1 research outputs found
Identification of a Small Molecule Yeast TORC1 Inhibitor with a Multiplex Screen Based on Flow Cytometry
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 doseāresponse 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 <i>in vitro</i> and <i>in vivo</i> with EC<sub>50</sub>'s of 150 nM and 3.9 Ī¼M,
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