High-throughput screening for mTORC1/mTORC2 kinase inhibitors using a chemiluminescence-based ELISA assay.
A high-throughput chemiluminescence and ELISA-based biochemical assay to identify mTORC1/mTORC2 kinase inhibitors is described. These mTOR complexes were isolated from HeLa whole cell lysate using mTOR antibodies and in-well immunoprecipitation. The integrity and purity of the mTORC1 and mTORC2 immunocomplexes were confirmed by western blotting. Full-length recombinant 4E-BP1 was used as a substrate and the catalytic activity was measured by detection of p4E-BP1 [T37/46] by a chemiluminescence method. The performance of this assay that can be used to identify dual mTORC1 and mTORC2 kinase inhibitors in a high-throughput 384-well format is described.
Bhagwat
Shripad V
SV
Department of Cancer Biology, OSI Pharmaceuticals Inc., Farmingdale, New York 11735, USA. Sbhagwat@osip.com
Kahler
Jennifer
J
Yao
Yan
Y
Maresca
Paul
P
Brooks
Maureen
M
Crew
Andy
A
Boisclair
Michael
M
Pachter
Jonathan A
JA
D000255:Adenosine Triphosphate / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007202:Indicators and Reagents; D049449:Luminescence; D000076222:Mechanistic Target of Rapamycin Complex 1; D009025:Morpholines / Q000494:pharmacology; D046912:Multiprotein Complexes; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011506:Proteins; D015203:Reproducibility of Results; D013379:Substrate Specificity; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000037:antagonists & inhibitors*
C493245:CRTC2 protein, human; D002867:Chromones; D007202:Indicators and Reagents; D009025:Morpholines; D046912:Multiprotein Complexes; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D011506:Proteins; D014157:Transcription Factors; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D000255:Adenosine Triphosphate; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases