Drug Design And Mechanism Studies Of Key Targets Based On Protein Dynamics

Biomacromolecules like proteins are basic components of life,and protein dynamics form the fundamental basis of protein function regulation,as well as play pivotal roles in keeping life activities in order.Protein dynamics determine their specific biological functions,and such dynamics can range from sidechain rotation of single residue and conformational changes of local loops and linkers to conformational dynamics inside single structural domain as well as rearrangements of multiple domains.Besides,a variety of post-translational modifications like phosphorylation,ubiquitination,acetylation and methylation,etc.together with mutations on specific residues and protein-protein interactions could regulate the structural dynamics of protein,which make protein dynamics network more complicated.From the point of theoretical simulation,protein dynamics could be investigated using different kinds of computational tools.These tools include molecular dynamics simulation and other enhanced sampling techniques,Monte Carlo simulation,and Markov State Models.On the other hand,experimental methods comprising nuclear magnetic resonance,hydrogen-deuterium exchange mass spectrometry,X-Ray crystallography and cryo-electron microscopy could also be used for characterizing protein dynamics.Protein dynamics bring new challenges and opportunities for drug discovery and design.Recently,with the development of high-performance computational servers and computational algorithms,molecular dynamics simulations have been applied for investigating protein conformational dynamics,which promote functional mechanism studies of important proteins,as well as advance the process of drug discovery.The development of bioactive molecules based on conventional substrate pockets of drug targets is widely used in the field of drug discovery.Generally,orthosteric pockets of protein may have clear pharmacophore features,which makes it easier to set up molecular docking-based or pharmacophore-based virtual screening.However,selectivity optimization of hit molecules could be challenging owing to the evolutional conservation of orthosteric pockets among protein subfamily.In contrast,protein dynamics could provide new clues for selectivity optimization of orthosteric compounds by utilizing the dynamic conformations of key residues adjacent to orthosteric pockets.Moreover,based on protein dynamics,discovering new allosteric pockets and designing corresponding allosteric compounds with new skeletons may solve the problem thoroughly.In the first part of this thesis,molecular dynamics simulation and structure-based drug design was integrated for developing and validating hit molecules with new scaffolds targeting histone lysine demethylase LSD1.As an important anti-tumor drug target,LSD1 inhibitors have attracted widespread attention in drug development.However,almost all inhibitors of LSD1 in clinical research are irreversible inhibitors like TCP which covalently binds to LSD1 cofactor FAD.Besides,reversible inhibitors reported so far lack chemical diversity.In this work,we performed hierarchical docking-based virtual screening targeting the substrate pocket of LSD1,and further in vitro LSD1 enzymatic assay validated a new scaffold molecule.On the other hand,extensive analysis of LSD1 crystal structures,and molecular dynamics simulation led to the discovery of a potential allosteric pocket near the substrate pocket of LSD1.Further virtual screening based on the allosteric pocket as well as in vitro enzymatic assay and complex crystal determination validated the first allosteric molecule,which may have great potential applications.Cyclin-dependent kinase 2(CDK2)and its activator Cyclin-E are overexpressed in many tumors,which is associated with the required resistance of CDK4/6 approved drugs.Although selective CDK2 inhibitors could play great roles in investigating CDK2 biological functions and overcoming CDK4/6 drug resistance,almost all inhibitors of CDK2 are nonselective.In the second part of the thesis,based on the pocket dynamics suggested by structural ensemble analysis and long time-scale molecular dynamics simulations of CDK2,we designed and synthesized a new class of CDK2 inhibitors based on a reported pan-CDKs inhibitor,which were subsequently validated by in vitro enzymatic and binding assays as well as complex crystal determination and RNAseq analysis.This work may provide clues for compound optimization driven by pocket dynamics and benefit for future CDKs inhibitor design.The third part of the thesis focuses on mechanism study about proton sensitivity of human N-methyl-D-aspartate receptor(NMDAR).NMDARs are critical for regulating synaptic plasticity and function as drug targets for anti-depressive drugs.Endogenous protons could regulate NMDAR activity with the specific mechanism remaining elusive.Based on the human GluN1-GluN2 A NMDAR cryo-EM structure at pH 7.8 provided by our collaborators,we conducted long time-scale molecular dynamics simulations.Both molecular dynamics simulations and cryo-EM conformations determined at different pH states indicated GluN2A-NTD would be the major proton sensor,which may give insight into further physiological and pathological function studies of NMDAR.In conclusion,inspired by protein dynamics,we conducted computer-aided drug design targeting important anti-tumor drug targets LSD1 and CDK2,in the meantime,we investigated the mechanism of proton sensitivity of NMDAR.Combining structure-based drug design strategies with molecular dynamics simulations,we illustrated the potential applications of protein dynamics in drug discovery and development from allosteric pocket discovery,compound optimization based on orthosteric pocket dynamics and dynamic regulation mechanism.