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BioAssay: AID 2023

Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae_specifically_ LAP4_MLPCN.

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 more ..
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 Tested Compounds
 Tested Compounds
All(324737)
 
 
Active(1090)
 
 
Inactive(322340)
 
 
Inconclusive(1308)
 
 
 Tested Substances
 Tested Substances
All(324844)
 
 
Active(1091)
 
 
Inactive(322445)
 
 
Inconclusive(1308)
 
 
AID: 2023
Data Source: NMMLSC (UNM_TOR_Pathway_HTS_in_Saccharomyces_cerevisiae_specifically..)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-10-26
Modify Date: 2011-04-05

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1090
Related Experiments
Show more
AIDNameTypeProbeComment
1908Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiaeSummary1 depositor-specified cross reference: Summary report for TOR screen project
488795Confirmatory Primary Screen Hits KU SAR Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically LAP4Confirmatory depositor-specified cross reference
488818Phenotypic Counterscreen Cherry Pick 3 SAR Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically s288cOther depositor-specified cross reference
488823Confirmatory Cherry Pick 3 SAR Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically LAP4Confirmatory depositor-specified cross reference
488829Phenotypic Counterscreen Primary Screen Hits KU SAR Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically s288cOther depositor-specified cross reference
504463Phenotypic Counterscreen Primary Screen Hits KU SAR Set2 Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically s288cOther depositor-specified cross reference
504469Confirmatory Primary Screen Hits KU SAR Set2 Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically LAP4Confirmatory depositor-specified cross reference
504472Gln3p-GFP Translocation AssayOther depositor-specified cross reference
504478Sch9 phosphorylation assay (AP)Other depositor-specified cross reference
1862Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae_specifically_RPL19AScreening same project related to Summary assay
1867Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae_specifically_MEP2Screening same project related to Summary assay
1870Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae_specifically_CIT2Screening same project related to Summary assay
1873Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae_specifically_LAP4Screening same project related to Summary assay
1887Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae_specifically_AGP1Screening same project related to Summary assay
2016Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae_specifically_ MEP2_MLPCN.Screening same project related to Summary assay
2025Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae_specifically_ RPL19A_MLPCN.Screening same project related to Summary assay
2029Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae_specifically_ CIT2_MLPCN.Screening same project related to Summary assay
2066Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae_specifically_ AGP1_MLPCN.Screening same project related to Summary assay
2270Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae, specifically RPL19A Cherry Pick CompoundsScreening same project related to Summary assay
2271Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae, specifically LAP4 Cherry Pick CompoundsScreening same project related to Summary assay
2272Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae, specifically MEP2 Cherry Pick CompoundsScreening same project related to Summary assay
2273Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae, specifically AGP1 Cherry Pick CompoundsScreening same project related to Summary assay
2274Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae, specifically CIT2 Cherry Pick CompoundsScreening same project related to Summary assay
2621Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae specifically LAP4 Second Cherry Pick CompoundsScreening same project related to Summary assay
2622Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae specifically MEP2 Second Cherry Pick CompoundsScreening same project related to Summary assay
2623Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae specifically RPL19A Second Cherry Pick CompoundsScreening same project related to Summary assay
2624Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae specifically CIT2 Second Cherry Pick CompoundsScreening same project related to Summary assay
2643Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae specifically AGP1 Second Cherry Pick CompoundsScreening same project related to Summary assay
2740Dose Response of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae specifically RPL19A based on MLPCN hitsConfirmatory same project related to Summary assay
2742Dose Response of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae specifically MEP2 based on MLPCN hitsConfirmatory same project related to Summary assay
2743Dose Response of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae specifically AGP1 based on MLPCN hitsConfirmatory same project related to Summary assay
2744Dose Response of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae specifically CIT2 based on MLPCN hitsConfirmatory same project related to Summary assay
2745Dose Response of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae specifically LAP4 based on MLPCN hitsConfirmatory same project related to Summary assay
2757Test compound autofluorescence in Saccharomyes cerevisiae specifically s288cOther same project related to Summary assay
488790Confirmatory Cherry Pick 3 SAR Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically AGP1Confirmatory same project related to Summary assay
488792Confirmatory Primary Screen Hits KU SAR Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically AGP1Confirmatory same project related to Summary assay
488801Confirmatory Primary Screen Hits KU SAR Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically MEP2Confirmatory same project related to Summary assay
488808Confirmatory Primary Screen Hits KU SAR Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically RPL19AConfirmatory same project related to Summary assay
488812Confirmatory Cherry Pick 3 SAR Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically MEP2Confirmatory same project related to Summary assay
488814Confirmatory Cherry Pick 3 SAR Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically RPL19AConfirmatory same project related to Summary assay
488825Confirmatory Cherry Pick 3 SAR Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically CIT2Confirmatory same project related to Summary assay
488827Confirmatory Primary Screen Hits KU SAR Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically CIT2Confirmatory same project related to Summary assay
504321Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae specifically MEP2 Third Cherry Pick CompoundsScreening same project related to Summary assay
504334Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae specifically AGP1 Third Cherry Pick CompoundsScreening same project related to Summary assay
504336Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae specifically RPL19A Third Cherry Pick CompoundsScreening same project related to Summary assay
504338Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae specifically LAP4 Third Cherry Pick CompoundsScreening same project related to Summary assay
504340Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae specifically CIT2 Third Cherry Pick CompoundsScreening same project related to Summary assay
504456Yeast TORC1 Bypass Cell Growth AssayOther same project related to Summary assay
504458Confirmatory Primary Screen Hits KU SAR Set2 Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically AGP1Confirmatory same project related to Summary assay
504461Confirmatory Primary Screen Hits KU SAR Set2 Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically CIT2Confirmatory same project related to Summary assay
504468Confirmatory Primary Screen Hits KU SAR Set2 Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically MEP2Confirmatory same project related to Summary assay
504470Confirmatory Primary Screen Hits KU SAR Set2 Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically RPL19AConfirmatory same project related to Summary assay
504471Rtg3-GFP translocation assayOther same project related to Summary assay
504473Flow cytometric HTS Screen of Saccharomyces cerevisiae GFP-tagged clones sensitive to rapamycinOther same project related to Summary assay
504479Expression of GFP-tagged protein in TOR C1 bypass strains and wild type Saccharomyces cerevisiaeOther same project related to Summary assay
504481Expression of GFP-tagged protein in Transducer mutant strains of Saccharomyces cerevisiaeOther same project related to Summary assay
Description:
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 report specifically addresses the LAP4 target in the MSN4 branch of the TOR pathway.
Protocol
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. Differential stain barcoding is used to discriminate between yeast strains in the multiplex using ratiometric labeling with Alexafluor 405 (violet laser excitation) and Alexafluor 633 (red laser excitation). Bar-coded yeast cell populations are discriminated then interrogated for changes in GFP expression (blue laser excitation).
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 for multiplexing with the violet and red alexafluors. Following staining, the yeast are combined and diluted into fresh media at 0.2 OD600. Aliquots of the multiplex are transferred into 384-well microtiter plates and library compounds are added at 10microM final concentration. The cells are incubated at for 3 hours at 30 degrees C with end-over-end rotation. Control wells contain the multiplex treated for 3 hours with 200nanogram/milliliter Rapamycin as a positive control and the multiplex is treated with an equal volume of DMSO as a solvent control. The cells in the multiplex 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 stained yeast strains. These gates are made on graphs of FL8 versus FL6 data (i.e., red versus violet laser excitation). 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 each target.
Calculations:
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.
Effects of innate fluorescence compounds resulted in shifting of yeast strains from their designated red versus violet. Tracking of these compounds were made by assessing if there were missing strains for a particular compound, and annotated in column PUBCHEM_COMMENT. For this upload of around 150,000 compounds there were around 2,700 fluorescent compounds.
Compounds were demeaned Active if the percent response was greater than 150, which corresponded closely to the average response plus 3 standard deviations of the entire screen, and for the LAP4 strain that was 100 + 3*13.0 = 139.
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 0.80 +/- 0.09.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1RESPONSE (10μM**)Percent response calculated as ratio to DMSO negative controlFloat%
2Z_PRIMEZprime calculated from rapamycin control and DMSO control per plateFloat

** Test Concentration.
Additional Information
Grant Number: 1R03 MH086450-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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