In-gel activity-based protein profiling of a clickable covalent ERK1/2 inhibitor

Mol Biosyst. 2016 Aug 16;12(9):2867-74. doi: 10.1039/c6mb00367b.

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.

MeSH terms

  • Cell Line
  • Click Chemistry*
  • Drug Discovery / methods
  • Enzyme Activation / drug effects
  • Humans
  • Inhibitory Concentration 50
  • Mitogen-Activated Protein Kinase 1 / antagonists & inhibitors
  • Mitogen-Activated Protein Kinase 1 / chemistry*
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3 / antagonists & inhibitors
  • Mitogen-Activated Protein Kinase 3 / chemistry*
  • Mitogen-Activated Protein Kinase 3 / metabolism
  • Models, Molecular
  • Molecular Conformation
  • Molecular Structure
  • Protein Kinase Inhibitors / chemistry*
  • Protein Kinase Inhibitors / pharmacology
  • Proteome
  • Proteomics* / methods

Substances

  • Protein Kinase Inhibitors
  • Proteome
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3