Identification of a small molecule yeast TORC1 inhibitor with a multiplex screen based on flow cytometry
| Autor: | Larry A. Sklar, Robbie Loewith, Margaret Werner-Washburne, Tuanli Yao, J. Jacob Strouse, Nicolas Panchaud, Chris Allen, Oleg Ursu, Marie-Pierre Péli-Gulli, Jun Chen, Jeffrey Aubé, Mark B. Carter, Susan M. Young, Huining Kang, Bruce S. Edwards, Jennifer E. Golden, Anna Waller, Blake R. Peterson, Cristian Bologa, Andrew Seeber, Claudio De Virgilio, Terry D. Foutz |
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| Jazyk: | angličtina |
| Rok vydání: | 2012 |
| Předmět: |
Saccharomyces cerevisiae
Green Fluorescent Proteins Clone (cell biology) TORC1 signaling Biochemistry Green fluorescent protein Flow cytometry 03 medical and health sciences 0302 clinical medicine ddc:570 Signal Transduction/drug effects medicine Saccharomyces cerevisiae Proteins/antagonists & inhibitors Humans Protein Kinase Inhibitors/analysis 030304 developmental biology 0303 health sciences biology medicine.diagnostic_test Cell growth General Medicine biology.organism_classification Flow Cytometry Molecular biology Yeast 030220 oncology & carcinogenesis Molecular Medicine Signal transduction Transcription Factors/antagonists & inhibitors Saccharomyces cerevisiae/drug effects |
| Zdroj: | ACS Chemical Biology, Vol. 7, No 4 (2012) pp. 715-22 ACS chemical biology ACS Chemical Biology |
| ISSN: | 1554-8929 |
| Popis: | 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 EC50'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. |
| Databáze: | OpenAIRE |
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