AID	Name	Type	Comment
488795	Confirmatory Primary Screen Hits KU SAR Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically LAP4	confirmatory
1908	Multiplex HTS Screen of TOR pathway GFP-fusion proteins in Saccharomyes cerevisiae	summary
488823	Confirmatory Cherry Pick 3 SAR Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically LAP4	confirmatory

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.

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). Analysis is performed using time-resolved acquisition of a single data file and analysis using HyperView (R) (Intellicyt, USA) software parses the data to produce annotated fluorescence summary data for each well and merges the flow cytometry data files 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 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 (16 in total).

Compounds were demeaned Active if the response was greater than the average response plus 3 standard deviations of the entire screen, and for the LAP4 strain that was 99 + 3*16.7.

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.

Note, due to the requirement of utilizing red and violet stains to distinguish the different yeast strains, when a compound was found to alter these distinctions, primary due to innate fluorescence, there was no response measured. And the compound was listed as "Inconclusive". Furthermore, these compounds have been annotated as "Potential_fluorescent_compound" in the PUBCHEM_COMMENT column.

Zprime was calculated from the average and standard deviations of the rapamycin control and DMSO control. For these sets of plates the average 0.55 +/- 0.18.

Grant Number: 1R03 MH086450-01