| Notch signalling pathway is highly conserved in the evolutionary process of multicellular organisms and is one of the major signalling pathways that mediate the direct cell-cell contact.Notch pathway also plays an important role in the cellular division,differentiation,proliferation,apoptosis,and the angiogenesis of tumor cells.Abnormal regulation of Notch pathway would cause malfunctional development of tissues and lead to the occurrence of tumor.Therefore,the regulation of Notch signalling pathway is considered as a potential effective target for tumor treatment.Currently,explicit targets in Notch pathway for the development of inhibitors mainly include Notch receptor and ligand,Adam,γ-secretase and transcription factor RBP-JK.Notch receptors and ligands can be targeted by some macromolecular monoclonal antibodies,however,which have high production cost and poor stability.Some inhibitors acting onγ-secretase and ADAM have unsatisfactory specificity due to the co-existence ofγ-secretase and ADAM in other signalling pathways.In addition,RBP-JK,a key transcription factor in the canonical Notch pathway,has been targeted by several small-molecule inhibitors with strong specificity,however,the number of candidate molecules is small,and they still remain in the laboratory and preclinical stages.Therefore,this study focuses on RBP-JK,and has used some virtual screening technologies to screen potential RBP-JK inhibitors from traditional Chinese medicine ingredients and some anti-tumor drugs.We have studied the anti-tumor mechanism of the screened candidate compounds,in terms of in vitro cell and in vivo animal models,and detailed molecular mechanisms.This dissertation is mainly divided into the following four parts:Part Ⅰ.IntroductionThis chapter mainly summarised the Notch signal pathway,the molecular docking and molecular dynamics simulation technologies.First of all,the mechanism of Notch signalling pathway and the related Notch pathway inhibitors were reviewed.Then we discussed the molecular docking and molecular dynamics simulation research progress and the application in drug screening.These reviews will provide theoretical,data and methodological support for this study.Part Ⅱ.Screening of RBP-jk inhibitors based on molecular docking and molecular dynamics simulationIn this chapter,molecular docking and molecular dynamics simulation techniques were used to screen the ingredients of anti-tumor traditional Chinese medicine and the marketed drugs.First,by analyzing the interaction mode of the NICD-MAML-RBP-jk-DNA complex and the docking results of NICD,MAML,DNA and RBP-jk,the active sites of RBP-jk was determined,including 37 amino acid residues:Lys44,Tyr46,Lys50,Arg51,PHE52,Cys86,PHE88,Met98,Lys130,Gly134,Ser136,Asp138,Ser151,Ser154,GLN158,Arg178,Arg180,Ser181,GLN182,PHE221,VAL223,MET243,PRO246,Lys271,GLN293,GLN347,Leu348,Gly350,Glu358,Asn367,Arg369,Arg378,Tyr381,Arg382,Cys383,Gly384,Glu385and ASN417.Also,these interaction and key residues within this active site were used as the reference for the analysis of docking results.Then,through all-atom docking,small molecule inhibitors were screened according to the following criteria:the docking binding energy of small molecule compounds with RBP-jk should be less than-8kcal/mol,the key residues interacted with candidate compound should be more than three,and the spatial structure of the compounds could occupy the active pocket.After that,21 small molecule compounds were screened.Furthermore,based on the key 37 active residues,21 compounds screened were docked and analyzed in the manner of restricted binding sites with the same criteria.Three molecular compounds such as Fidaxomicin,Schaftoside and Acarbose were identified.Nest,virtual investigation was conducted to examine the binding ability of three candidate compounds to RBP-jk.The results showed that the maximum number of molecules that could stably bound to the active pocket were 2,3 and 2,respectively,indicated that they could effectively occupy the binding pocket.The RBP-jk-small molecule complex was further used as a receptor which was used to verify the ability of each compound to block the binding between RBP-jk and DNA.The results showed that the binding position of the DNA bound complex significantly deviated from the normal site of RBP-jk and DNA,confirming that all three compounds could block the binding of RBP-jk to DNA.Finally,candidate compounds were investigated by molecular dynamics simulation,and the results showed that the binding energy of fidaxomycin,schaftoside and acarbose to RBP-jk protein were 154.142±12.316k J/mol,-167.584±15.725 k J/mol,-163.674±8.845 k J/mol,respectively,which indicated the strong binding interactions between compounds and RBP-jk protein.Therefore,Fidaxomicin,Schaftoside side and Acarbose were finally identified as small-molecule weight candidate inhibitors of Notch pathway.Part Ⅲ.Study on in vitro antitumor activity of RBP-jk inhibitorsIn this chapter,we used human breast cancer cell line MCF-7 and mouse breast cancer cell line 4T1 to investigate the in vitro antitumor activity of Fidaxomicin,Schaftoside and acarbose,respectively.The results showed that the IC50values of Fidaxomicin on MCF-7 cells and 4T1 cells were 53.4μM and 32.3μM,respectively,which demonstrated a certain anti-tumor effect in a dose-dependent way.However,no obvious antitumor effects were observed with other two compounds in the concentration range of 1-400μm M,which might be due to the fact that these two compounds could not enter the cellular nucleus to exert their functions.Then we investigated the cellular distribution of the candidate compound Fidaxomycin.Using laser confocal scanning microscopy,it was found that some of the Fidaxomycin entered the nucleus after 0.5h of incubation.With the prolonged incubation time,the amount of drug accumulated in nucleus was also increased,verifying that Fidaxomycin could enter the nucleus.In addition,q RT-PCR was used to detect the levels of downstream genes in Notch pathway,including Notch1,Hes1,Hes5 and HEY1,after 24h and 48h of Fidaxomicin administration.The results showed that Notch1,Hes1,Hes5,Hey1 and other genes were significantly down-regulated after24h and 48h incubation,suggesting that Fidaxomicin could inhibit the expression of related genes in Notch pathway.Also,Western Blotting technology was used to detect the expression levels of downstream Hes1 and Hes5 protein in the Notch pathway after 24h and 48h drug treatment.The results showed that the expression levels of Hes1 and Hes5 proteins were not significantly changed after 24h of Fidaxomicin treatment;however,after 48 hours treatment,the protein expressions of Hes1 and Hes5 were significantly down-regulated.These results revealed the action mechanism of Fidaxomicin,confirming that it could induce the apoptosis of tumor cells by inhibiting Notch pathway.Part Ⅳ.In vivo antitumor activity of small molecule inhibitors of Notch pathwayThe antitumor activity of Fidaxomicin was investigated in 4T1 tumor-bearing mice at various doses,including a high dose（50 mg/kg）,medium dose（25 mg/kg）and low dose（5 mg/kg）.Results revealed that the inhibitory efficacies of Fidaxomicin at each low-dose,medium-dose and high-dose Fidaxomicin groups were 31.77%,56.56%and 83.19%,respectively,indicating the dose-dependent anti-tumor activity of Fidaxomicin.As a marketed drug,it has little effect on the body weight of mice and high safety.To further study its mechanism,Western blotting technology was used to detect the protein levels of Notch downstream molecules HES1 and HES5 in tumor tissues.Compared with the control group,the administration of Fidaxomicin could significantly down-regulate the protein levels of downstream HES1 and HES5.In addition,the expression levels of Hes1 and Hes5 proteins were decreased in a dose-dependent manner,showing that Fidaxomicin could be used as a Notch inhibitor with effective anti-tumor capability.In summary,our study finally screened and verified a Notch inhibitor Fidaxomicin,which was investigated through virtual screening,in vivo and in vitro evaluation of anti-tumor activity,and the detection of gene and protein expression levels.These results collectively presented a Notch pathway inhibitor candidate,and also provided a method for the future computational screening of Notch pathway inhibitors. |
