Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2

Abstract

Modulating the dynamics of protein kinases expands the inhibitory mechanisms for small molecules. NMR measurements of the MAP kinase, ERK2, have shown that activation by dualphosphorylation induces global motions involving exchange between two states, "L'' and "R".We show that ERK inhibitors Vertex-11e and SCH772984 exploit the small energetic difference between L and R to shift the equilibrium in opposing directions, while inhibitor GDC-0994 and ATP analogue AMP-PNP retain L⇌R exchange. An X-ray structure of active 2P-ERK2 complexed with AMP-PNP reveals a shift in the Gly-rich loop along with domain closure to position the nucleotide in a more catalytically productive conformation relative to inactive 0P-ERK2:ATP. X-ray structures of 2P-ERK2 complexed with Vertex-11e or GDC-0994 recapitulate this closure, which is blocked in a complex with a SCH772984 analogue. Thus, the L R shift in 2P-ERK2 is associated with movements needed to form a competent active site. Solution measurements by hydrogen-exchange mass spectrometry (HX-MS) reveal distinct binding modes for Vertex-11e, GDC-0994 and AMP-PNP to active vs inactive ERK2, where the extent of HX protection matches their degree of R-state formation. In addition, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop, suggesting that L⇌R exchange involves coupling between the activation loop and the active site. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity towards 2P-ERK2. We conclude that global motions in ERK2 promote productive nucleotide binding, and couple with the activation loop to allow control of dephosphorylation by conformation-selective inhibitors.

Probing the signaling requirements for naive human pluripotency by high-throughput chemical screening

“…Furthermore, western blotting revealed increased p-ERK levels in AXGY, GXGY, and FXGY relative to PXGY (Figure 4J). Notably, GDC-0994 is known not to alter the phosphorylation of cellular ERK1/2 (Basken et al, 2018;Pegram et al, 2019). We conclude that inhibition of FGFR, RAF, MEK, or ERK can sustain bona fide features of naive human pluripotency despite variable levels of ERK phosphorylation.…”

Targeting KRAS-mutant stomach/colorectal tumours by disrupting the ERK2-p53 complex

“…shERK1 knockdown had little or no effect in both cases, suggesting that this is an ERK2-specific event. Next, we repeated this experiment with two ERK2 kinase inhibitors, AZD0364 and SCH772984 (Pegram et al, 2019; Ward et al, 2019). Cell viability data showed that both AZD0364 and SCH772984 significantly reduced the viability of KRAS G12D mutant GSU cells, but their inhibitory effects were significantly reduced by TP53 gene knockout (Figure 4B).…”