title: Chemical genetics of rapamycin-insensitive TORC2 in S. cerevisiae.
abstract: Current approaches for identifying synergistic targets use cell culture models to see if the combined effect of clinically available drugs is better than predicted by their individual efficacy. New techniques are needed to systematically and rationally identify targets and pathways that may be synergistic targets. Here, we created a tool to screen and identify molecular targets that may synergize with new inhibitors of target of rapamycin (TOR), a conserved protein that is a major integrator of cell proliferation signals in the nutrient-signaling pathway. Although clinical results from TOR complex 1 (TORC1)-specific inhibition using rapamycin analogs have been disappointing, trials using inhibitors that also target TORC2 have been promising. To understand this increased therapeutic efficacy and to discover secondary targets for combination therapy, we engineered Tor2 in S. cerevisiae to accept an orthogonal inhibitor. We used this tool to create a chemical epistasis miniarray profile (ChE-MAP) by measuring interactions between the chemically inhibited Tor2 kinase and a diverse library of deletion mutants. The ChE-MAP identified known TOR components and distinguished between TORC1- and TORC2-dependent functions. The results showed a TORC2-specific interaction with the pentose phosphate pathway, a previously unappreciated TORC2 function that suggests a role for the complex in balancing the high energy demand required for ribosome biogenesis.
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biosciences, QB3, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, San Francisco, CA 94158, USA.
authors.forename: Joseph I
authors.initials: JI
authors.lastname: Kliegman
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08540, USA.
authors.forename: Dorothea
authors.initials: D
authors.lastname: Fiedler
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biosciences, QB3, San Francisco, CA 94158, USA; School of Computer Science and Informatics, University College Dublin, Dublin 4, Ireland.
authors.forename: Colm J
authors.initials: CJ
authors.lastname: Ryan
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08540, USA.
authors.forename: Yi-Fan
authors.initials: YF
authors.lastname: Xu
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08540, USA.
authors.forename: Xiao-Yang
authors.initials: XY
authors.lastname: Su
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08540, USA.
authors.forename: David
authors.initials: D
authors.lastname: Thomas
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biosciences, QB3, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, San Francisco, CA 94158, USA.
authors.forename: Max C
authors.initials: MC
authors.lastname: Caccese
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biosciences, QB3, San Francisco, CA 94158, USA.
authors.forename: Ada
authors.initials: A
authors.lastname: Cheng
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biosciences, QB3, San Francisco, CA 94158, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Shales
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08540, USA.
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Rabinowitz
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biosciences, QB3, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA.
authors.forename: Nevan J
authors.initials: NJ
authors.lastname: Krogan
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biosciences, QB3, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, San Francisco, CA 94158, USA. Electronic address: kevan.shokat@ucsf.edu.
authors.forename: Kevan M
authors.initials: KM
authors.lastname: Shokat
mesh_terms: D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004843:Epistasis, Genetic; D017353:Gene Deletion; D057166:High-Throughput Screening Assays / Q000379:methods*; D010427:Pentose Phosphate Pathway; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology*
chemical_list: D018797:Cell Cycle Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D020123:Sirolimus; C083324:TOR1 protein, S cerevisiae; C081135:TOR2 protein, S cerevisiae