Development of a homogeneous time-resolved fluorescence assay for high throughput screening to identify Lck inhibitors: Comparison with scintillationproximity assay and streptavidin-coated plate assay
N. Ohmi et al., Development of a homogeneous time-resolved fluorescence assay for high throughput screening to identify Lck inhibitors: Comparison with scintillationproximity assay and streptavidin-coated plate assay, J BIOMOL SC, 5(6), 2000, pp. 463-470
This study details the development of a homogeneous time-resolved fluoresce
nce (HTRF) high throughput screening assay to identify inhibitors of Lck, H
TRF was compared with scintillation proximity and streptavidin-coated plate
assays, Because of the differences in the sensitivity of detection of phos
photyrosine 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 inhibi
tory compounds were independent of assay format. Furthermore, filtration ex
periments revealed that phosphorylation of a biotinyl poly-Glu,Ala, Tyr pep
tide substrate was less than autophosphorylation of the Lck enzyme; this wa
s 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, the
reby minimizing the effects of autophosphorylation, Higher ATP concentratio
n 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.