Test compound autofluorescence in Saccharomyes cerevisiae specifically s288c
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: 9
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.
Compounds that have been assessed as potential actives from the primary screen and repeated in a single point concentration from cherry pick plate are evaluated in a dose response assay. These dose response assays are set up as single plex assays, meaning only one target protein per well. The parental strain, s288c, for all the GFP expressing strains was utilized to assess the potential innate fluorescence of the test compounds. This s288c strain does not express GFP, thus any measured changes in 520 nanometer emission from this strain are due to compound autofluorescence.
Data were collected in single plex mode, meaning data from s288c strain was collected separately from all other strains.
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. Then 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 hit compounds found from primary screen are added in dose response concentrations ranging from 0.001 to 33 microM, 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 fluorescence emission at 520 nanometer (same as used for GFP expression assessment) 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. HyperView applies this gate 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) and Prism (R) (GraphPad, USA) template file constructed specifically for the assay to calculate the percent response.
Calibration beads were used to convert the measured raw median channel fluorescence (MCF) to Molecules of Equivalent Soluble Fluorophores (MESF) by first assessing the linear correlation of the 5 different calibration bead levels of known MESF, then by using the following equation to convert the measured MCF:
kMESFSample = SlopeCalibration * RawMCFSample
where kMESFSample is kilo (1000) MESF from Sample and SlopeCalibration is the slope from linear fit of kMESF versus MCF of the calibration beads, and RawMCFSample is the raw MCF of the sample. By making the measurements standardized by the calibration beads, values of MCF collected from different machines could be compared. These kMESFSample values are reported in this upload.
PUBCHEM_ACTIVITY_SCORE are based on the magnitude of difference of the kMESFSample for DMSO control to the 33 microM test sample by the following equation:
SCORE = kMESFSample at 33 microM - kMESFDMSO
And compounds were demeaned active if the kMESF_Span or SCORE is greater than 25.
** Test Concentration.
Data Table (Concise)