Evaluation of FOXO1 Target Engagement Using a Single-Cell Microfluidic Platform

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.