Abstract: Abstract: Objective To explore a novel and highly specific small-molecule 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 TNFβ based on the crystal structure of the TNFβ-TNFR1 complex. The effect of the small-molecule 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 8.19μM. 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-molecule 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.

Key words: Key words: TNFβ, Small molecular inhibitor, Cell apoptosis, Signal pathway, Cell cycle