Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae, specifically LAP4 Cherry Pick Compounds
The target of rapamycin, TOR, is a ser/thr protein kinase evolutionarily conserved from yeast to man [Wullschleger, et al. 2006]. TOR functions in two distinct protein complexes, TOR complex 1 (TORC1) and TORC2 [Cafferkey, et al. 1993; Stan, et al. 1994]. Curiously, only TOR in TORC1 is bound and inhibited by the lipophilic macrolide rapamycin [Kunz, et al. 1993; Helliwell, et al. 1998; Zhang, et more ..
BioActive Compounds: 246
University of New Mexico Assay Overview:
Assay Support: 1R03 MH086450-01
Project Title: Chemical Screen of TOR pathway GFP fusion proteins in S. cerevisiae
PI: Maggie Werner-Washburne
Center PI: Larry Sklar
Assay Implementation: Jun Chen, Chris Allen, Susan Young, Anna Waller, Mark Carter
Assay Background and Significance:
The target of rapamycin, TOR, is a ser/thr protein kinase evolutionarily conserved from yeast to man [Wullschleger, et al. 2006]. TOR functions in two distinct protein complexes, TOR complex 1 (TORC1) and TORC2 [Cafferkey, et al. 1993; Stan, et al. 1994]. Curiously, only TOR in TORC1 is bound and inhibited by the lipophilic macrolide rapamycin [Kunz, et al. 1993; Helliwell, et al. 1998; Zhang, et al. 2006]. Although the signaling events up- and downstream of TORC2 (which regulates spatial aspects of growth) have yet to be elucidated in detail, it is well established that TORC1 is a central hub of a signaling network that couples cues from hormones and growth factors (in mammalian cells), energy and stresses, and the abundance of nutrients, to cell growth and proliferation. Very recent work has elucidated many details of the signaling events upstream of TORC1 as well as downstream targets of TORC1. Importantly, in this context, most negative regulators of mammalian TORC1 (mTORC1)have been previously identified as tumor suppressor gene products, while many positive regulators of mTORC1 have been identified as proto-oncoproteins and/or are found at elevated levels in tumor-derived cell lines [De Virgilio, et al. 2006a; De Virgilio, et al. 2006b].
The purpose of this HTS screen is to identify small molecule modulators of protein targets in the pathway containing the target of rapamycin (TOR), a multi-protein complex (TORC1 and TORC2) that is highly conserved from yeast to man. The screen will detect structurally distinct, but functionally rapamycin-like compounds (rapalogs) by probing four major TOR pathways using the following targets in a multiplex format:
*RPL 19A: YAK kinase branch
*LAP4: MSN4 branch
*MEP2 and AGP1: GLN3 branch
*CIT2: RTG branch
Each of these target proteins (RPL 19A, LAP4, MEP2, AGP1, CIT2) are GFP (Green Fluorescent Protein) tagged, thus the expression of the proteins can be tracked by monitoring the GFP fluorescence.
This AID reports the responses elicited from the single concentration confirmation of the primary screening hits. These cherry picked compounds were assessed in a slightly altered protocol compared to the primary screening protocol. The strains were collected in single plex manner, therefore eliminated the need for having dual staining to distinguish the different strain populations. However, one color staining was utilized in order to address the potential response difference that might occur between the different generational populations (during the 3 hour incubation at least one generation of cells can be produced).
The yeast cell-based multiplex assay is constructed using 5 strains from the Yeast-GFP Collection (Invitrogen, USA), representing 4 distinct branches of the TOR pathway: YAK Kinase, MSN4, GLN3 and RTG. Data were collected in single plex mode, meaning data from each individual strain were separately. The strains, however, were labeled with Alexafluor 633 (red laser excitation) in order to discriminate between generational populations, i.e., separate mothers and daughters of the same strain. The yeast cell populations are discriminated then interrogated for changes in GFP expression (blue laser excitation).
Similar to the primary screening campaign, the assay is performed in a total volume of 10.1 microliters in 384-well microtiter plates. The strains are grown separately overnight in synthetic complete liquid media in a shaking incubator at 30 degrees C, and then stained with the red alexafluor. Following staining, the each individual yeast strain was diluted into fresh media at 0.2 OD600. Aliquots of the yeast are transferred into 384-well microtiter plates and library compounds are added at 10microM final concentration. The cells are incubated for 3 hours at 30 degrees C with end-over-end rotation. Control wells contain the yeast strain treated for 3 hours with 400nanogram/milliliter Rapamycin as a positive control and the yeast strain with an equal volume of DMSO as a solvent control. The cells are interrogated for GFP expression levels using established high-throughput flow cytometric methodologies at the UNMCMD. Sample analysis is conducted with the HyperCyt(R) (Intellicyt, USA) high throughput flow cytometry platform. The HyperCyt system interfaces a flow cytometer and autosampler for high-throughput microliter-volume sampling from 384-well microtiter plates [Kuckuck, et al. 2001]. Flow cytometric data are collected on a Cyan Flow Cytometer (Dako, USA).
Data analysis of the original flow cytometric data was done using HyperView (R) (Intellicyt, USA) software. The data are gated on forward scatter versus side scatter to distinguish the single yeast population. Additional gating are made to separate the different generational populations based on stained yeast strains. These gates are made on graphs of FL8 versus FL6 data (i.e., red versus violet laser excitation). One gate was applied to include All cells, another gate applied to only the Mother generation (i.e., population with higher Alexafluor 633 staining), and a final gate applied to the Daughter generation (i.e., population with lower Alexafluor 633 staining). HyperView applies these different gates and parses the time-resolved data file to produce annotated fluorescence summary data for each well, which are merges with compound worklist files generated by HyperSip(R) (Intellicyt, USA) software. The parsed data are then processed through an Excel (R) (Microsoft, USA) template file constructed specifically for the assay to calculate the percent response and segregate data for gate population.
For each of the gated populations (All, Mother, Daughter), the following calculations were made. Percent Response is calculated in comparison to negative control (wells with DMSO) using the following equation:
%Response = 100*RawMCF_Sample/PlateAve_RawMCF_NCntrl
where RawMCF_Sample is the median channel fluorescence measured from the well with sample compound and PlateAve_RawMCF_NCntrl is the average of the median channel fluorescence measured from all the negative control wells on the plate. Note, in order to eliminate effects of potential spillover from innate fluorescent compounds into the control wells, the plate average of the negative control wells are only of the control wells that are within 1 standard deviation of all 16 control wells on the plate.
Compounds were demeaned Active if the percent response was greater than 150.
PUBCHEM_ACTIVITY_SCORE was calculated from the %Response:
SCORE = %Response - 100
Thus any %Responses less than 100% were given SCORE values of 0. An Active compound has a PUBCHEM_ACTIVITY_SCORE greater than 50.
If there were less than 70 events measured for on yeast strain, then the compound was listed as "Inconclusive".
Zprime was calculated from the average and standard deviations of the rapamycin control and DMSO control. For these sets of plates the average 0.81 +/- 0.08.
Comparison can be made with the Response value calculated from the All gate versus those from the Daughter and Mother gates. And from these comparisons it is evident that distinction between the generational populations are not required, hence for future dose response data collection the single plex yeast data do not require staining.
** Test Concentration.
Data Table (Concise)