Molecular Dynamics Simulation Survey Of The Drug Resistance Mechanisms And Structural Activation Of ALK

Protein kinase is the largest family of proteins in the human body.Protein tyrosine kinases regulate most of the signaling pathways in cells by catalyzing the phosphorylation of tyrosine residues in a variety of substrate proteins,making them important in physiological processes such as cell growth,metabolism,differentiation,and apoptosis.Protein tyrosine kinases have become a target for many cancers.Anaplastic lymphoma kinase(ALK)is a receptor-type tyrosine kinase belonging to the insulin receptor superfamily,whose abnormal expression,such as gene mutation,rearrangement and amplification,is closely related to a variety of human malignancies,such as anaplastic lymphoma,non-small cell lung cancer,neuroblastoma,diffuse large B-cell lymphoma,inflammatory muscle fibers,and Blastoma,etc.Activation of anaplastic lymphoma kinase can induce a series of downstream cellular signaling pathways that regulate tumor cell growth,differentiation,and migration.Due to the nature of its oncogenes,anaplastic lymphoma kinase has become a new target for antitumor therapy.ALK's targeted therapies have achieved good results in the clinic,but the emergence of drug resistance problems has limited their use in the treatment of tumors.Therefore,it is very important to explore the mechanism of drug resistance and understand its abnormal activation mechanism for targeted therapy of tumors.At present,the research on ALK is mostly at the level of cells and clinical trials.The precise study of the relationship between ALK structure and function at the atomic level will play a non-negligible role in the targeted therapy of tumors.In this paper,a variety of molecular dynamics simulation methods were used to systematically study the inhibitory mechanisms of several clinically discovered inhibitors of ALK gene mutation.At the same time,ALK gene mutations and ATPinduced ALK allosterics were discussed in depth.The main contents of the paper are as follows:1.Molecular dynamic investigation of resistance mechanisms induced by three mutations in ALKAlectinib,a highly selective anaplastic lymphoma kinase(ALK)next-genetation inhibitor,has demonstrated promising antitumor activity in patients with ALK-positive non-small cell lung carcinomas(NSCLC).However,the therapeutic benefits of alectinib is inescapable hampered by the development of acquired resistant mutations.Despite the ample experimental mutagenesis data,the molecular origin and the structural motifs for alectinib binding affinity deficiencies are still ambiguous.Here,molecular dynamics(MD)simulations and molecular mechanics generalized born surface area(MM-GBSA)calculation approaches were employed to elucidate the resistance mechanisms of alectinib induced by the mutations I1171 N,V1180L,and L1198 F.The MD results reveal that the studied mutations could trigger the dislocation of alectinib as well as the conformational changes at the inhibitor binding site,thus induce the interactional changes between alectinib and mutants.The most influenced regions are the ligand binding entrance and the hinge region,who are considered to be the dominant binding motifs accounts for the binding affinity loss in mutants.The “key and lock mechanism” between the ethyl group at position 9 of alectinib and a recognition cavity in the hinge region of ALK is presented to illustrate the major molecular origin of drug resistance.Our results provide mechanistic insight into the effect of ALK resistance mutations to alectinib,which could contribute to further rational design of inhibitors to combat the acquired resistance.2.Insight on mutation-induced resistance to ALK inhibitor ceritinib from molecular dynamics simulationsCertinib,an advanced anaplastic lymphoma kinase(ALK)next-generation inhibitor,has been proved excellent antitumor activity in the treatment of ALKassociated cancers.However,the accumulation of acquired resistance mutations compromise the therapeutic efficacy of ceritinib.Despite abundant mutagenesis data,the structural determinants for reduced ceritinib binding in mutants remains elusive.Focusing on the G1123 S and F1174 C mutations,we applied molecular dynamics(MD)simulations to study possible reasons for drug resistance caused by these mutations.The MD simulations predict that the studied mutations allosterically impact the configurations of the ATP-binding pocket.An important hydrophobic cluster is identified that connects P-loop and the ?C-helix which has effects on stabilizing the conformation of ATP-binding pocket.It is suggested in this study that the G1123 S and F1174 C mutations can induce the conformational change of P-loop thereby causing the reduced ceritinib affinity and causing drug resistance.3.Conformational Transition of Key Structural Features Involved in Activation of ALK Induced by two Neuroblastoma Mutations and ATP Binding: Insight from Accelerated Molecular Dynamics SimulationsDeregulated kinase activity of Anaplastic Lymphoma Kinase(ALK)has been observed to be implicated in developing of tumor progress.The activation mechanism of ALK is proposed to be similar with other receptor tyrosine kinase(RTK),but the distinct static X-ray crystal conformation of ALK suggests its unique conformational transition.Herein,we have illustrated the dynamic conformational property of wild type ALK as well as the kinase activation equilibrium variation induced by two neuroblastoma mutations(R1275Q and Y1278S)and ATP binding by performed enhanced sampling accelerated Molecular Dynamics(aMD)simulations.The results suggested that the wild type ALK is mostly favored in the inactive state,whereas the mutations and ATP binding promoted clear shift toward the active-like conformation.The R1275 Q mutant stabilize the active conformation by rigidified the ?C-in conformation.The Y1278 S mutant promotes the activation at the expense of a ?-stacking hydrophobic cluster which plays critical role in the stabilization of inactive conformation of native ALK.ATP produces a more compact active site and thereby facilitates the activation of ALK.Taken together,these findings not only elucidate the diverse conformations in different ALKs,but also can shed light on new strategies for protein engineering and structural based drug design for ALK.