Moleculal Modeling Study On The Interaction Mechanism Between Kinases And Thie Inhibitors

Discovery of targets related to serious human diseases and drug development are of great importance in the process of target therapeutics. Protein kinases represent one of the most important targets in pharmaceutical research, especially in oncology drug discovery, because of their role in regulation of physiological mechanisms, including cell proliferation, differentiation and metabolism. Interruption of the signalling pathways with specific inhibitors prevents cell activation and proliferation and may be useful in treating various diseases especially for cancer. An emerging strategy for the development of potent and selective kinase-targeted drugs is of great interest. With the development of computational technology and information science, it has been recognized that molecular simulation can help to investigate the mechanisms of ligand-protein or protein-protein interactions. Computer-aided drug design (CADD) can be used as a helpful complementary tool to experiment. Various molecular simulation methods have been applied in the design and development of therapeutic drugs targeting protein kinase, such as quantitative structure-activity relationship (QSAR), molecular docking, pharmacophore model, molecular dynamics simulation, free energy calculation, and quantum mechanics/molecular mechanics (QM/MM). Based on the structure of kinase and its inhibitors, this dissertation uses several techniques (QSAR, molecular docking, molecular dynamic simulation and binding free energy calculation) to study the mechanisms of interactions between kinase targets and their inhibitors. From the ligand-based perspective, we correlated molecular structure features to their bioactivity, gaining insights into the key structural features affecting activity; from the receptor-based perspective, we studied the interactions between the protein kinases and their inhibitors in the binding process and explored the mechanism of inhibition by small-molecule inhibitors with different activity.In Chapter1, we present a general introduction of protein kinase and its inhibitors, including protein kinase classification, structure, kinase inhibitor structure and classification etc. A brief introduction of the molecular simulation method was also given, such as3D-QSAR, molecular docking, molecular dynamic simulation and free energy calculation. We also describe and overview the recent progress in the study of protein and protein inhibitors using these methods.V600EB-RAF is the most frequent oncogenic protein kinase mutation in melanoma and is a promising target to treat malignant melanoma. In Chapter2, a molecular modeling study combining QM-Polarized ligand docking, molecular dynamics, free energy calculation and three dimensional quantitative structure-activity relationships (3D-QSAR) was performed on a series of pyridoimidazolone compounds as the inhibitors of V600EB-RAF to understand the binding mode between the inhibitors and V600EB-RAF and the structural requirement for the inhibiting activity.3D-QSAR models including CoMFA and CoMSIA were developed from the conformations obtained by QM-Polarized ligand docking strategy. The obtained models have a good predictive ability in both internal and external validation. Furthermore, molecular dynamics simulation and free energy calculations were employed to determine the detailed binding process and to compare the binding mode of the inhibitors with different activities. The binding free energies calculated by MM/PBSA gave a good correlation with the experimental biological activity. The decomposition of free energies by MM/GBSA indicates the van der Waals interaction is the major driving force for the interaction between the inhibitors and V600EB-RAF. The hydrogen bond interactions between the inhibitors with Glu501and Asp594of the v600EB-RAF help to stabilize the DFG-out conformation. The results from this study can provide some insights into the development of novel potent EB-RAF inhibitors.The p38MAP kinase is a promising target for anti-inflammatory treatment. The classical kinase inhibitors Imatinib and Sorafenib as well as BI-1and BIRB-796were reported to bind in the DFG-out form of human p38a, known as Type II or allosteric kinase inhibitors. Although DFG-out conformation has attracted great interest in design of Type II kinase inhibitors, the structural requirements for binding and mechanism of stabilization of DFG-out conformation remain unclear. As allosteric inhibition is important to the selectivity of kinase inhibitor, in Chapter3, the binding modes of Imatinib, Sorafenib, BI-1and BIRB-796to p38a were investigated by molecular dynamics (MD) simulation. Binding free energies were calculated by molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) method. The predicted binding affinities can give a good explanation of the activity difference of the studied inhibitors. Furthermore, binding free energies decomposition analysis and further structural analysis indicate that the dominating effect of van der Waals interaction drives the binding process and key residues such as Lys53, Gly71, Leu75, Ile84, Thr106, Met109, Leu167, Asp168, and Phe169play important roles by forming hydrogen bond, salt bridge and hydrophobic interactions with the DFG-out conformation of p38a. Finally, we also conducted a detailed analysis of BI-1, Imatinib and Sorafenib binding to p38a in comparison with BIRB-796exploited for gaining potency as well as selectivity of p38inhibitors. These results are expected to be useful for future rational design of novel Type â…¡ p38inhibitors.Because of the high conservation of ATP-binding sites in all kinases, the quest for selective kinase inhibitor has been increasingly urgent in recent years. The Aurora kinase family represents attractive targets in cancer therapy and several small molecule inhibitors targeting Aurora kinase are undergoing clinical trials. Among them, MLN8054has been proved to be a selective Aurora-A inhibitor, and it is currently being evaluated in phase I trial for patients with advanced solid tumors. But the detailed selectivity mechanism of MLN8054towards Aurora-A over Aurora-B is still lacking. In Chapter4, this selectivity mechanism was investigated by molecular dynamics simulation and free energy calculation. The predicted binding conformation and binding affinities of MLN8054to Aurora-A and Aurora-B suggest that there exists stronger interaction between MLN8054and Aurora-A through an induced DFG-up conformation. Further analyses on the structure and free energy decomposition can provide some information about the structural basis for selectivity mechanism. Binding of MLN8054to Aurora-A induces the conformation of activation loop adopts an unusual DFG-up conformation and makes the hydrophobic pocket of active site opened and thus increases the interaction between MLN8054and the residue Va1279. The residue Glu177in Aurora-B has electrostatic repulsion to MLN8054, while the corresponding Thr217in Aurora-A has favorable interaction with MLN8054. The conformation change and the difference between the binding pocket for Aurora-A and B are key factors responsible for the selectivity. The results could be helpful for the rational design of selective inhibitors of Aurora-A kinase.The formation of p38MAPK and MAPK-activated protein kinase2(MK2) signaling complex is physiologically relevant to cellular responses such as the pro inflammatory cytokine production. The interaction between p38a isoform and MK2is of great importance for this signaling. In Chapter5, molecular dynamics simulation, domain motion analysis and binding free energy calculation have been performed on the MK2-p38a signaling complex to investigate the protein-protein interaction (PPI) between the two proteins. Dynamic domain motion analyses based on principal component analysis (PCA) were performed to analyze the conformational changes between the unbound and bound states of proteins during the interaction. The activation loop, aF-I helices, and loops among a helices in the C-lobe of MK2are found to be highly flexible and exhibit significant changes upon p38a binding. The molecular mechanics Poisson-Boltzmann and generalized-Born surface area (MM-PB/GBSA) methods are used to calculate binding free energies of MK2-p38a. This calculation indicates that the electrostatic interaction contributes critically to the specificity, rather than to MK2-p38a binding affinity. The contribution of each residue at the interaction interface to the binding affinity of MK2with p38a was also analyzed by free energy decomposition. Several important residues responsible for the protein-protein interaction were also identified.