| BackgroundHepatocellular carcinoma(HCC)is the most common and high mortality rate of malignant tumors,the highest in the world of malignant tumor mortality of 3,a serious threat to human health and life.China is located in the high incidence area of HCC,epidemiological data show that HCC ranked second in the mortality of malignant tumors in China.The liver resection and radiofrequency ablation(RFA)is a common treatment for early HCC,in patients with advanced disease can be by transcatheter arterial chemoembolization(TACE),radiotherapy and chemotherapy means,but in postoperative patients with easy to relapse and to radiotherapy.Chemotherapy is easy to produce drug resistance.The prognosis is very poor.Molecular target to antitumor drugs in reducing the traditional chemotherapy drugs of systemic side effects and improve the curative effect has certain superiority.Therefore,HCC molecular target to drug development in control of HCC tumor proliferation,delay recurrence and metastasis and improve the quality of life of patients and has a very important significance.Silent information regulator 1(silent information regulator1,the SIRT1)with that of yeast Sir2 homolog is a class dependent nicotinamide adenine dinucleotide(NAD)is a class III histone to acetylase.SIRT1 localization in the nucleus,early embryonic content rich and in mature tissues widely expressed,through the histone and non histone to acetylation regulates gene expression,involved in cellular senescence,apoptosis,differentiation,stress tolerance and energy metabolism of various important physiological activities.In recent years,many studies showed that SIRT1 with a variety of tumor occurrence and development are closely related,in particular the relationship between SIRT1 and HCC got the wide attention of researchers.Clinical findings,SIRT1 in HCC patients with abnormal high expression and prognosis prediction.Further research shows that SIRT1 in HCC cell proliferation and tumor growth playthe key role,and the role in a wide range,complicated molecular mechanism.At present,SIRT1,which is a new target of anti liver cancer drugs has not been reported.Therefore,to STRTl target for small molecule inhibitors is innovative anti liver cell cancer drug development direction of.ObjectiveThis project based on the receptor of SIRT1,select the EX527,nicotinamide and SIRT1 protein binding sites,through high-throughput virtual screening and molecular simulation methods screened small molecule inhibitors of docking scoring higher,based on,further screening and optimization,design,synthesis,by inhibiting the overexpression of SIRT1 in hepatoma cells,hepatoma cells proliferation,on its anti hepatoma activity were in vitro and in vivo effects on experimental,for screening and transformation model of the SIRT1 inhibitors for the target of the anti hepatoma activity lay a good foundation.The main contents1.Virtual screening based on SIRT1 receptor(1)Small molecule database Bank Chem and Drug download small molecular compound library,protein database PDB download SIRT1 and ligand complex crystal structure(PDB:4I5I).(2)Using molecular docking and other computer virtual screening methods,the use of molecular docking software Studio3.0 SYBYL2.0,Discovery,molecular docking scoring,and SIRT1 active pocket to select the highest matching degree of small molecule inhibitors.(3)Determination of the binding affinity of small molecule inhibitors to SIRT1 receptors and to determine the potential activity of small molecule inhibitors.2.The activity of anti hepatic carcinoma in vitro(1)CCK-8 assay HepG2,MDA-231 KM3,K562,Ca Ski,A549,He La,Jurkat cells by small molecule inhibitors of 48 hours after treatment with the proliferation inhibition rate,initial validation of small molecule inhibitors are able to inhibit the growth of tumor cells,and screened on what kind of tumor cell proliferation inhibition was the most obvious.(2)In HepG2 cells as target cells,CCK-8 method comparison to small molecule inhibitors and EX527,nicotinamide and sirtinol,SIRT1 inhibitors on HepG2 cell proliferation inhibition rate.(3)CCK-8 assay for detection of SIRT1 expression in HepG2 cells,SIRT1 expression of Huh-7 cells and human liver L02 cells by small molecule inhibitors,the positive control drug EX527 48 h treatment proliferation inhibition rate and initial validation of virtual screening of small molecule inhibitors of SIRT1 specific.(4)After a positive control for drugs and small molecule inhibitors EX527 treated HepG2 cells after 48 h were Annexin V-FITC / PI double staining to detect induction of apoptosis of HepG2 cells.(5)Effect of blot 48 h method on the expression of SIRT1 and related proteins in HepG2 cells treated with different concentrations of small molecule inhibitors Western.3.The activity of anti hepatic carcinoma in vivo(1)40 nude mice of 5-7w age were selected and inoculated into tumor.The mice model of human hepatocellular carcinoma HepG2 and Huh-7 nude mice were randomly divided into two groups: control group and administration group,small molecule inhibitor was injected into the abdominal cavity,and the growth of tumor was observed daily.(2)Killed nude mice,tumor removal,and inhibitory rate of tumor was calculated and selected the tumor paraffin.Immunohistochemical method was used to detect the SIRT1,P450 protein expression.Results1.In SIRT1(PDB ID: 4I5I)as a target,based on ADME / T and Chem Properties from Drug Bank database screened 83,745 compounds,and then use surflex Dock screening and molecular modeling to obtain verification SIRT1 inhibitors that are small molecule inhibitors of T;molecular dynamics simulation T binding of small molecule inhibitors bind to the active site after SIRT1 free energy,T prove small molecules with more stable binding SIRT1.2.By CCK-8 proliferation inhibition assay.The results showed that: small molecule inhibitors of t can inhibit the growth of tumor cells,especially high expression of SIRT1 in HepG2 cell proliferation inhibition was the most obvious,and with the other three SIRT1 inhibitors,inhibition of small molecule inhibitors of T on HepG2 was obvious concentration and time dependence.3.Using Annexin V-FITC / PI double staining,flow cytometry results showed that:different concentrations of small molecule inhibitors of T and the positive control drug EX527 have HepG2 cells induced apoptosis,but the small molecule inhibitors ofT treatment apoptosis rate much higher than the positive control EX527.4.Western Blot method confirmed that the expression of SIRT1 protein in HepG2 cells changes with the concentration of small molecule inhibitor T,and small molecule inhibitor T has a certain effect on the expression of related protein.5.With high expression of SIRT1 HepG2 and low expression of SIRT1 in human hepatoma cell line Huh-7 cells as target cells were inoculated into nude mice to establish tumor models in vivo,the daily observation of tumor growth.The results showed that small molecule inhibitors t of nude mice model of HepG2 tumor has more significant inhibitory activity,and tumor of nude mice model of Huh-7 no inhibitory activity.6.Executed in nude mice,tumor removal and the tumor inhibition rate was calculated.The results show that small molecule inhibitors of T of nude mice model of HepG2 tumor was significantly higher than that in nude mice model of Huh-7.7.Select the tumor paraffin section,immunohistochemical method to detect SIRT1,P450 protein expression,results show that: HepG2 xenograft model in nude mice to treatment group of SIRT1 protein expression was lower than that of the control group,and P450 protein expression had no significant difference,Huh-7 nude mice model to the drug group and control group of SIRT1,P450 protein expression showed no significant difference.ConclusionThis topic based on the receptor of SIRT1,select the EX527,nicotinamide and SIRT1 protein binding sites,through high-throughput virtual screening and molecular simulation methods and screening of small molecule inhibitors t not only in vitro tests can effectively inhibit the proliferation of human hepatoma HepG2 cells and induce the apoptosis.In vivo tumor of nude mice model of HepG2 growth also has significant inhibitory activity and Western blot assay confirmed that small molecule inhibitors t can specific hepatocellular carcinoma cell line HepG2 was inhibited SIRT1 expression,so small molecular compound t is expected as antitumor drugs,especially against HCC molecular targeted drugs. |
