A chemical genetic screen for mTOR pathway inhibitors based on 4E-BP-dependent nuclear accumulation of eIF4E.
The signal transduction pathway wherein mTOR regulates cellular growth and proliferation is an active target for drug discovery. The search for new mTOR inhibitors has recently yielded a handful of promising compounds that hold therapeutic potential. This search has been limited by the lack of a high-throughput assay to monitor the phosphorylation of a direct rapamycin-sensitive mTOR substrate in cells. Here we describe a novel cell-based chemical genetic screen useful for efficiently monitoring mTOR signaling to 4E-BPs in response to stimuli. The screen is based on the nuclear accumulation of eIF4E, which occurs in a 4E-BP-dependent manner specifically upon inhibition of mTOR signaling. Using this assay in a small-scale screen, we have identified several compounds not previously known to inhibit mTOR signaling, demonstrating that this method can be adapted to larger screens.
Livingstone
Mark
M
Department of Biochemistry and McGill Cancer Centre, McGill University, Montreal, QC H3A 1A3, Canada.
Larsson
Ola
O
Sukarieh
Rami
R
Pelletier
Jerry
J
Sonenberg
Nahum
N
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D055808:Drug Discovery / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D039561:Eukaryotic Initiation Factor-4E / Q000235:genetics / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009025:Morpholines / Q000494:pharmacology; D010750:Phosphoproteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002867:Chromones; C499527:Eif4ebp1 protein, mouse; C491255:Eif4ebp2 protein, mouse; D004791:Enzyme Inhibitors; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D047908:Intracellular Signaling Peptides and Proteins; D009025:Morpholines; D010750:Phosphoproteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C546842:MTOR protein, human; C546843:mTOR protein, mouse; D017346:Protein Serine-Threonine Kinases; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus