kinase , fluorescence , chromatography , microbiology and biotechnology , biology , biotin , protein kinase a , receptor , ecology , physics , quantum mechanics
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 IC 50 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 K m 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.