Virtual Screening And Antibacterial Activity Of Lead Compounds Targeting To Penicillin- Binding Protein 3 (PBP3) Of P.Aeruginosa

Against the increasingly serious drug resistance of P.aeruginosa(?A) to P-lactam antibiotics, Simulating the bacteriostatic mechanism of β-lactam antibiotics, we have a large-scale virtual screening by targeting to the penicillin-binding protein 3(PBP3) of PA, and obtained approximately 200 lead compounds that have new structure, these lead compounds provided material basis for the development of new antibacterial agents. Selected a representative lead compound to synthesize to verify the results of virtual screening. We also have a preservation experiment to verify the preservation capability of the lead compound to fresh pork. The main conclusions are as follows:1. A large-scale virtual screening was carried out using UCSF dock6.5. The target was the PBP3 of PA. The drug-like subset of the ZINC database which containing 1.04 million small molecules was used for the virtual screening. Finally, we get approximately 200 lead compounds through two round screening of Grid score and Amber score. By querying some database including the American Chemical Abstracts (CA), we find no studies were reported about these compounds’antibacterial ability. What’s more, these new compounds have completely different structure with the β-lactam antibiotics.2. We have an organic synthesis for the lead compound whose serial number is ZINC00310847, and also its five derivatives. The result of the bacteriostatic test indicate that these six compounds all have a certain antibacterial activity to both gram negative and positive, the minimum inhibitory concentration (MC) is between 200μg/mL and 400μg/mL, which is better than the control group of polyphenols (800-1600μg/mL) but is far worse than the control group of ampicillin sodium (12.5μg/mL). The experimental results of MTT indicate that in the concentration range of 0.2-3.2 mg/ml, lead compounds and its derivatives were non-toxic to the mouse hepatocytes.3. The lead compound was labeled with a fluorescent agent and then incubated with PBP3, then have a SDS-PAGE, we found the location of fluorescent band is consistent with PBP3 band, which indicate that the lead compound can be combined well with the PBP3 protein. Draw 1 mL PA that growth in good condition(OD6oo=0.6) into the 250 mL erlenmeyer flask which contain 99 mL MH broth, then add lead compound to the erlenmeyer flask and makes its concentrations can just restrain the growth of PA. Continue to foster the PA in 37℃, shakers for 2 h, then extracted strain, prepare the sample, through TEM observation, we found the cell wall of PA which was handle by the lead compound was broken seriously, this indicate that the lead compound can broken cell wall of PA. Integrated this two experimental results, we can guess that the antibacterial mechanism of lead compound is most likely to interference peptidoglycan biosynthesis by integration with PBP3 closely and resulting in the bacteria’s death by destroying its cell wall.4. The preservation experiment results show that all indicators of lead compound group were significantly better than blank control group(P<0.05), the lead compound group were also good for tea polyphenols group in the indicators of aerobic bacterial count, pH, drip loss, TVB-N, but in the indicators of color value, tea polyphenols is better than lead compound. Use the SPSS software to have a statistical analysis, we find there were no significant difference (P>0.05) between lead compound group and tea polyphenols group in the preservation results.