title: In-gel activity-based protein profiling of a clickable covalent ERK1/2 inhibitor.
abstract: In-gel activity-based protein profiling (ABPP) offers rapid assessment of the proteome-wide selectivity and target engagement of a chemical tool. Here we demonstrate the use of the inverse electron demand Diels Alder (IEDDA) click reaction for in-gel ABPP by evaluating the selectivity profile and target engagement of a covalent ERK1/2 probe tagged with a trans-cyclooctene group. The chemical probe was shown to bind covalently to Cys166 of ERK2 using protein MS and X-ray crystallography, and displayed submicromolar GI50s in A375 and HCT116 cells. In both cell lines, the probe demonstrated target engagement and a good selectivity profile at low concentrations, which was lost at higher concentrations. The IEDDA cycloaddition enabled fast and quantitative fluorescent tagging for readout with a high background-to-noise ratio and thereby provides a promising alternative to the commonly used copper catalysed alkyne-azide cycloaddition.
authors.affiliation: Astex Pharmaceuticals, 436 Cambridge Science Park, Cambridge, CB4 0QA, UK. Tom.Heightman@astx.com.
authors.forename: Honorine
authors.initials: H
authors.lastname: Lebraud
authors.affiliation: Astex Pharmaceuticals, 436 Cambridge Science Park, Cambridge, CB4 0QA, UK. Tom.Heightman@astx.com.
authors.forename: David J
authors.initials: DJ
authors.lastname: Wright
authors.affiliation: Astex Pharmaceuticals, 436 Cambridge Science Park, Cambridge, CB4 0QA, UK. Tom.Heightman@astx.com.
authors.forename: Charlotte E
authors.initials: CE
authors.lastname: East
authors.affiliation: Astex Pharmaceuticals, 436 Cambridge Science Park, Cambridge, CB4 0QA, UK. Tom.Heightman@astx.com.
authors.forename: Finn P
authors.initials: FP
authors.lastname: Holding
authors.affiliation: Astex Pharmaceuticals, 436 Cambridge Science Park, Cambridge, CB4 0QA, UK. Tom.Heightman@astx.com.
authors.forename: Marc
authors.initials: M
authors.lastname: O'Reilly
authors.affiliation: Astex Pharmaceuticals, 436 Cambridge Science Park, Cambridge, CB4 0QA, UK. Tom.Heightman@astx.com.
authors.forename: Tom D
authors.initials: TD
authors.lastname: Heightman
mesh_terms: D002460:Cell Line; D057930:Click Chemistry; D055808:Drug Discovery / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D020128:Inhibitory Concentration 50; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D015394:Molecular Structure; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D020543:Proteome; D040901:Proteomics / Q000379:methods
chemical_list: D047428:Protein Kinase Inhibitors; D020543:Proteome; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3