Confirmatory Cherry Pick 3 SAR Dose Response Multiplex in TOR pathway GFP-fusion proteins for Saccharomyes cerevisiae, specifically LAP4
Assay Implementation: Jun Chen, Stephanie Sedillo, Anna Waller, Annette Evangelisti, Cristian Bologa, Oleg Ursu, Mark Carter ..more
BioActive Compound: 1
University of New Mexico Assay Overview:
Assay Support: 1R03 MH086450-01
Project Title: Chemical Screen of TOR pathway GFP fusion proteins in S. cerevisiae
Assay Provider: Maggie Werner-Washburne, UNM
Screening Center/ PI: UNMCMD/ Larry Sklar
Lead Biologist: Jun Chen
Chemistry Center/ PI: University of Kansas Specialized Chemistry Center/ Jeff Aube
Chemistry Center Lead: Jennifer Golden, Blake Peterson
Assay Implementation: Jun Chen, Stephanie Sedillo, Anna Waller, Annette Evangelisti, Cristian Bologa, Oleg Ursu, 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 HTS screen of the MLSMR is a multiplex assay 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 detects structurally distinct, but functionally rapamycin-like compounds (rapalogs) by probing four major TOR pathways using the following targets in a multiplex format:
RPL19A: YAK kinase branch
LAP4: MSN4 branch
MEP2 and AGP1: GLN3 branch
CIT2: RTG branch
The HTS identified 1090 active compounds for this target out of 324,737 tested (AID 2023). These compounds were evaluated in counterscreen assays (AID 2757) and actives were included in a confirmatory dose response multiplex in which a total of 364 actives were identified out of 2346 tested (AID 2271 and 2621 from two separate cherry pick selections). In the assay reported here, a confirmatory SAR assay of powder re-supply for all five targets was performed in dose response. 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 RGT. 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 masculineC,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 at10muM final concentration. The cells are incubated at for 3 hours at 30 masculineC with end-over-end rotation. Control wells contain the multiplex treated for 3 hours with 200ng/mL 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 into a single data file and analysis using HyperView (R) (Intellicyt, USA) software to merge the flow cytometry data files with compound worklist files generated by HyperSip(R) (Intellicyt, USA)software. The raw data are parsed in IDLeQuery to produce annotated fluorescence summary data for each well. 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.
PUBCHEM_ACTIVITY_SCORE was calculated based on an EC50 cutoff of 34 microM, using the following equation:
SCORE = 100*(1-EC50/34)
Test compounds were considered active if the EC50 was equal to or less than 33 microM.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)