Evaluation of FOXO1 target engagement using a single-cell microfluidic platform

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Title: Evaluation of FOXO1 target engagement using a single-cell microfluidic platform
Authors: Klug, D
Osman, S
Bendtsen, C
Peel, S
Yrlid, L
Muthas, D
Simpson, J
Willison, K
Item 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.
Issue Date: 9-Nov-2021
Date of Acceptance: 30-Sep-2021
URI: http://hdl.handle.net/10044/1/92554
DOI: 10.1021/acs.analchem.1c02808
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/Funder: Engineering & Physical Science Research Council (EPSRC)
Funder's Grant Number: EP/C54269X/1
Keywords: Drug Discovery
Microfluidics
Proteins
Analytical Chemistry
0301 Analytical Chemistry
0399 Other Chemical Sciences
Publication Status: Published
Embargo Date: 2022-10-24
Online Publication Date: 2021-10-25
Appears in Collections:Chemistry
Biological and Biophysical Chemistry
Faculty of Natural Sciences