Screening New Therapeutic Targets And Designing Anti-tumor Peptide-drug Conjugate For Hepatocellular Carcinoma

Objective:Hepatocellular carcinoma(HCC)is one of the malignant tumors with high mortality worldwide,whose incidence rate is about 70%?85%of all cases of primary liver cancer.The main treatment policies of HCC include surgical treatment,radiotherapy and drug treatment,among which the first choice is surgical treatment.However,due to the early stage of HCC without obvious symptoms,more than 50%of patients missed the most treatable stage.Therefore,targeted drug therapy,interventional therapy and radiotherapy are also very important in the control and treatment of HCC.The development of HCC has experienced many complex processes,including the change of intracellular signal pathway,the change of tumor proliferation metabolism system and the formation of local tumor microenvironment.Based on the changes of gene expression profile and intracellular signaling pathway in the genesis of HCC,potential therapeutic targets can be selected.According to the protein crystal structure of HCC therapeutic target,we can design the targeted drugs.According to the theory of drug receptor,the corresponding target drugs can be designed based on the 3D structure of the target protein or its ligand.Based on the peptide mimics of ligands,the target peptides that are compatible with specific proteins can be obtained by using the computer simulation design technology to carry out reasonable structural modification.After the design of the synthesis route,the target peptide compounds can be obtained by chemical synthesis.The purpose of this research is to screen the potential therapeutic targets of HCC,and further design the targeted peptide coupled drugs that can be combined with this target.The main contents are divided into the following two parts:(1)Based on the big data analysis of biological information,the new targets of HCC treatment were selected:the candidate targets CTAG2 and PRSS3 were selected by analyzing the data of HCC differentially expressed genes and the correlation of clinicopathological parameters;(2)Peptide-drug conjugate(PDC)design and activity research of targeted prss3:Based on 3D spatial structure of PRSS3,the targeted peptide WZ-1 was obtained by computer-aided drug design.The synthesis route of peptide WZ-1 coupled with nitrogen mustard benzoate was designed,and the PDC targeting prss3 was synthesized.Methods:1.Transcriptome data acquisition and differential gene screening of HCC:RNA-Seq data and corresponding clinical data used in this study were downloaded from TCGA database,including 374 HCC samples and 50 pairs of paracancerous matched samples.EdgerR downloaded from Bioconductor is used to screen differential expression genes(DEGs)RNA differential expression in HCC and paracancerous tissues in TCGA database were analyzed.Then,R language was used to visualize the differentially expressed genes.Volcano map and thermogram were plotted.2.Construction of PPI:By analyzing the differential genes with STRING database,a PPI network was constructed to obtain the corresponding TSV files;The network analysis was carried out by using the software of Cytoscape,the TSV file was imported,the origin node and the target node were set,and then four topological analysis methods CC?DMNC?MNC?degree were adopted to building the sub-networks.Finally the candidate target genes CTAG2?NTS?GNG4?GAST?PRSS1and PRSS3 were obtained.3.Correlation analysis of candidate target genes expression and clinicopathological parameters:the correlation of candidate target genes expression with poor prognosis and clinical case parameters was analyzed by TCGA database,and the expression of CTAG2in tumor tissues and adjacent tissues of patients with HCC was detected by immunohistochemistry.4.Gene set enrichment analysis:c2.cp.kegg.v6.1.symbols.gmt data sets were obtained from MsigDB data base of GSEA website.According to the expression of CTAG2/PRSS3,statistical analysis of default weighted enrichment of RNA-seq data from tumor tissues of patients in TCGA LIHC was carried out.The main parameters of enrichment results were NES>1.5,P<0.05.5.Computer aided molecular drug design:based on 3D spatial structure of candidate target PRSS3,binding site of ligand and PRSS3 was automatically recognized by MOE software to determine the key anchor points.Then,the rational drug design was carried out through the optimization of peptide conformation,molecular docking and binding energy analysis.6.Fmoc solid-phase synthesis:Using insoluble solid resin as carrier,peptide targeting PRSS3 was synthesized from C-terminal to N-terminal by SPPS method.Fmoc was used to protect the amino parts of Thr?Gly?Pro?Cys?Arg?Leu?Asp?Ile,then prolonged peptides by using the characteristics of Fmoc which is easy to be removed under weak base condition.Using Fmoc-e-acp-oh as raw material,through linear synthesis,the peptide sequence continues to condense,and then reacts with nitrogen mustard benzoate to form the final product.Finally,the peptide wz-1-biphenylbutyrate-n-mustard targeting PRSS3 was obtained after treated with resin cracking liquid.7.RP-HPLC method for determination the content:The content of peptide WZ-1was determined by RP-HPLC,the sample of which was studied by linear relation test,stability test,precision test.8.Cell vigor detection:MTT assay was used to detect the killing effect of peptide wz-1 biphenylbutyrate nitrogen mustard on tumor cells.9.Statistical analysis:Kaplan Meier curve(log-rank test)was used for survival analysis,and univariate and multivariate analysis were used in Cox regression analysis.Immunohistochemistry data was analyzed with Wilcoxon rank sum test.The correlation between the expression level of CTAG2 and PRSS3 and the clinicopathologicalparameters of HCC was analyzed by Pearson Chi square(?~2)test or Fisher exact test.Allthe above statistical tests were completed with SPSS software version 16.0(SPSS,Inc.,Chicago,IL,USA).All the analysis were significant difference(P<0.05).Results:1.Screening new therapeutic targets of HCC based on big data analysis of biological information(1)Data analysis of differential genes and determination of candidate target genes:The results showed that there were 4750 up-regulated DEGs in tumor tissues and DEGs of paracancerous tissue of patients with liver cancer in TCGA database.The results showed that these DEGs were closely related to transcriptional regulation and transcriptional co suppressor activity.The results of PPI analysis indicate that six genes including CTAG2,NTS,GNG4,GAST,PRSS1 and PRSS3 can be used as candidate target genes for further study.(2)Correlation between the expression of CTAG2 and PRSS3 with clinicopathological parameters in HCC.The expression of the six candidate target genes in TCGA database was significantly higher than that in the adjacent tissues.According to the result of univariate Cox regressive analysis,the TNM stage(P<0.001),AFP high expression(P=0.010)and CTAG2 high expression(P=0.035)was considered to be significantly related to the prognosis of patients,which indicated that the overall survival time of patients in ctag2 highly expressed group is marked shortened.Furthermore,The expression of CTAG2 was significantly correlated with age(P=0.003),T stage(P=0.028),TNM stage(P=0.028)and AFP stage(P=0.045).The expression of PRSS3 was significantly with age at diagnosis(P=0.012)but was not correlated with gender,T stage,TNM stage and AFP stage.(3)CTAG2 and PRSS3 related-KEGG signaling pathway analysis:GSEA enrichment was applied to analyze KEGG signal pathway,CTAG2 overexpression samples enriched to homologous recombination,cell cycle,DNA replication,base excision repair,pentose phosphate pathway,mismatch repair,nucleotide excision repair.PRSS3 overexpression group was significantly enriched in KEGG signaling pathways including DNA replication,cell cycle,the intestinal immune network generated by IgA,mismatch repair and cytokine receptor interaction.The results suggested that the KEGG signaling pathway of CTAG2 and PRSS3 might be related to the abnormal proliferation of tumor cells.2.Peptide coupled drug design and activity research of target PRSS3.(1)Determination of the key anchor point of interaction between PRSS3 and its protein inhibitor:MOE was used to refine the structure of the PRSS3 protein complex and to identify the site of interaction between the inhibitor and PRSS3.The analysis result showed that the Pro13?Cys14,Arg15,Asp17 residues of bovine trypsin inhibitor variants was the key point of the interaction,which meant that the inhibitor was embedded into the binding pocket of PRSS3 by hydrogen bond and hydrogen ion bond of these amino acids.(2)Design and molecular docking of PRSS3 targeting peptide:The peptide fragment of the key binding site between PRSS3 inhibitor and receptor was used as the template of peptide sequence(TGPCRADI).According to that,18 peptide sequences containing 8 amino acids were designed.Using MOE to connect the designed peptide with PRSS3 receptor,the results showed that the binding of 6 simulated peptides were significantly better than that of template peptide sequence(S score<-40).Among the 6peptides,the peptide WZ-1 can be well embedded in the binding pocket of PRSS3 with the lowest S-score,so the peptide sequence was selected for synthesis.(3)Synthesis route design of peptide WZ-1 coupling drug:using Fmoc combinational strategies,the carboxyl groups of resin and Ile were covalently linked.Then the compound was condensed with the carboxyl group of Asp.The peptide chain was extended according to the cycle of deprotection,condensation and washing.The condensation reaction was carried out by the active ester with the combination of HOAt and DIC as coupling agents.Finally,linker and nitrogen mustard benzoate were linked to peptide WZ-1 by linear synthesis to obtain peptide coupled drugs.(4)Synthesis of peptide WZ-1 coupled drugs:We selected Wang resin as the carrier in the solid-phase reaction and defined that the ratio of amino acid to resin was 4:1.The peptide WZ-1 biphenylbutyrate nitrogen mustard was synthetized by amino acids with protective groups including Fmoc-Ile-OH?Fmoc-Asp(otBu)-OH?Fmoc-Cys(Trt)-OH?Fmoc-Thr(tBu)-OH.MS was used to confirm the structure and 96%purity was detected by analytical liquid phase.(5)Methodological validation of the content of peptide WZ-1 biphenylbutyrate nitrogen mustard:the test solution of polypeptide WZ-1 biphenylbutyrate nitrogen mustard was stable within 48 hours at room temperature;Linear regression of peak area with sample concentration showed that the linear relationship between peak area and concentration(0.50?1.5mg/ml)was good(linear regression equation:y=8680x+109.29,the correlation coefficient r:0.9996).(6)Cytotoxicity of peptide WZ-1 biphenylbutyrate nitrogen mustard:MTT results showed that the peptide WZ-1 biphenylbutyrate nitrogen mustard had cytotoxic effect on human cancer cell line.Conclusion:1.Two candidate target genes,CTAG2 and PRSS3,were screened by data mining combined with correlation study of clinicopathological parameters.2.The high expression of CTAG2 is closely related to the poor prognosis of HCC.However,there is no 3D structure of CTAG2 available at present.PRSS3 is highly expressed in HCC,and the related KEGG signal pathway analyzed by GSEA is related to tumor proliferation.3.Through virtual computing,the peptide wz-1 combines the 3D spatial structure of PRSS3 with key anchors such as Pro,Cys and Arg.4.The peptide WZ-1 coupling chlorambucil is a PDC compound whose targeting site is peptide WZ-1.5.The synthesized peptide WZ-1-biphenylbutyrate nitrogen mustard can be used as a new anti-tumor targeting drug candidate for its cytotoxic effect on hepatoma cell.