Protein Kinase Selective And Small Molecule Inhibitors Jak2 Kinase Regulatory Mechanism Of Computational Chemistry Research

The theory of computational chemisty is on the basis of physics, and the theoretical computational technologies can be widely used to model and simulate the biomolecule systems. In the protein function and structure study, the computational approaches can provide a series of tools, including the homology modeling for protein three-dimensional structure prediction, the protein-protein docking algorithm for complex structures prediction, and the molecular dynamics (MD) simulations for protein flexibility and function analysis. In the field of computational drug design, the structure-based virtual screening has been widely applied. In this thesis, two efficient computational protocols were developed to address two interesting biological questions, individually. One is a physics-based approach to computationally modeling the kinase inhibitor selectivity profile. The other is a novel informatics-guided protein-protein docking strategy for ab initio modeling protein complex structures.The rational design of selective kinase inhibitors is a great challenge in the kinase drug discovery. We described a physics-based approach to computationally modeling the kinase inhibitor selectivity profile. Briefly, our computational modeling procedure consists the following steps:1. Selecting the representative kinase crystal structure in an active conformation;2. Predicting the ligand binding pose using comparative docking;3. Sampling the binding site conformation with the presence of the docked ligand;4. Scoring the resulted complex structure using the MM-GB/SA scoring function. We retrospectively assessed this protocol by computing the binding profiles of17well-known kinase inhibitors against143kinases. The averaged EF20value of2.81and the averaged PI value of0.35indicated a reasonable prediction performance of our computational approach in ranking order of the kinase targets for a given inhibitor. This automatic workflow allowed rapid evaluation of compounds of interest against the entire structural kinome. Next, we predicted the binding profile of the chemotherapy drug mitoxantrone (MX), and chose the predicted top five kinase targets for in vitro kinase assays. Remarkably, mitoxantrone was shown to possess low nanomolar inhibitory activity against PIM1kinase and to inhibit the PIM1-mediated subtrates phosphorylation in cancer-cells. We further determined the crystal complex structure of PIM1bound with mitoxantrone, which revealed the structural and mechanistic basis for a novel mode of PIM1inhibition. Although mitoxantrone’s mechanism of action had been originally thought to act through DNA intercalation and type Ⅱ topoisomerase inhibition, we provided for the first time evidence that PIM1kinase inhibition may contribute to mitoxantrone’s therapeutic efficacy and specificity.The function of protein kinases are highly regulated in the cell by their dimerization, supramolecular assemblies, other regulatory domains or interacting proteins. For example, the pseudokinase domain (JH2) of Janus Kinase2(JAK2) regulates the activity of its adjacent kinase domain (JH1) althought the exact molecular mechanisms are not fully understood. We first applied MuInf algorithm, one method can identify protein sites exhibited correlated torsional motions, to predict the potential protein interface. The primary models from MutInf predicted interfaces were refined by a hierarchical protein-protein docking and molecular dynamics simulations refinement procedure to systematically improve the quality of these complex models. We applied this novel strategy to model the pseudokinase domain-kinase domain complex structure of the JAK2. Based on this model, a detailed JAK2JH2-mediated auto-inhibition mechanism was proposed, where JH2traped the activation loop of JH1in an inactive conformation and blocked the movement of kinase a C helix through critical hydrophobic contacts and extensive electrostatic interactions. These stabilizing interactions were less favorable in JAK2-V617F, a gain-of-function mutation within JH2was found in the majority of patients with myeloproliterative neoplasms. Notably, several predicted binding interfacial residues in JH2were confirmed to hyperactivate JAK2kinase activity in site-directed mutagenesis and BaF3/EpoR cell transformation studies. Although there might exist other JH2-mediated mechanisms to control JH1, our JH1-JH2structural model represented a verifiable working hypothesis for further experimental studies to elucidate the role of JH2in regulating JAK2in both normal and pathological settings. This step-wise computational strategy we devised may be easily adopted for studying novel protein-protein interactions in a general manner.