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Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling
Published on: September 20, 2016
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Chaperone mediated detection of small molecule target binding in cells
Kelvin F Cho1, Taylur P Ma2, Christopher M Rose2
1Department of Biochemical and Cellular Pharmacology, Genentech Inc., South San Francisco, CA, 94080, USA.
Nature Communications
|January 25, 2020
Summary
HIPStA is a new high-throughput assay to measure small molecule drug binding to target proteins in cells. This method aids drug discovery by assessing target engagement and identifying new drug targets.
Area of Science:
- Biochemistry and Molecular Biology
- Pharmacology and Drug Discovery
- Cellular Signaling
Background:
- Quantitatively measuring small molecule-protein interactions is vital for understanding cellular signaling and advancing drug discovery.
- Existing target engagement assays have limitations in application and interpretation.
- A need exists for high-throughput, versatile methods to assess drug binding to endogenous proteins within cells.
Purpose of the Study:
- To introduce HIPStA (Heat Shock Protein Inhibition Protein Stability Assay), a novel high-throughput method for assessing small molecule target engagement in cells.
- To demonstrate HIPStA's ability to measure drug binding to diverse target classes, including receptor tyrosine kinases, nuclear hormone receptors, and cytoplasmic protein kinases.
- To showcase HIPStA's utility in identifying novel drug targets when combined with quantitative mass spectrometry.
Main Methods:
- HIPStA leverages changes in protein turnover, specifically the stabilization effect of drug-target binding when heat shock protein 90 (HSP90) is inhibited.
- Quantitative fluorescence imaging is employed to measure drug binding across different target classes.
- The method is validated by pairing it with quantitative mass spectrometry for target identification.
Main Results:
- HIPStA successfully measured drug binding to receptor tyrosine kinases, nuclear hormone receptors, and cytoplasmic protein kinases.
- The assay demonstrated high-throughput capability for assessing target engagement.
- Integration with mass spectrometry identified previously unknown targets of a receptor tyrosine kinase inhibitor.
Conclusions:
- HIPStA provides a complementary and high-throughput approach to quantitatively measure small molecule target engagement in cells.
- The method is versatile, applicable to various protein target classes, and aids in mechanistic studies and drug discovery.
- HIPStA, particularly when coupled with mass spectrometry, offers a powerful tool for target identification and validation.
