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Identification of a small molecule yeast TORC1 inhibitor with a flow cytometry-based multiplex screen
Chen, Jun Young, Susan M. Allen, Chris Seeber, Andrew Péli-Gulli, Marie-Pierre Panchaud, Nicolas Waller, Anna Ursu, Oleg Yao, Tuanli Golden, Jennifer E.
Chen, Jun
Young, Susan M.
Allen, Chris
Seeber, Andrew
Péli-Gulli, Marie-Pierre
Panchaud, Nicolas
Waller, Anna
Ursu, Oleg
Yao, Tuanli
Golden, Jennifer E.
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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 GFPtagged
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 an analogous manner to 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 EC50s 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.
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2012-04-20
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ACS Chem Biol.
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Chen, J., Young, S. M., Allen, C., Seeber, A., Péli-Gulli, M.-P., Panchaud, N., … Sklar, L. A. (2012). Identification of a small molecule yeast TORC1 inhibitor with a flow cytometry-based multiplex screen. ACS Chemical Biology, 7(4), 715–722. http://doi.org/10.1021/cb200452r