Objective

The MAPK signalling pathway is activated through oncogenic mutations in RAS or RAF in over 30% of cancers. The therapeutic potential of inhibiting this pathway has been demonstrated by the clinical efficacy of RAF and MEK inhibitors in the treatment of BRAF mutant melanoma. However, the response to these inhibitors is often short lived due to the emergence of resistance. The majority of resistance mechanisms result in increased ERK signalling which drives cell survival and proliferation even in the presence of RAF and MEK inhibitors. The direct targeting of ERK is therefore an attractive approach to overcome the limitations of RAF or MEK inhibitors. The majority of ERK inhibitors currently in clinical development block phosphorylation of downstream substrates of ERK such as RSK, but do not modulate the phosphorylation of ERK by MEK. We hypothesise that additional inhibition of ERK phosphorylation by MEK could lead to sustained knockdown of MAPK signalling and therefore improved efficacy.

We initiated a fragment-based approach to develop ERK kinase inhibitors that also modulate the phosphorylation of ERK by MEK. A Pyramid screen was conducted by using a combination of high throughput X-ray crystallography and biophysical assays. Initial fragment hits from these methods were evaluated for potency in a time-resolved fluorescence (TRF) ERK kinase activity assay. Structure-guided optimisation and growth of the initial hinge binding fragment led to the discovery of a selective ERK inhibitor which inhibits ERK catalytic activity with low nanomolar potency. Using an in vitro MEK-ERK cascade assay we demonstrated that the lead compound also inhibits the phosphorylation of ERK by MEK without directly inhibiting MEK. Further characterisation demonstrated potent suppression of pRSK and pERK in vitro and in vivo in a range of MAPK driven models. Furthermore, once daily oral dosing of the lead compound conferred significant anti-tumor activity in a range of MAPK driven models, including Colo205 (BRAF-mutant colorectal) tumor xenografts. These data support the further optimisation of this series of compounds for clinical development.