[Establishment of homogeneous time-resolved fluorescence immunoassay for high throughput screening of protein tyrosine kinase inhibitors].
OBJECTIVE
To establish an in vitro homogeneous time-resolved fluorescence immunoassay method for high throughput screening of protein tyrosine kinase (PTK) inhibitors.


METHODS
Specific fluorescence signals at 670 and 612 nm were measured by multifunctional microplate reader when the fluorescence was emitted through a resonance energy transfer between fluorescent materials (EuK and XL-665). The inhibitory activity of Sunitinib, a standard PTK inhibitor, on vascular endothelia growth factor receptor 2 (VEGFR-2) kinase activity was investigated.


RESULTS
A homogeneous time-resolved fluorescence immunoassay was established for high throughput screening of PTK inhibitor. In this system, the concentrations of VEGFR-2, adenosine triphosphate (ATP) and poly-peptide substrate were 5 ng/microl, 100 micromol/L and 1 micromol/L, respectively. Sunitinib inhibited VEGFR-2 kinase activity with an IC50 value of 86.7 nmol/L, which was close to the values tested using other methods.


CONCLUSIONS
The homogeneous time-resolved fluorescence immunoassay we established can be easily used for high throughput screening of PTK inhibitors.
Li
Xu-gui
XG
School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China.
Wang
Guang-fa
GF
Zhang
Jun-yan
JY
Wu
Shao-yu
SY
Xu
Wei
W
Wu
Shu-guang
SG
Zhang
Jia-jie
JJ
D015200:Fluoroimmunoassay / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D007211:Indoles / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011758:Pyrroles / Q000494:pharmacology; D000077210:Sunitinib; D013997:Time Factors; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors / Q000378:metabolism
D007211:Indoles; D010455:Peptides; D047428:Protein Kinase Inhibitors; D011758:Pyrroles; D011505:Protein-Tyrosine Kinases; D040301:Vascular Endothelial Growth Factor Receptor-2; D000077210:Sunitinib