Small Molecule Drug Discovery Of CD73 And USP28

Extracellular membrane-bound CD73 catalyzes the dephosphorylation of monophosphate（AMP）to adenosine（ADO）with strong immunosuppressive effects.Increased level of extracellular ADO will suppress immune cell proliferation,there by upgrading activity of CD4+CD25+regulatory T cells（Treg cells）,suppressing the immune function of T cells.Therefore,blockade of CD73 can reduce ADO in the tumor microenvironment and reverse tumor immune escape.In recent years,CD73has become a very potential cancer therapeutic target.Inhibitors of CD73 has the potential to be used in combination with other tumor immunodrugs or chemotherapeutic drugs to treat cancer synergistically.The first part of our work is the discovery and optimization of novel CD73inhibitors.Our cooperative research group firstly obtained a novel hit compound C1with a certain activity(IC₅₀ value of 4.32±1.13μM)with High-throughput screening,then we explored its inhibitory activity to CD73 with HPLC and optimized various parts of the hit compound by chemically synthesizing a series of compounds.Among the compounds,2 of them have higher inhibiton rate at 20μM than that of the positive compound APCP,and the IC₅₀ activity of compound C39 reaches 0.48±0.03μM,which is about ten times higher than that of compound C1.We further carried out enzyme kinetics experiments on compound C39,and determined its fast-slow protein binding action type,enzyme binding reversibility and enzyme inhibition type,resulting that this compound is a reversible competitive inhibitor of CD73.The research contributes to our understanding the overall Structure activity relationship and enzyme inhibition type information of this novel small molecule inhibitor of CD73,which can provide a deeper insight into related drug development in the future.The second part of our work is to explore and optimize small molecule inhibitors of USP28.The biological function of the ubiquitin-specific protease USP28 is extremely important and complex.By regulating the stability of oncoproteins and tumor suppressors,USP28 can affect tumor progression.USP28 can affect the stability of FBW7,which further affects the stability of oncoproteins such as Cyclin E,c-Myc,c-Jun and Notch.Therefore,USP28 is expected to be a potential target for the treatment of cancers including lung squamous cell carcinoma,colorectal cancer and non-small cell lung cancer.Through High-throughput screening,our collaborating reseachers found that a marketed drug,Vismodegib,can down-regulate USP28(IC₅₀ 4.41±1.08μM),and obtained information of its binding mode to USP28 by Crystal Diffraction Method.Our work intends to make full use of the information of the binding mode to explore the Structure and activity relationship for the compounds and to optimize the properties of the compounds.A total of 24 analogs of Vismodegib were obtained by chemical synthesis,and the enzyme activity inhibition rate was measured with Ub-AMC hydrolysis experiment.The inhibition rate of compound P17 at 10μM was higher than that of Vismodegib.Based on the experimental results obtained in our work,a valuable analysis of the Structure and activity relationship between Vismodegib and USP28 can be made,which will lay foundation for further optimization.