Evaluation of FOXO1 target engagement using a single-cell microfluidic platform
File(s)
Accepted version
Author(s)
Type
Journal Article
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.
Date Issued
2021-11-09
Date Acceptance
2021-09-30
Citation
Analytical Chemistry, 2021, 93 (44), pp.14659-14666
ISSN
0003-2700
Publisher
American Chemical Society
Start Page
14659
End Page
14666
Journal / Book Title
Analytical Chemistry
Volume
93
Issue
44
Copyright Statement
© 2021 American Chemical Society. This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.analchem.1c02808
Sponsor
Engineering & Physical Science Research Council (EPSRC)
Identifier
https://pubs.acs.org/doi/10.1021/acs.analchem.1c02808
Grant Number
EP/C54269X/1
Subjects
Drug Discovery
Microfluidics
Proteins
Analytical Chemistry
0301 Analytical Chemistry
0399 Other Chemical Sciences
Publication Status
Published
Date Publish Online
2021-10-25