Discovery And Mechanism Studies Of Natural Products Targeting TEAD-YAP Complex

Hippo signaling pathway is a cell-inhibiting growth pathway that regulates tissue regeneration and organ size by inhibiting cell hyperproliferation and promoting apoptosis,and plays a key role in early embryonic development,organ size control and regeneration,and tissue stability maintenance.Over the past two decades,numerous studies have found that abnormalities in the Hippo signaling pathway are closely related to a variety of diseases,including malignant tumors.YAP protein is the most important effector molecule in this pathway.When the Hippo pathway is abnormal,YAP enters the nucleus and forms a heterodimeric complex with TEADs to activate downstream gene transcription.The TEAD-YAP inhibitors have been reported to have disadvantages such as unclear molecular mechanism,weak activity,and poor drug-forming properties,all of which are in the pre-clinical research stage.Therefore,the discovery and design of small molecule inhibitors targeting TEAD-YAP is still a hot research topic to be solved.In the second and third chapters of the thesis,we first establish and optimize a high-throughput screening platform for targeting TEAD1-YAP interaction inhibitors based on fluorescence polarization technology.Based on this,we screen the natural product libraries built by the laboratory.Then,using AlphaScreen,nuclear magnetic resonance,surface plasmon resonance and other techniques,the compounds with better activity were screened for false positive exclusion and binding experiments.Finally,the non-covalent seed compound DC_TEF1 was selected for further characterization.We found that DC_TEF1 binds to the TEAD1 palmitic acid substrate pocket and inhibits TEAD1 palmitoyltransferase activity.The molecular docking analysis binding mode showed that DC_TEF1 is spatially close to the cysteine conserved at the substrate site,and there is the possibility of designing a covalent bond.Under the guidance of the combination mode,we designed and grafted the covalent reaction“warhead”by chemical synthesis method,and obtained the covalent inhibitor DC_TEF6 with significantly improved activity.The high-resolution protein spectrum confirmed that DC_TEF6 was covalently bound to the Cys359 of TEAD1 protein.In the fourth chapter,we studied the effect of DC_TEF6 on TEAD-YAP interaction at the cellular level,and selected the HCT116 colon cancer cell model to investigate the anti-tumor activity of DC TEF6.Through HEK293T intracellular pull down,homogeneous time-resolved fluorescence and luciferase reporter assays,we demonstrate that compound DC_TEF6 disrupts TEAD-YAP interaction at the cellular level and affects TEADs transcriptional activity.On this basis,we examined the effects of DC_TEF6 on proliferation,migration and apoptosis of colon cancer cells by performing cell viability assay,Wound Healing assay and apoptosis assay on HCT116 cells.Transcription levels of several target genes associated with TEAD-YAP were detected by qPCR.Preliminary mechanism studies indicate that DC_TEF6 can inhibit the interaction of TEAD-YAP at the cellular level and affect the transcriptional activity of TEADs,thereby down-regulating the transcription levels of CTGF,Cvr61,CDX2.Cyclin E and C-Myc,and finally inhibiting the proliferation and migration of colon cancer cells and promoting antitumor activity of apoptosis.The work of this paper found a novel small molecule inhibitor of TEAD-YAP complex,which provides a new idea for the discovery of TEAD-YAP inhibitor.