Discovery of novel, dual mechanism ERK inhibitors by affinity selection screening of an inactive kinase

J Med Chem. 2014 Nov 13;57(21):8817-26. doi: 10.1021/jm500847m. Epub 2014 Oct 22.


An affinity-based mass spectrometry screening technology was used to identify novel binders to both nonphosphorylated and phosphorylated ERK2. Screening of inactive ERK2 identified a pyrrolidine analogue 1 that bound to both nonphosphorylated and phosphorylated ERK2 and inhibited ERK2 kinase activity. Chemical optimization identified compound 4 as a novel, potent, and highly selective ERK1,2 inhibitor which not only demonstrated inhibition of phosphorylation of ERK substrate p90RSK but also demonstrated inhibition of ERK1,2 phosphorylation on the activation loop. X-ray cocrystallography revealed that upon binding of compound 4 to ERK2, Tyr34 undergoes a rotation (flip) along with a shift in the poly-Gly rich loop to create a new binding pocket into which 4 can bind. This new binding mode represents a novel mechanism by which high affinity ATP-competitive compounds may achieve excellent kinase selectivity.

MeSH terms

  • Affinity Labels
  • Anilides / metabolism*
  • Anilides / pharmacology
  • Crystallography, X-Ray
  • Inhibitory Concentration 50
  • MAP Kinase Signaling System / drug effects*
  • Mass Spectrometry / methods
  • Mitogen-Activated Protein Kinase 1 / antagonists & inhibitors*
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Phosphorylation
  • Protein Kinase Inhibitors / chemical synthesis*
  • Protein Kinase Inhibitors / pharmacology
  • Pyrrolidines / metabolism*
  • Pyrrolidines / pharmacology
  • Structure-Activity Relationship


  • 1-(2-(4-(4-acetylphenyl)piperazin-1-yl)-2-oxoethyl)-N-(3-chloro-4-hydroxyphenyl)pyrrolidine-3-carboxamide
  • Affinity Labels
  • Anilides
  • Protein Kinase Inhibitors
  • Pyrrolidines
  • Mitogen-Activated Protein Kinase 1