Anti-tumor Drug Screening And Mechanism Studies Based On Activation Of Non-canonical P53 Signaling Pathway

P53 has been a star molecule for more than 40 years,since it was first discovered in1979,and has been a compelling cancer target.As a transcription factor,p53 has a short half-life in the unstimulated state.Under external stimuli,they are activated and regulate the expression of downstream target genes,thus initiating a variety of regulatory procedures to resist cell proliferation.At present,the drug development of p53 pathway activator has been advancing,and the drug development of wild-type p53 is gene therapy first,but there are certain limitations,the difficulty lies in how to improve the efficacy and safety.Compared with direct activation of p53 protein,it is easier to interfere with the function of its main negative regulator MDM2,which is also a current research hotspot.MDM2 not only acts as a downstream target gene of p53,but also acts as a negative regulator of p53.By directly binding with p53,it can induce ubiquitination degradation and inhibit the p53 signaling pathway,thus forming the MDM2-p53 signaling pathway between p53 tumor suppressor protein and MDM2 cancer protein.Currently,studies on the interference of MDM2 oncoprotein function are mainly divided into three directions:targeted inhibition of p53-MDM2 interaction,targeted inhibition of MDM2 activity,and targeted DEGRADATION of MDM2.Due to the bottleneck of drug development in the first two directions,md M2-targeted degradation has become an important research direction in this field.USP7(Ubiquitin Specific Protease 7)is one of the most studied deubiquitination enzymes.It can deubiquitination MDM2 and p53,and MDM2 shows a higher binding affinity than p53 without external pressure.USP7 binds prior to MDM2 to activate the p53 signaling pathway.At the same time,USP7 is highly expressed in a variety of cancers and contributes to the occurrence and development of cancer.Inhibition of USP7 kinase activity can promote MDM2 protein degradation,thus activating p53 signaling pathway to inhibit tumor cell proliferation.In summary,this paper,with the inhibition of MDM2 protein expression as the starting point,is divided into five parts to screen anti-tumor drugs and study the mechanism of USP7-MDM2-p53 signaling pathway.Part I:This part was based on the pharmacophore model of USP7 6VN2 crystal structure for virtual screening and biological evaluation,and was used to discover small molecule inhibitors targeting USP7 catalytic active sites.The TS-4 was screened from Specs database and home-Lab database(215,480 small molecule compounds)and was found to have USP7 inhibitory activity.Preliminary in vitro studies disclosed its antiproliferative activity on human colon cancer cell lines(HCT116and RKO),compared with normal colon cell lines(CCD841Co N).Furthermore,molecular dynamics simulation revealed the binding pattern between USP7 with the TS-4.The TS-4 formed stable interactions with Tyr224,Phe409 and Tyr514,which were critical to enhance its biological activity.Overall,this study suggested that compound TS-4 would be a promising candidate for the development of new USP7 inhibitors.Part Ⅱ:This part was based on the results of previous pharmacophore model screening to explore whether natural flavonoid compounds could affect USP7 kinase activity and activate p53 signaling pathway.From home-lab 160 flavonoids,28 flavonoids were screened to inhibit USP7 kinase activity and 32 flavonoids activated p53 luciferase reporter system.Among them,4 flavonoids met these two screening conditions.However,it was found that only Gardenin could significantly increase p53 protein level and activate downstream signaling pathway(p21 target protein expression level was increased),and inhibited the growth of HCT116 colon cancer cells most significantly.Furthermore,the binding mode between Gardenin B and USP7 was verified by molecular docking and molecular dynamics simulation.Howerver,it was regrettable that gardenin B could not cause the decrease of MDM2 protein expression.After all,the drug was not a single regulation of protein expression in cells,there were multiple pathways interaction.In order to better explore the biological effects of other flavonoid compounds by the two screening systems,we found that 4’,7-dimethylnaringin and hinokiflavone could significantly reduce MDM2 protein expression and strongly increase p53 protein expression at the same time.which indicated that they could be used as small molecule inhibitors of MDM2 protein for the later in-depth mechanism research.Part Ⅲ:Studies on the efficacy and pharmacology of 4’,7-dimethylnaringin showed that it could strongly inhibit the proliferation of HCT116 cells,and further found that it relies on wild-type p53 to exert anti-tumor mechanism,and finally induces apoptosis and G2/M cycle arrest of HCT116 cells.It was further demonstrated that although 4’,7-dimethylnaringin down-regulated MDM2 protein expression in dose-dependent and time-dependent manners.Then,it could not affect mdm2 m RNA transcription level,but inhibited MDM2 protein synthesis after transcription process.Finally,we tried to analyze possible direct targets through CETSA,DARTS and bioinformatics aided target prediction,so as to provide certain data basis for target researches in the later stage.Overall,the study demonstrated that 4’,7-dimethylnaringin could activate the p53 pathway by down-regulating MDM2,and was a potential natural small molecule compound for the treatment of colon cancer.Part Ⅳ:The results of pharmacological studies showed that hinokiflavone was a new natural product inhibitor of MDM2.More specifically,we found that hinokiflavone directly inhibited mdm2 gene transcription,leading to m RNA level decrease,and finally MDM2 protein expression was decreasing.The reduction of MDM2 protein induced by hinokiflavone in turn activated the p53 signaling pathway,which induced cell apoptosis and G2/M cell cycle arrest,thus reducing the survival of HCT116 cells.Then,through literature investigation,Mol Target prediction,KEGG pathway analysis and GO analysis suggested that ESR1 was a key factor in the decrease of mdm2 m RNA caused by hinokiflavone.Finally,molecular docking and molecular dynamics simulations were used to verify the binding pattern of ESR1 and hinokiflavone.In summary,our study found that hinokiflavone was a feasible natural MDM2 inhibitor by down-regulating mdm2 m RNA level and activating the p53 signaling pathway,which could promote drug development against tumor herbal medicine.Part V:In order to better understand the differences of the biflavonoids derivatives mentioned in PartⅣ,it was found that ginkgo biflavonoids(bilobetin,isoginkgetin and ginkgetin)had the same effect on MDM2 protein expression deceasing and p53 protein expression increasing.There were odd about these compounds was that although they increased p53 protein levels,they could not activate the p53 downstream signaling pathway(the expression levels of p53 target proteins such as BAX,p21 and PUMA were not upregulated).We used q PCR,EMSA and Ch IP experiments to prove whether the function of p53 protein as a transcription factor was inhibited.The results showed that bilobetin and ginkgetin could lead to the failure of p53 function to play the role of transcription factor,but the inhibition of isoginkgetin on p21 occurred in the post-transcriptional protein synthesis.For the MDM2 protein expression,we concluded that bilobetin and ginkgetin affected the transcription process and reduced the synthesis of MDM2 protein,while isoginkgetin affected the post-transcription synthesis process.We measured their cytotoxic effects on p53 wildtype(WT),mutated(MUT),or deleted(NULL)colon cancer cells and found that p53 WT played a key role in cell death.In order to find out why ginkgo biflavonoids exerted antitumor effect in p53-dependent wild-type status,we further found that they could slightly block G2/M cell cycle,induce cell early apoptosis,and promote the production of reactive oxygen species(ROS).It was subsequently demonstrated that increased iron content and decreased glutathione in cells resulted in another type of programmed cell death-ferroptosis.Through RNAseq analysis and ferroptosis database analysis,we focused on the HMGB1 protein that promotes iron death,and found that obvious nuclear shift occurred after treatment with ginkgo biflavonoids,which provided a certain reference significance for exploring the relationship between ginkgo bioflavonoids and ferroptosis.At the same time,we proved in vivo and in vitro that ginkgetin combined with 5-FU could better inhibit tumor growth.In general,ginkgo biflavonoids could bring more drug efficacy and had a good promotion effect on the development of natural product drugs against cancer.To sum up,most natural anticancer products have a wider range of targets than anticancer compounds found to target a single target,and natural products derived from edible and medicinal plants have shown strong chemoprophylaxis and chemotherapy activity for cancer in preclinical and clinical studies.4’,7-dimethylnaringin,hinokiflavone,bilobetin,isoginkgetin and ginkgetin stood out in our screening process as MDM2 inhibitors,and the unique mechanism of ginkgo biflavonoids is worth further study.