Modulating multi-functional ERK complexes by covalent targeting of a recruitment site in vivo

Tamer S Kaoud,Mohamed F Radwan,William H Johnson,Andrea Piserchio,Mangalika Warthaka,Ranajeet Ghose,Jacey R Pridgen,Ramakrishna Edupuganti,Sabrina X Van Ravenstein,Jihyun Park,Pengyu Ren,Rachel M Sammons ,Clint D.j Tavares ,Diana Zamora‐Olivares ,Kenneth Y Tsai,Eric V Anslyn,Micael Cano,Matthew Harger,Nancy D Ebelt,Kevin N Dalby

doi:10.1038/s41467-019-12996-8

Abstract

Recently, the targeting of ERK with ATP-competitive inhibitors has emerged as a potential clinical strategy to overcome acquired resistance to BRAF and MEK inhibitor combination therapies. In this study, we investigate an alternative strategy of targeting the D-recruitment site (DRS) of ERK. The DRS is a conserved region that lies distal to the active site and mediates ERK–protein interactions. We demonstrate that the small molecule BI-78D3 binds to the DRS of ERK2 and forms a covalent adduct with a conserved cysteine residue (C159) within the pocket and disrupts signaling in vivo. BI-78D3 does not covalently modify p38MAPK, JNK or ERK5. BI-78D3 promotes apoptosis in BRAF inhibitor-naive and resistant melanoma cells containing a BRAF V600E mutation. These studies provide the basis for designing modulators of protein–protein interactions involving ERK, with the potential to impact ERK signaling dynamics and to induce cell cycle arrest and apoptosis in ERK-dependent cancers.

Highlights

The targeting of ERK with ATP-competitive inhibitors has emerged as a potential clinical strategy to overcome acquired resistance to BRAF and MEK inhibitor combination therapies
We found that BI-78D3 impedes the ability of a constitutively active form of the ERK kinase, MKK1 (MKK1G7B)[38] to phosphorylate ERK1 and ERK2 in vitro and impedes the ability of activated ERK1 and ERK2 to phosphorylate v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1; a construct including the residues 1–138 that is necessary and sufficient for ERK-mediated phosphorylation, was used39) (Fig. 1b)
We found that C159 forms a covalent bond with the C5 carbon of the 1,2,4-triazol-3one ring of BI-78D3 to form a relatively stable tetrahedral adduct

Summary

Introduction

The targeting of ERK with ATP-competitive inhibitors has emerged as a potential clinical strategy to overcome acquired resistance to BRAF and MEK inhibitor combination therapies. BI-78D3 promotes apoptosis in BRAF inhibitor-naive and resistant melanoma cells containing a BRAF V600E mutation These studies provide the basis for designing modulators of protein–protein interactions involving ERK, with the potential to impact ERK signaling dynamics and to induce cell cycle arrest and apoptosis in ERKdependent cancers. Recent evidence supports the possibility that inhibitors of ERK, for example, the ATP-competitive inhibitor ulixertinib, may exhibit acceptable safety profiles[22], and can suppress the emergence of resistance and overcome acquired resistance to BRAF and MEK inhibitors[22,23]. Long-term exposure of cells to the ATP-competitive inhibitor of ERK, SCH77298423 leads to acquired resistance, corresponding to a mutation, G186D, in the DFG motif of ERK126 and an in vitro analysis revealed other potential resistance mutations, mainly located within the ATP-binding site[27]

Methods

Results

Conclusion