title: Identification of a small molecule yeast TORC1 inhibitor with a multiplex screen based on flow cytometry.
abstract: TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high-throughput flow cytometry multiplexed screen using five GFP-tagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast. Each GFP-tagged clone was differentially color-coded, and the GFP signal of each clone was measured simultaneously by flow cytometry, which allows rapid prioritization of compounds that likely act through direct modulation of TORC1 or proximal signaling components. A total of 255 compounds were confirmed in dose-response analysis to alter GFP expression in one or more clones. To validate the concept of the high-throughput screen, we have characterized CID 3528206, a small molecule most likely to act on TORC1 as it alters GFP expression in all five GFP clones in a manner analogous to that of rapamycin. We have shown that CID 3528206 inhibited yeast cell growth and that CID 3528206 inhibited TORC1 activity both in vitro and in vivo with EC(50)'s of 150 nM and 3.9 μM, respectively. The results of microarray analysis and yeast GFP collection screen further support the notion that CID 3528206 and rapamycin modulate similar cellular pathways. Together, these results indicate that the HTS has identified a potentially useful small molecule for further development of TOR inhibitors.
authors.affiliation: Center for Molecular Discovery, University of New Mexico, Albuquerque, 87131, United States. juchen@salud.unm.edu
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
authors.forename: Susan M
authors.initials: SM
authors.lastname: Young
authors.forename: Chris
authors.initials: C
authors.lastname: Allen
authors.forename: Andrew
authors.initials: A
authors.lastname: Seeber
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Péli-Gulli
authors.forename: Nicolas
authors.initials: N
authors.lastname: Panchaud
authors.forename: Anna
authors.initials: A
authors.lastname: Waller
authors.forename: Oleg
authors.initials: O
authors.lastname: Ursu
authors.forename: Tuanli
authors.initials: T
authors.lastname: Yao
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Golden
authors.forename: J Jacob
authors.initials: JJ
authors.lastname: Strouse
authors.forename: Mark B
authors.initials: MB
authors.lastname: Carter
authors.forename: Huining
authors.initials: H
authors.lastname: Kang
authors.forename: Cristian G
authors.initials: CG
authors.lastname: Bologa
authors.forename: Terry D
authors.initials: TD
authors.lastname: Foutz
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Edwards
authors.forename: Blake R
authors.initials: BR
authors.lastname: Peterson
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Aubé
authors.forename: Margaret
authors.initials: M
authors.lastname: Werner-Washburne
authors.forename: Robbie J
authors.initials: RJ
authors.lastname: Loewith
authors.forename: Claudio
authors.initials: C
authors.lastname: De Virgilio
authors.forename: Larry A
authors.initials: LA
authors.lastname: Sklar
mesh_terms: D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006801:Humans; D047428:Protein Kinase Inhibitors / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000187:drug effects*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors*; D015398:Signal Transduction / Q000187:drug effects; D014157:Transcription Factors / Q000037:antagonists & inhibitors*
chemical_list: D047428:Protein Kinase Inhibitors; D029701:Saccharomyces cerevisiae Proteins; C561842:TORC1 protein complex, S cerevisiae; D014157:Transcription Factors; D049452:Green Fluorescent Proteins