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Issue 26, 2016
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Cellular thermal shift and clickable chemical probe assays for the determination of drug-target engagement in live cells

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Abstract

Proof of drug-target engagement in physiologically-relevant contexts is a key pillar of successful therapeutic target validation. We developed two orthogonal technologies, the cellular thermal shift assay (CETSA) and a covalent chemical probe reporter approach (harnessing sulfonyl fluoride tyrosine labeling and subsequent click chemistry) to measure the occupancy of the mRNA-decapping scavenger enzyme DcpS by a small molecule inhibitor in live cells. Enzyme affinity determined using isothermal dose response fingerprinting (ITDRFCETSA) and the concentration required to occupy 50% of the enzyme (OC50) using the chemical probe reporter assay were very similar. In this case, the chemical probe method worked well due to the long offset kinetics of the reversible inhibitor (determined using a fluorescent dye-tagged probe). This work suggests that CETSA could become the first choice assay to determine in-cell target engagement due to its simplicity.

Graphical abstract: Cellular thermal shift and clickable chemical probe assays for the determination of drug-target engagement in live cells

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Supplementary files

Article information


Submitted
17 May 2016
Accepted
18 May 2016
First published
19 May 2016

Org. Biomol. Chem., 2016,14, 6179-6183
Article type
Communication

Cellular thermal shift and clickable chemical probe assays for the determination of drug-target engagement in live cells

H. Xu, A. Gopalsamy, E. C. Hett, S. Salter, A. Aulabaugh, R. E. Kyne, B. Pierce and L. H. Jones, Org. Biomol. Chem., 2016, 14, 6179
DOI: 10.1039/C6OB01078D

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