Download MendeleyIn-gel activity-based protein profiling of a clickable covalent ERK1/2 inhibitor.
Lebraud, H., Wright, D.J., East, C.E., Holding, F.P., O'Reilly, M., Heightman, T.D.(2016) Mol Biosyst 12: 2867-2874
- PubMed: 27385078
- DOI: https://doi.org/10.1039/c6mb00367b
- Primary Citation of Related Structures:
5LCJ, 5LCK - PubMed 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.
Organizational Affiliation:
Astex Pharmaceuticals, 436 Cambridge Science Park, Cambridge, CB4 0QA, UK. [email protected].
