Virtual Screening, Synthesis And Application Of Lead Compounds Targeting To Penicillin-Binding Protein 1b (PBP1b) Of Escherichia Coli

Escherichia Coli is a kind of pathogens-common seen in food and clinics. Penicillin-binding protein 1b (PBP1b) of E. coli-plays a key role in cell wall synthesis and its survival. In this study, molecular docking software DOCK6.5 was used for the virtual screening of lead compounds targeting to E. coli. The lead compounds with new structures and high affinities were synthesized and then subjected to activity and mechanism research. Finally, the lead compounds with high antibacterial activities were used for the preservation of chilled meat.Results:1. DOCK6.5 was used for the virtual screening of leads targeting to PBPlb of E. coli against the ZINC database containing 1,040,000 compounds. Grid score was adopted for the first round of screening and 60,000 compounds with scores below -30 kcal/mol were obtained. These compounds were then subjected to the second round of screening where amber score was used for the evaluation of binding affinity. Two hundred compounds with amber scores below-20 kcal/mol were obtained. After structure and interaction analysis, four compounds, namely ZINC03866448, ZINC02496118, ZINC13276500, ZINC 13277245, were selected as lead compounds for further studies..2. According to the principle of operable and economic in the synthesis, we selected one of candidate compound to synthesize. The synthesis included three stages, namely substitution, deprotection, and adding anhydride. The lead compound and three derivatives were synthesized and then purified by a silica gel column. Nuclear magnetic resonance and mass spectrometry were used to determine the molecular weight, structure and other information of the four compounds.3. Constant broth dilution method of Clinical and Laboratory Standards Institute (CLSI) was adopted to test the antibacterial activities of the four compounds. These compounds showed strong antibacterial activities and their minimal inhibitory concentrations (MICs) against five kinds of test bacteria, such as Escherichia Coil, Pseudomonas Aeruginosa, Listeria Monocytogenes and Staphylococcus Aureus, were 175-225 μg/mL. The antibacterial mechanism was then researched by PBP1b-binding assay. The fluorescence-labeled lead compound could bind to penicillin-binding protein 1b of E.coli and make the PBP1b band in SDS-PAGE gel emitting fluorescence at 590 nm of light. The antibacterial mechanism was further verified by cell wall detection. With TEME observation, the cell wall of E.coli was found to be damaged after lead compound’s treatment. The results indicated that the lead compound could bind to PBPlb of E. coli and interfere with its cell wall synthesis.4. The lead compound was then used for the preservation of chilled meat. The total number of colonies, TVB-N, pH, TBA value, hydrogen sulfide, color, and other indicators were tested. Potassium Sorbate was used as positive control. MTT assay was also performed to evaluate the potential toxicity of the lead compound on liver cells. The result showed that the lead compound was better than Potassium Sorbate on the control of total number of colonies and on the keep of color value. However, there were no significant differences on the indicators of TVB-N, pH, drip loss, and H2S. The lead compound had poor antioxidant activity as well. MTT assay showed that the lead compound had no toxic to liver cells.