Study On The Inhibitory Effect Of Isoquercitrin On Coa Activity Of Staphylococcus Aureus Coagulase

Staphylococcus aureus(S.aureus)is an opportunistic pathogen which can cause various diseases such as skin and soft tissue infections,sepsis,endocarditis,osteomyelitis and sepsis.Antibiotics are currently the main treatment used in clinical treatment of S.aureus infections,mainly by exerting bacteriostatic or bactericidal effects to achieve therapeutic effects,but at the same time also give bacteria a strong selection pressure,triggering the emergence of resistant strains,such as methicillinresistant S.aureus(Methicillin-resistant Staphylococcus aureus,MRSA).The emergence and spread of MRSA present a major challenge for anti-infective treatment.Therefore,there is an urgent need to conduct research on new anti-infective drugs that have a different mechanism of action from traditional antibiotics.Anti-virulence therapiesis a new way to fight bacterial infections,mainly by inhibiting the expression or activity of virulence factors to play an anti-infective effect.Because the virulence factors produced by bacteria are not necessary for bacterial growth,the selection pressure on pathogens will be lower than traditional antibiotics,thereby reducing the development of drug resistance.Coagulase(Coa)is a virulence factor secreted by S.aureus,it can activate prothrombin,convert fibrinogen into fibrin and promote blood coagulation,the formed fibrin network can help S.aureus escape immune phagocytosis.Coa plays an essential role in the pathogenesis of diseases,so inhibitors of Coa may become potential drugs for the treatment of S.aureus infections.In this study,small molecule inhibitors of Coa was constructed using a test tube coagulation experiment.And a natural flavonoid compound,isoquercitrin,was identified from a variety of small molecule compounds.The results of growth curve experiment showed that isoquercitrin(256 ?g/ml)had no effect on the growth of S.aureus Newman in vitro.Test tube coagulation experiments and agarose plate coagulation experiments demonstrated that isoquercitrin inhibits Coa coagulation activity of Coa in a concentration-dependent manner.Thermal Shift Assay proved thatisoquercitrin can significantly improve the thermal stability of Coa protein,indicating that isoquercitrin can directly bind to Coa.In order to elucidate the mechanism of the combination of the two,we further used molecular docking and molecular dynamics simulation to explore the binding mode.Based on the result of molecular modeling,we mutated the possible key amino acid residues,and verified them by test tube coagulation and fluorescence quenching experiments,we finally found that isoquercitrin binds to Coa through Asp-181,Tyr-188,thereby affecting the interaction of Coa with prothrombin.Finally,we constructed a mouse pneumonia infection model to evaluate the therapeutic effect of isoquercitrin on S.aureus pneumonia infection.The results shown that Coa plays an important part in the pathogenesis of S.aureus pneumonia.Isoquercitrin can prolong the survival time of infected mice and relieve pathological damage to the lungs of mice.This study provides a meaningful attempt to develop novel anti-infective drugs with different mechanisms of action from traditional antibiotics,and may provide a new direction for the design and screening of Coa inhibitors.