Lead Discovery And Mechanism Studies Of Post-Translational Modification Related Proteins

Biomacromolecules especially proteins form the fundamental basis of the constitution of nature that play the pivotal role in life activities.In eukaryotes,the function of proteins is dynamically regulated by post-translational modifications,such as phosphorylation,acetylation,methylation,glycosylation,fatty acylation and ubiquitination.A variety of dynamic,reversible post-translational modifications with specific distribution form important molecular basis for the establishment of biological function networks and the diversity of multi-cellular organisms,which is also the essential part of the process for the maturation of biological macromolecules and a frontier scientific issue in life science.Recent studies have shown that the abnormal expression and dysfunction of post-translational modifying proteins is closely related to the occurrence and progression of many diseases including cancers,nervous system diseases,endocrine diseases and cardiovascular diseases.Many targeted drugs like kinase inhibitors and HDAC inhibitors have been approved for the treatment of malignant diseases especially cancers,which demonstrated the feasibility of the strategy.However,the development of drugs that target other key post-translational modifying proteins such as lipid-modifying proteins lags behind.Many targets are considered as“difficult”targets like HATs,KDMs and phosphatases.Therefore,there is urgent need to establish new methodologies and drug discovery platforms,which could shed light on novel target drug discovery.The first part of this thesis is the discovery and development of the covalent inhibitors targeting the autopalmitoylation pocket of transcription factor family TEADs.In this work,we established a structure-based virtual screening platform and chemical biology validation platform based on CuAAC bio-orthogonal reactions,which solved the problem of the lack of high-throughput screening methods against the target in wet lab.Through virtual screening and a series of biophysical and biochemical validation,we identified the novel non-covalent inhibitor DC-TEADin01.Based on the non-covalent scaffold,through rational medicinal chemistry optimization,we obtained the highly potent and selective TEADs covalent inhibitor DC-TEADin02.DC-TEADin02is the most potent and selective TEAD inhibitors ever reported(IC₅₀=197±19 nM).In colon cancer xenograft model,DC-TEADin02 significantly inhibited tumor growth with minimal effect on body weight of mice,which is also the first TEAD inhibitor ever reported with in vivo activity.This work demonstrates the feasibility of targeting protein lipidation in cell signaling,which is of great value in the era of targeted cancer therapy.The second part of this thesis is the discovery and design of TEAD3 subtype selective inhibitor.In this part of the work,we firstly obtained the pan-TEAD inhibitor with the novel chemotype through scaffold hopping.Then we quickly finished hit validation based on the previously established validation platform.Notably,through iterative medicinal chemistry optimization,we obtained the selective TEAD3 inhibitor,the subtype that has never been explored before in drug discovery campaigns.Additionally,the selective inhibitor could significantly inhibit the embryo development and retard the growth rate of juvenile fish.This work demonstrates the important role of TEAD3 in regenerative medicine and may serve as good examples of how computational approaches cooperating with rational optimization could efficiently promote the drug discovery progress,which could be easily extended to develop other TEAD subtype-selective inhibitors.The third part of the work of this thesis is the discovery of the allosteric regulator of histone demethylase LSD1,a very important target for future therapeutic innovation.However,the current available inhibitor ORY-1001 covalently binds to the cofactor FAD and shows poor selectivity and severe toxicity,which limits further clinical translation.In this work,we performed extensive conformation sampling of LSD1/CoREST and identified a novel allosteric site by molecular dynamics simulation.Further virtual screening campaigns led to the discovery of first allosteric LSD1inhibitor.We disclosed the mechanism of action of the compound by enzymatic assays and complex crystal structure determination.Further structure-based medicinal chemistry modifications led to the identification of more potent LSD1 inhibitors,which hold enormous potential in future applications.The fourth part of this thesis focuses on the discovery of lead compounds that target the histone acetylation readers BPTF and SMARCA2,which belong to the non-BET histone acetylation reader family.Although scientists made breakthrough progress in the development of BET inhibitors,the drug discovery and development targeting non-BET proteins lags behind.In this work,we made use of structure-based virtual screening methodology and ALPHAScreen-based high-throughput screening technique leading to the identification of hit compounds targeting BPTF and SMARCA2,respectively.A series of biophysical assays demonstrated the direct binding between hit compounds and related targets.Further 2D similarity-based analogue search and molecular docking studies disclosed the mechanism of action of the series of compounds.In sum,we identified DCB29 and DCSM06-05 as bona fide BPTF and SMARCA2 inhbitors with novel chemotypes,which may lay foundation for the following medicinal chemistry optimization.To sum up,based on the comprehensive analysis of the current status of drug development targeting post-translational modifying proteins,we make use of site-oriented modification and allosteric regulation strategies and theoretical simulation-driven chemical biology methodologies to conduct drug discovery against the TEAD autopalmitoylation pocket,the histone demethylase LSD1 and the non-BET family protein,which are cutting edge techniques in the field of drug discovery and development.Most importantly,these effective chemical probes would serve as useful tools to interrogate the biological function of post-translational modification related proteins.