Objective To explore a novel and highly specific small-molecule TNFβinhibitor by using computer-aid?ed 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 targeted TNFβbased on the crystal structure of the TNFβ-TNFR1 com?plex. The effect of the small-molecule compound against TNFβ-induced cytotoxicity of L929 cells was detected by MTT as?say, and the efficacy of the compound to inhibit TNFβ-induced apoptosis of L929 cells was determined by flow cytometry as?say. 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 assay and Ultra VIEW VOX 3D Live Cell Imaging System. Results No.35 compound (named as C35 thereafter) could effectively inhibit TNFβ-induced cell death in a dose dependent manner, 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 revealed that C35 could affect TNFβ-induced apoptotic pathway by blocking the activation of Caspase 3, and markedly reduce L929 cell apoptosis induced by TNFβ. Conclusion A novel TNFβsmall-molecule inhibitor was identified by combining computer-aided virtual screening with functional assays, and which could block TNFβ-triggered apoptotic pathway and efficiently inhibit the cell death in?duced by TNFβ.

To improve the expression level and catalytic efficiency of (R)-carbonyl reductase from Candida parapsilosis in Escherichia coli, we optimized the mRNA secondary structure of (R)-carbonyl reductase gene in translation initiation region (from +1 to +78), and constructed the corresponding variant. The formation of hairpin structure was significantly reduced and the Gibbs free energy was dramatically decreased from -9.5 kcal/mol to -5.0 kcal/mol after optimization. As a result, the expression level of (R)-carbonyl reductase in the variant was increased by 4-5 times and its specific activity in cell-free extract was enhanced by 61.9% compared to the wild-type strain. When using the whole cells as catalyst and 2-hydroxyacetophenone as substrate with a high concentration of 5.0 g/L, the variant showed excellent performance to give (R)-1-phenyl-1, 2-ethanediol with optical purity of 93.1% enantiomeric excess and a yield of 81.8%, which were increased by 27.5% and 40.5% respectively than those of the wild-type. In conclusion, the optimization of mRNA secondary structure in translation initiation region can overcome the steric hindrance of translation startup, promote translation smoothly to acquire high expression of target protein, and favor protein folding correctly to efficiently improve the enzyme specific activity and biotransformation function.
Alcohol Oxidoreductases ; biosynthesis ; chemistry ; genetics ; Base Sequence ; Biocatalysis ; Candida ; enzymology ; Catalysis ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Mutant Proteins ; genetics ; Nucleic Acid Conformation ; Peptide Chain Initiation, Translational ; RNA, Messenger ; chemistry ; Recombinant Proteins ; biosynthesis ; genetics ; Stereoisomerism