title: Cellular thermal shift and clickable chemical probe assays for the determination of drug-target engagement in live cells.
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.
authors.affiliation: Worldwide Medicinal Chemistry, Pfizer, 610 Main St., Cambridge, Massachusetts 02139, USA. lyn.jones@pfizer.com.
authors.forename: Hua
authors.initials: H
authors.lastname: Xu
authors.forename: Ariamala
authors.initials: A
authors.lastname: Gopalsamy
authors.forename: Erik C
authors.initials: EC
authors.lastname: Hett
authors.forename: Shores
authors.initials: S
authors.lastname: Salter
authors.forename: Ann
authors.initials: A
authors.lastname: Aulabaugh
authors.forename: Robert E
authors.initials: RE
authors.lastname: Kyne
authors.forename: Betsy
authors.initials: B
authors.lastname: Pierce
authors.forename: Lyn H
authors.initials: LH
authors.lastname: Jones
mesh_terms: D057930:Click Chemistry; D004305:Dose-Response Relationship, Drug; D004722:Endoribonucleases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D015394:Molecular Structure; D011799:Quinazolines / Q000737:chemistry / Q000494:pharmacology*; D013441:Sulfinic Acids / Q000737:chemistry; D013696:Temperature; D014443:Tyrosine / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; C000623384:PF-06652474; D011799:Quinazolines; D013441:Sulfinic Acids; D014443:Tyrosine; C048899:sulfuryl fluoride; D004722:Endoribonucleases; C485332:DcpS protein, human