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A high-throughput, cell-based screening method for siRNA and small molecule inhibitors of mTORC1 signaling using the
Gregory R Hoffman1, Nathan J Moerke, Max Hsia
1Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. greg_hoffman@hms.harvard.edu
Abstract:
The mTORC1 pathway is a central regulator of cell growth, and defective mTORC1 regulation plays a causative role in a variety of human diseases, including cancer, tumor syndromes such as the tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), and metabolic diseases such as diabetes and obesity. Given the importance of mTORC1 signaling in these diseases, there has been significant interest in developing screening methods suitable for identifying inhibitors of mTORC1 activation. To this end, we have developed a high-throughput, cell-based assay for the detection of rpS6-phosphorylation as a measure of mTORC1 signaling. This assay takes advantage of the "In Cell Western" (ICW) technique using the Aerius infrared imaging system (LI-COR Biosciences). The ICW procedure involves fixation and immunostaining of cells in a manner similar to standard immunofluorescence methods but takes advantage of secondary antibodies conjugated to infrared-excitable fluorophores for quantitative detection by the Aerius scanner. In addition, the cells are stained with an infrared-excitable succinimidyl ester dye, which covalently modifies free amine groups in fixed cells and provides a quantitative measure of cell number. We present validation data and pilot screens in a 384-well format demonstrating that this assay provides a statistically robust method for both small molecule and siRNA screening approaches designed to identify inhibitors of mTORC1 signaling.
Insights
Researchers developed a new high-throughput assay to measure mTORC1 signaling by detecting rpS6-phosphorylation. This method aids in discovering drugs targeting diseases like cancer and diabetes.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The mechanistic target of rapamycin complex 1 (mTORC1) pathway is crucial for cell growth regulation.
- Dysregulation of mTORC1 signaling is implicated in various human diseases, including cancer, tuberous sclerosis complex (TSC), lymphangioleiomyomatosis (LAM), diabetes, and obesity.
- Identifying inhibitors of mTORC1 activation is a significant therapeutic goal.
Purpose of the Study:
- To develop a high-throughput, cell-based screening assay for quantifying mTORC1 signaling activity.
- To validate the assay's robustness for identifying inhibitors of mTORC1 activation.
Main Methods:
- Utilized the In Cell Western (ICW) technique with an Aerius infrared imaging system.
- Employed immunostaining for rpS6-phosphorylation as a readout for mTORC1 signaling.
- Incorporated a cell number normalization method using an infrared-excitable succinimidyl ester dye.
Main Results:
- The developed ICW assay quantitatively measures rpS6-phosphorylation, a direct indicator of mTORC1 activity.
- Validation data and pilot screens in a 384-well format demonstrated statistically robust results.
- The assay is suitable for both small molecule and siRNA screening approaches.
Conclusions:
- The novel cell-based ICW assay provides a reliable and efficient method for screening mTORC1 signaling inhibitors.
- This assay facilitates drug discovery efforts for diseases associated with mTORC1 dysregulation.
- The technique offers a quantitative and robust platform for high-throughput screening in cancer and metabolic disease research.
