The ERK pathway is one of the most important signaling cascades involved in
tumorigenesis. So far, eight noncovalent inhibitors of RAF and
MEK kinases in the ERK pathway have been approved by the FDA for the treatment of
cancers; however, their efficacies are limited due to various resistance mechanisms. There is an urgent need to develop novel targeted covalent inhibitors. Here we report a systematic study of the covalent ligandabilities of the ERK pathway
kinases (ARAF, BRAF, CRAF, KSR1, KSR2, MEK1, MEK2, ERK1, and ERK2) using constant pH molecular dynamics titration and pocket analysis. Our data revealed that the hinge GK (gate keeper)+3
cysteine in RAF family
kinases (ARAF, BRAF, CRAF, KSR1, and KSR2) and the back loop
cysteine in MEK1 and MEK2 are reactive and ligandable. Structure analysis suggests that the type II inhibitors belvarafenib and
GW5074 may be used as scaffolds for designing pan-RAF or CRAF-selective covalent inhibitors directed at the GK+3
cysteine, while the type III inhibitor
cobimetinib may be modified to label the back loop
cysteine in MEK1/2. The reactivities and ligandabilities of the remote
cysteine in MEK1/2 and the DFG-1
cysteine in MEK1/2 and ERK1/2 are also discussed. Our work provides a starting point for medicinal chemists to design novel covalent inhibitors of the ERK pathway
kinases. The computational protocol is general and can be applied to the systematic evaluation of covalent ligandabilities of the human cysteinome.