The Mechanism Of Abnormal Activation Of MEK1 Induced By Dehydroalanine Modification And The Effect Of FDA-Proved Inhibitors

Mitogen-activated protein kinase kinase 1(MAPKK,MEK1)is a key kinase in the mitogen-activated protein kinase(MAPK)signaling pathway.Abnormal protein activation caused by residue mutations in MEK1 is closely related to malignant growth and metastasis of various cancers,such as melanoma,and is an important target for cancer treatment.Four FDA-approved drugs,Trametinib,Cobimetinib,Binimetinib,and Selumetinib,target MEK1.Recently,it has been shown that dehydroalanine(Dha)modification can also lead to abnormal activation of MEK1,with potential to promote tumor development.In this study,we used computational biology methods to investigate the effect of Dha modification on the protein structure of MEK1,explored the mechanism of abnormal activation caused by Dha modification,and predicted the inhibitory effects of existing FDA-approved MEK1 inhibitors on Dha modification-induced abnormal activation of MEK1.First,we constructed wild-type MEK1 protein(non-activated MEK1 protein),phosphorylated MEK1 protein(normally activated MEK1 protein),and Dhamodified MEK1 protein(abnormally activated MEK1 protein),and performed microsecond-level all-atom molecular dynamics simulations(MD)for each of them.The research results showed that,similar to the conformational changes in the normal activation process,after being modified by Dha,MEK1 moved its activation loop towards the C-terminus,opened the catalytic pocket,and exposed the active site,which led to the abnormal activation of MEK1.Specifically,by analyzing RMSF and hedgehog plots,we found that Dha modification caused significant changes in the position and conformation of the C-helix,Pro-rich region,and activation loop of MEK1,leading to the opening of the catalytic pocket and exposure of the active site.Quantitative calculation of the catalytic pocket volume further confirmed that the catalytic pocket had enlarged,consistent with the observed structural changes.Structural changes were accompanied by changes in the interactions between internal residues of the molecule,among which hydrogen bond analysis showed that the enhanced hydrogen bond interaction between the activation loop and Glu114,Glu233,Tyr240,and Arg305 contributed significantly to the movement of the activation loop.Next,based on Well-Tempered Metadynamics enhanced sampling simulations,we obtained representative Dha-modified MEK1 protein structures and docked four FDA-approved small molecule drugs targeting MEK1.Further structural analysis revealed that the Selumetinib inhibitor caused the activation loop of Dha-modified MEK1 to unwind,leading to the closure of its active pocket,thereby inhibiting the catalytic activity of MEK1 abnormal activation.On the other hand,Cobimetinib,Trametinib,and Binimetinib had little effect on the conformation of Dha-modified MEK1 protein.In addition,hotspot residue analysis and hydrogen bond analysis showed that Thr226,Arg227,Phe209,and Asp190 were important for the binding of Selumetinib to Dha-modified MEK1.In summary,this study has revealed the mechanism of MEK1 abnormal activation caused by Dha modification,which is due to the movement of the activation loop,leading to the opening of the active site and the continuous abnormal activation of MEK1.The Selumetinib inhibitor blocks the activity of Dhamodified MEK1 by affecting the secondary structure of the activation loop,causing it to transition from an alpha helix to a loop and blocking the active site.Our research will help uncover the mechanism of Dha modification-induced abnormal activation of MEK1 and provide clues for the development of corresponding inhibitors.