Lebraud et al.. “In-gel activity-based protein profiling of a clickable covalent ERK1/2 inhibitor .” Mol. BioSyst., 2016,12, 2867-2874 DOI: 10.1039/C6MB00367B .

Summary

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.

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Lebraud et al.. “In-gel activity-based protein profiling of a clickable covalent ERK1/2 inhibitor .” Mol. BioSyst., 2016,12, 2867-2874 DOI: 10.1039/C6MB00367B .