Acumen qHTS Assay for Inhibitors of the mTORC1 Signaling Pathway in MEF (Tsc2-/-, p53-/-) Cells: Hit Validation
The mammalian target of rapamycin (mTOR) is an intracellular serine/threonine kinase Aberrant regulation of mTOR signaling plays a causative role in a number of human diseases, including cancer, benign tumors such as lymphangioleiomyomatosis (LAM) and Tuberous Sclerosis Complex (TSC), and metabolic diseases such as diabetes and obesity. This project aims to identify inhibitors of ribosomal more ..
BioActive Compounds: 168
The mammalian target of rapamycin (mTOR) is an intracellular serine/threonine kinase Aberrant regulation of mTOR signaling plays a causative role in a number of human diseases, including cancer, benign tumors such as lymphangioleiomyomatosis (LAM) and Tuberous Sclerosis Complex (TSC), and metabolic diseases such as diabetes and obesity. This project aims to identify inhibitors of ribosomal protein S6 (rpS6) phosphorylation. The phosphorylation of rpS6 is a key event downstream of mTOR activation. The assay uses a phospho-specific antibody of phospho-rpS6 to detect inhibitors of mTOR activation. Mutations in the Tsc1 and Tsc2 genes are the underlying genetic lesions responsible for TSC, causing constitutive activation of the mTORC1 pathway and leading to profound effects on cell metabolism. Aberrant metabolism in cancer has recently received significant attention as a target to develop therapeutic agents in the treatment of cancer.1 As Tsc2-/- cells have lost the ability to respond to energy and growth factor deprivation, these cells are unable to properly down regulate cellular biosynthetic processes in response to metabolic stress, and are thus extremely sensitive to glucose deprivation-induced death.2 A central goal of this project is to explore the unique cell survival and metabolic properties of cells with Tsc1 or Tsc2 by screening for compounds that selectively target the Tsc2-/- MEFs by screening both Tsc2-/- and WT MEF cell lines. We hope to better understand these aspects of TSC physiology in order to exploit them for therapeutic benefit.
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: NS059428-01
Assay Submitter (PI): Blenis, John; Harvard University
For quantitative high throughput analysis of mTORC1 signaling, we developed a cell-based assay for immuno-fluorescence based detection of rpS6 phosphorylation on the Acumen Explorer that will be used a platform for the primary small molecule screen. Mouse embryonic fibroblasts (MEFs) are cultured in 1536-well plates using DMEM + 10% FBS. Twenty-four hrs after plating, test compounds (solubilized in DMSO) are delivered and incubated with the cells for 24 hrs at 37 degree Celsius. Following the incubation, media with compound is aspirated and the cells are fixed in 4% paraformaldehyde (PFA) containing PBS + 0.2% TX-100. Next, PFA is aspirated, cells are washed two times with PBS plus 0.2% TX-100 and the cells are blocked for 1 hr at room temperature (blocking buffer: LiCOR Blocking Buffer plus 0.2% TX-100). This is followed by aspiration and staining with Alexa-488 conjugated-phospho-rpS6 Ser235/236 antibody for 2 hours at room temperature (Cell Signaling Technology # 4803, 1:50 dilution in blocking buffer). Cells are washed two times with PBS plus 0.2% TX-100, and stained with 10 uM propidium iodide (PI) for 20-25 minutes for cell number measurement (1:500 dilution of Hoescht staining is also added for IN Cell imaging). The plates are scanned on the Acumen Explorer laser scanning instrument (TTP Labtech) using the 488nm laser for simultaneous excitation of the Alexa-488 and PI. The fluorescence emission from the Alexa-488 conjugated rpS6 phospho-235/236 antibody staining is selected using a 530-585 nm band pass filter. Fluorescence emission from the PI stain is selected using a 575-640 nm band pass filter. The normalized phospho-S6 staining is calculated as the ratio of the integrated Alexa 488 intensity divided by the integrated PI intensity for the single concentration rapamycin control (7.5 nM) and DMSO columns. Inactive and active kinase populations will be separated by their distinct histogram populations. Inhibition of the pathway is defined based on the normalized phospho-S6 intensity in rapamycin treated cells. Active cells are defined as those with a normalized phospho-S6 staining above this threshold, and each treatment is reported as the % positive cells in the well, where thresholds are defined by selecting wells with the highest and lowest signal and differentiating between the % non-active and % active populations. In addition to the pathway specific phospho-S6 endpoint, the number of PI stained cells from the Acumen is recorded as a measure of cell viability. The primary screen was performed with a MEF cell line in a defined genetic background, Tsc2-/-, p53-/- cell line stably transfected with an empty vector control (Tsc2-/- MEF).
Compounds which are inhibitors are determined from active and inactive kinase populations by calculating the ratio of the integrated Alexa 488 intensity divided by the integrated PI intensity for the single concentration rapamycin control (7.5 nM) and DMSO columns.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)