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A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments
Published on: August 6, 2013
Evaluation of FOXO1 Target Engagement Using a Single-Cell Microfluidic Platform
Suhuur Osman1, Claus Bendtsen2, Samantha Peel2
1Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, 80 Wood Lane, London, W12 0BZ, United Kingdom.
Abstract:
The cellular thermal shift assay (CETSA) has been used extensively since its introduction to study drug-target engagement within both live cells and cellular lysate. This has proven to be a useful tool in early stage drug discovery and is used to study a wide range of protein classes. We describe the application of a single-cell CETSA workflow within a microfluidic affinity capture (MAC) chip. This has enabled us to quantitatively determine the active FOXO1 single-molecule count and observe FOXO1 stabilization and destabilization in the presence of three small molecule inhibitors, including demonstrating the determination of EC50. The successful use of the MAC chip for single-cell CETSA paves the way for the study of precious clinical samples owing to the low number of cells needed by the chip. It also provides a useful tool for studying any underlying population heterogeneity that exists within a cellular system, a feature that is usually masked when conducting ensemble measurements.
Insights
This study introduces a single-cell cellular thermal shift assay (CETSA) on a microfluidic affinity capture (MAC) chip. This method quantifies active FOXO1 levels and drug-target engagement, enabling precise EC50 determination.
Area of Science:
- Biochemistry
- Pharmacology
- Biotechnology
Background:
- Cellular thermal shift assay (CETSA) is a key technique for studying drug-target engagement in early drug discovery.
- CETSA is versatile, applicable to various protein classes in both live cells and lysates.
- Existing methods often mask cellular heterogeneity due to ensemble measurements.
Purpose of the Study:
- To apply a single-cell CETSA workflow using a microfluidic affinity capture (MAC) chip.
- To quantitatively determine the active FOXO1 single-molecule count.
- To observe FOXO1 stabilization/destabilization with small molecule inhibitors and determine EC50.
Main Methods:
- Integration of single-cell CETSA with a microfluidic affinity capture (MAC) chip.
- Quantitative analysis of active FOXO1 at the single-molecule level.
- Treatment with three small molecule inhibitors to assess drug-target interactions.
Main Results:
- Successful quantitative determination of active FOXO1 single-molecule counts.
- Observation of FOXO1 stabilization and destabilization in response to inhibitors.
- Demonstration of EC50 determination for small molecule inhibitors targeting FOXO1.
Conclusions:
- The MAC chip enables single-cell CETSA, advancing drug discovery research.
- This approach is suitable for precious clinical samples due to low cell requirements.
- It facilitates the study of cellular population heterogeneity, often missed in bulk assays.
