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

• Published in: Organic & biomolecular chemistry 2016-01, Vol.14 (26), p.6179-6183
• Main Authors: Xu, Hua, Gopalsamy, Ariamala, Hett, Erik C, Salter, Shores, Aulabaugh, Ann, Kyne, Robert E, Pierce, Betsy, Jones, Lyn H
• Format: Article
• Language: English
• Subjects: Click Chemistry; Dose-Response Relationship, Drug; Endoribonucleases - antagonists & inhibitors; Endoribonucleases - metabolism; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Fluorescent Dyes - chemistry; HEK293 Cells; Humans; Molecular Structure; Quinazolines - chemistry; Quinazolines - pharmacology; Sulfinic Acids - chemistry; Temperature; Tyrosine - chemistry
• ISSN: 1477-0520; 1477-0539
• DOI: 10.1039/c6ob01078d

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 (ITDRF CETSA ) and the concentration required to occupy 50% of the enzyme (OC 50 ) 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. Proof of drug-target engagement in physiologically-relevant contexts is a key pillar of successful therapeutic target validation.