Microarray-based method for monitoring yeast overexpression strains reveals small-molecule targets in TOR pathway.
Identification of the cellular targets of small-molecule hits in phenotypic screens is a central challenge in the development of small molecules as biological tools and potential therapeutics. To facilitate the process of small-molecule target identification, we developed a global, microarray-based method for monitoring the growth of pools of yeast strains, each overexpressing a different protein, in the presence of small molecules. Specifically, the growth of Saccharomyces cerevisiae strains harboring approximately 3,900 different overexpression plasmids was monitored in the presence of rapamycin, which inhibits the target of rapamycin (TOR) proteins. TOR was successfully identified as a candidate rapamycin target, and many additional gene products were implicated in the TOR signaling pathway. We also characterized the mechanism of LY-83583, a small-molecule suppressor of rapamycin-induced growth inhibition. These data enabled functional links to be drawn between groups of genes implicated in the TOR pathway, identified several candidate targets for LY-83583, and suggested a role for mitochondrial respiration in mediating rapamycin sensitivity.
Butcher
Rebecca A
RA
Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
Bhullar
Bhupinder S
BS
Perlstein
Ethan O
EO
Marsischky
Gerald
G
LaBaer
Joshua
J
Schreiber
Stuart L
SL
D000634:Aminoquinolines / Q000494:pharmacology*; D000935:Antifungal Agents / Q000494:pharmacology; D015195:Drug Design; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D040081:Protein Array Analysis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology*
D000634:Aminoquinolines; D000935:Antifungal Agents; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D029701:Saccharomyces cerevisiae Proteins; D020123:Sirolimus; C041715:6-anilino-5,8-quinolinedione; C083324:TOR1 protein, S cerevisiae