Virtual Screening And Anti-mrsa Activity Of Small Molecular Inhibitors Against PBP2A

Background:PBPs are some membrane proteins located on the bacterial cell membrane,which catalyze the trans peptide reaction in the construction of cell wall,so they play an important role in the bacterial cell cycle.Its normal existence is the necessary condition for bacteria to maintain normal morphology and function.β-lactam antibiotics inhibit the biosynthesis of bacterial cell wall by binding with PBPs,causing bacterial cell death and thus play a bactericidal role.In addition to four types of PBP（PBP 1,PBP 2,PBP 3and PBP 4）,MRSA can also express penicillin binding protein 2a（PBP2a）.The affinity between PBP2a andβ-lactam antibiotics is very low,and many kinds of antibiotics can’t bind with PBP2a.When the high-affinity PBPs are inhibited,PBP2a can continue to perform the transpeptidase function,complete cell wall synthesis,and maintain the growth and reproduction of bacteria,thus exhibiting multi-drug resistance.Therefore,it is urgent to develop potential specific PBP2a inhibitors to overcome the multi-drug resistance of most current antibiotics.In recent years,virtual screening for drug discovery is becoming an essential tool in assisting fast,cost-efficient lead discovery and optimization.Rational and structure-based drug design is more efficient than the traditional method of drug discovery because this method examines the molecular basis of a disease and uses the three-dimensional structure of the biological target.Multiple virtual screening strategies have been widely used in the discovery of antimicrobial agents.Aim:In this study,we employed a strategy that combined virtual screening with biological evaluation,aimed to discover novel inhibitors of PBP2a from a commercial database and provide anti-MRSA drug lead compounds targeting PBP2a.Methods:In vitro,a hybrid virtual screening method constructed by Discovery Studio 2017,consisting of drug-likeness evaluation（Lipinski’s Rule of Five and ADMET）and rigid（Lib Dock）and semi-flexible（CDOCKER）docking-based virtual screenings,was used for retrieving novel PBP2a inhibitors from commercially available chemical databases.To determine whether the screened compounds were the most optimal compounds against PBP2a,antibacterial activity and cytotoxicity studies were performed.The affinity of the final hit compounds to bind to PBP2a was further confirmed by surface plasmon resonance（SPR）experiments and molecular dynamics（MD）simulation.And the combination mode between hit compound and PBP2a was also analyzed.Hit 2,with best anti-PBP2a activity,was selected to study its antibacterial effect on MRSA clinical isolates.The MIC and MBC values of hit 2 on these MRSA clinical isolates were detected.Intluences of hit 2 on m RNA expressions related to the cell wall synthesis and lysis of MRSA were measured.In vivo,MRSA 05 clinical isolate strain was used to infect mice to establish lethal model and sublethal model.The protective effect and of hit 2 on the survival of mice challenged with a lethal was studied.The effect of hit 2 on the amount of bacteria in the blood of mice infected with sublethal MRSA 05.The acute toxicity of hit 2 in mice observed after intraperitoneal injection.And the pathological changes of important organs were detected by HE stainingReults:In vitro,11 compounds were selected from the final hits and subsequently shifted to experimental studies.Among these compounds,hit 2,hit 3,and hit 10 exhibited excellent anti-MRSA activity（MIC ranging from 2 to 64μg/m L）and weak toxicity(IC₅₀>20μΜfor L-02 cell line)in vitro.SPR and MD experiment results suggested that the binding of the three hit compounds to PBP2a were favorable,with a K_Dvalue quantity class at 10^-7power M.An inter-complex interaction study showed that all hit compounds adapted well to the allosteric site of the PBP2a protein.MIC and MBC results showed that hit 2 had universal applicability to MRSA clinical isolates in vitro,and had the best antibacterial effect on MRSA 05.The MIC₅₀,MIC₉₀,MBC₅₀and MBC₉₀value of hit 2 on MRSA were 3.4、9.6、44.1 and 126.1μg/m L respectively.The time bactericidal curve showed that hit 2 could kill MRSA 05 in a dose-dependent manner.Electron microscopy and RT-PCR showed that hit 2 could break MRSA cell wall synthesis without affecting the expression of peptidoglycan synthesis related genes（agr、fem X、mec A、pbp2、pbp3、pbp4、murC、murE、vra R、mrp）,phosphoteichoic acid synthesis related genes（tag A、tag B、tag D、tag G、tag X）and cell wall hydrolysis related regulatory genes（ssa A、sae R、murD、sar A、lyt M）,indirectly confirmed that its antibacterial mechanism may be related to the binding of PBP2a.The mouse model of MRSA 05 infection was established in vitro.It was found that the concentration of 3.0×10¹⁰CFU/kg of MRSA 05 could be used as the sublethal model mice,and the concentration of 7.0×10¹⁰CFU/kg could be used as the lethal model mice.The antibacterial test in vivo confirmed that hit 2 had a significant protective effect on mice challenged with lethal dose of MRSA 05,and could significantly reduce the amount of bacteria in the blood of mice infected with sublethal dose of MRSA 05,and its antibacterial ability was equivalent to that of vancomycin.Acute toxicity test confirmed that hit 2 had good therapeutic safety,and the therapeutic concentration was far less than the median lethal dose.HE staining showed that some important organs such as the heart,liver,spleen,lung and kidney of mice had no obvious damage under the concentration of 20mg/kg.Conclusion:The hybrid screening strategy based on virtual screening and activity evaluation can be an effective method for screening PBP2a inhibitors.Hit 2 is a promising inhibitor of PBP2a,which can be used as the anti-MRSA drug lead compound.These results provide important information for the further study of novel anti-MRSA agent.