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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.

Analytical Chemistry
|October 25, 2021
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Summary

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.

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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.