Discovery Of Small Molecule Inhibitors For Extracellular Signal-regulated Kinase 1/2(ERK1/2)

Mitogen-activated protein kinase(MAPK) pathway, which is stimulated by extracellular stimuli, controls vital cellular processes like proliferation, migration and anti-apoptosis via a RAS-RAF-MEK-ERK cascade and plays a central role in conferring cancer malignancy. Mutations occurring in MAPK pathway components comprise 80% genetic aberrations in cancer, suggesting great opportunities in targeting MAPK pathway for cancer therapy. However, current strategies in targeting this pathway remain being challenged. In spite of the extremely high mutation frequency and decades of efforts invested in this target, there still lacks effective approach to inhibit mutated RAS. Clinically approved inhibitors targeting MAPK pathways include BRAF inhibitors and MEK inhibitors, whose clinical benefits have been largely restricted by frequently occurred acquired resistance. ERK kinases, in particular ERK1/2, are the only identified downstream kinase of MEK, converge MAPK pathway, and control multiple cellular events via directly phosphorylating over 300 substrates. ERK1/2 inhibition can effectively block MAPK pathway signaling and also overcome most of acquired resistance to BRAF or MEK inhibitors. As such, targeting ERK1/2 is believed to provide privileged opportunities for MAPK pathway inhibition. Several ERK1/2 inhibitors have been reported but all of them are still at early clinical or preclinical stages.This study aimed to establish a platform that integrated molecular, cell-based and animal models to support the discovery of novel ERK1/2 inhibitors. Specially, a fluorescence resonance energy transfer(FRET) based enzymatic assay was used for measuring the kinase activity of ERK1/2. As for the cell-based assay, a panel of human tumor cell lines harboring K-RAS or B-RAF mutations was screened using a Sulforhodamine B(SRB) colorimetric assay to identify ERK-dependent subset. Intracellular MAPK signaling cascades change was detected using immunoblotting. ERK-dependent human cell lines were orthotopically implanted into Balb/c nude mice for the assessment of in vivo activity. In particular, ADME properties were evaluated at early stage to solve the discrepancy between enzymatic and cellular activities and to gain a glimpse of the pharmacokinetic profile. This platform allowed us to assess over 400 rationally designed chemicals based on known ERK inhibitors and molecular modeling, and identified quite a few compounds with potent enzymatic activity with IC50 s below 50 nM. By assessing these active compounds, we solved the discrepancy between enzymatic and cellular activity and optimized the pharmacodynamics properties of candidate compounds. These efforts led to the discovery of a lead compound that was ready for further evaluation..In conclusion, this study established a comprehensive platform for the discovery of ERK1/2 inhibitors, which allowed us to discover a lead compound exhibiting improved enzymatic, cellular activity as well as pharmacodynamics properties compared with known ERK1/2 inhibitors. These findings paved the way for the development of ERK1/2 inhibitors as anticancer drugs.