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

Abstract

Conformational selection by small molecules expands inhibitory possibilities for protein kinases. Nuclear magnetic resonance (NMR) measurements of the mitogen-activated protein (MAP) kinase ERK2 have shown that activation by dual phosphorylation 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. 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 analog. 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 interactions for Vertex-11e, GDC-0994, and AMP-PNP with active vs. inactive ERK2, where the extent of HX protection correlates with R state formation. Furthermore, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity toward 2P-ERK2. We conclude that global motions in ERK2 reflect conformational changes at the active site that promote productive nucleotide binding and couple with changes at the activation loop to allow control of dephosphorylation by conformationally selective inhibitors.

Structural mechanism of inhibitor-resistance by ERK2 mutations

“…However, when ERK2 acquires inhibitor-resistant mutations, such as Y36H and G37C, the inhibitors no longer exhibit enzyme inhibition. Both the inhibitors bind to the ATPbinding pocket of ERK2 (Chaikuad et al, 2014;Pegram et al, 2019). In the ERK2-inhibitor complex structure, the core regions of both the inhibitors (Figure 4) were bound to the central region of the ATP-binding pocket where the adenosine base of the ATP molecule is positioned, and extension 1 and 2 regions interact with peripheral regions of the pocket.…”

“…ERK remains inactive until two amino residues within the activation loop are phosphorylated by mitogen-activated kinase (MEK) in response to extracellular growth factors. The ERK signaling pathway is involved in regulating cell proliferation, differentiation, and survival (Chaikuad et al, 2014;Goetz et al, 2014;Tong and Seeliger, 2015;Jaiswal et al, 2018;Pegram et al, 2019;Smorodinsky-Atias et al, 2020). It has been found that the tumor is resistant to ERK inhibitor treatment when the patient is continuously administered a small molecule inhibitor.…”

“…Nevertheless, although inhibitors targeting ERK have had dramatic success in cancer treatment, reactivation of ERK signaling due to tolerance presents a difficult problem for many patients (Garraway and Jänne, 2012). In addition, the mechanism of obtaining ERK inhibitor resistance and signal reactivation is still unknown (Pegram et al, 2019). Therefore, understanding the regulatory mechanisms of ERK and the effect of ERK mutations on inhibitor binding is an important goal.…”

Mutations That Confer Drug-Resistance, Oncogenicity and Intrinsic Activity on the ERK MAP Kinases—Current State of the Art

“…Not only that Erks are incapable of autophosphorylation as opposed to most ePKs, several other structural features also distinguish them from common ePKs. For example, although, like most ePKs, Erks seem to reside in equilibrium between two conformations (termed L and R; [74]) these conformations differ from the classical active and inactive conformations of ePKs. For example, no conformational change in the DFG motif (from 'out' to 'in') is apparent in the structure.…”

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 37,38 . 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 5b).…”