Discovery of Novel, Dual Mechanism ERK Inhibitors by Affinity Selection Screening of an Inactive Kinase

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 <b>1</b> that bound to both nonphosphorylated and phosphorylated ERK2 and inhibited ERK2 kinase activity. Chemical optimization identified compound <b>4</b> 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 <b>4</b> 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 <b>4</b> can bind. This new binding mode represents a novel mechanism by which high affinity ATP-competitive compounds may achieve excellent kinase selectivity