Screening And Identification Of A Novel Small-molecular TNFβ Inhibitor

Cloning, Expression and Identification of Recombinant Human Tumor Necrosis Factor βObjective Construct different prokaryotic expression vectors of TNFβ and induce the expression of the targeted protein, and build a basis for the interaction between hTNFβ and the screened small molecular inhibitor.Methods Construct two different prokaryotic expression vector:PAYZ-STII-hTNFβ-6his and pET44-hTNFβ-6his.Tranform them into 16C9 and BL21(DE3) competent cells respectively, and then induce the expression of hTNFβ via different methods.Detect and identify the target protein via SDS-PAGE and Western Blot technique.Compare the total amount,the soluble part and the inclusion body proportion of the expressed protein product.Denature the incusion body with 8M urea and renature the protein through dilution renaturation technique successfully.Test the biological activity of the purified hTNFβ on L929 cell line via MTT.Results SDS-PAGE and Western Blot results suggested that a large proportion of the expressed product was inclusion body, the soluble proportion of the protein was less.Use the method of dialysis to renature the denatured protein by reducing the concentration of urea step by step.The protein begun to precipitate during the process of dialysis and the acquired amount of renatured product was very little.The protein finally successfully renature via dilution renaturation.Examined its biological activity on L929 cell line.The renatured protein can induce the apoptosis of L929 cell.Conclusion Succeed in constructing PAYZ-STII-hTNFP-6his and pET44-hTNFβ-6his and obtain hTNFβ with biological activity via denaturation and renaturation technique of the inclusion body.Screening and Identification of A Novel Small-molecular TNFP InhibitorObjective To explore a novel and highly specific small-molecular TNFβ inhibitor by the use of computer-aided virtual screening and cell-based assays in vitro.Methods Computer-aided drug design and virtual screening were used to design and identify chemical compounds that target TNFP based on the crystal structure of the TNFP-TNFR1 complex. The effect of the small-molecular compound against TNFβ-induced cytotoxicity of L929 cell was detected by MTT assay, and the efficacy of the compound to inhibit TNFβ-induced apoptosis of L929 cell was determined by flow cytometry assay. The impact of the compound on L929 cell cycle was examined by Propidium Iodide (PI) staining and flow cytometry, and the influence of the compound on TNFβ-triggered signal pathway was analyzed by Western Blot and ULtra VIEW VOX 3D Live Cell Imaging System.Results 965 compounds were identified to closely mimic the spatial structure of the docking template, and 105 compounds among them were selected as leading compounds based on their binding energy, structural diversity and potential for future drug development. In further study, those compounds were examined their ability to inhibit TNFβ-induced cytotoxicity in L929 cell line with three different doses. Currently available results suggested that No.35 compound (named as C35 thereafter) could effectively inhibit TNFβ-induced cell death in a dose dependent way, and the Half-maximum Inhibition Concentration (IC50) was 8.19μmol/L. Furthermore, C35 had lower cytotoxicity and minimal effect on L929 proliferation. Here we further reveal that C35 could affect TNFβ-induced apoptotic pathway by blocking the activation of Caspase 3, and markedly reduced L929 cell apoptosis induced by TNFβ.Conclusion A novel TNFβ small-molecular inhibitor was identified by combining computer-aided virtual screening with functional assays, and it could block TNFβ-triggered apoptotic pathway and efficiently inhibit the cell death induced by TNFβ.