Cellular thermal shift and clickable chemical probe assays for the determination of drug-target engagement in live cells. Issue 26 (24th May 2016)

• Record Type: Journal Article
• Authors: Xu, Hua; Gopalsamy, Ariamala; Hett, Erik C.; Salter, Shores; Aulabaugh, Ann; Kyne, Robert E.; Pierce, Betsy; Jones, Lyn H.
• Is Part Of: Organic & biomolecular chemistry. Volume 14:Issue 26(2016)
• Page Start: 6179
• Page End: 6183
• Publication Date: 2016-05-24
• Subjects: Chemistry, Organic -- Periodicals; Bioorganic chemistry -- Periodicals; Chemistry, Physical organic -- Periodicals; 547
• DOI: 10.1039/c6ob01078d
• Languages: English
• ISSNs: 1477-0520

Abstract

Abstract : Proof of drug-target engagement in physiologically-relevant contexts is a key pillar of successful therapeutic target validation. 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.