Screening Of Inhibitors Of African Swine Fever Virus DUTPase And Preliminary Evaluation Of Its Enzymatic Activity

African Swine Fever(ASF)is a contagious viral disease caused by African Swine Fever Virus(ASFV),which can infect domestic and wild boars of all ages,with an acute infection mortality rate of up to 100%,bringing unprecedented mortality to the swine industry Challenge,so far there is no vaccine or specific drug for ASF.Therefore,effective anti-ASF virus drugs are needed.ASFV dUTPase(dUTPase)is a homotrimer composed of E165 R,dUTPase can degrade 2’-deoxyuridine 5’-triphosphate(dUTPase)in the cytoplasm,reducing the error of uracil incorporation into viral DNA,which is beneficial ASFV propagates infection and replicates in porcine macrophages.Therefore,the screening of drugs that inhibit ASFV dUTPase activity is of great significance for the comprehensive prevention and treatment of ASF.In this study,according to the binding sites of ASFV dUTPase and substrates,the FDA library of ASFV dUTPase was virtual screened by watvina.Combined with the docking results,drugs with better binding to the active site of ASFV dUTPase and higher docking scores were selected as ASFV candidate inhibitors of dUTPase,Secondly,through molecular dynamics simulation,RMSD,RMSF,Rg,SASA,H-bone,FEL and DCCM were calculated and analyzed,and then the binding stability and dynamic interaction between the inhibitor and ASFV dUTPase were analyzed.Boltzmann surface area(MM/PB(GB)SA)method was used to calculate the binding free energy of ASFV dUTPase and the inhibitor,and further evaluate the inhibitory effect of the inhibitor on ASFV dUTPase.Finally,the inhibitory effect of the inhibitor on ASFV dUTPase was verified by in vitro enzyme activity inhibition experiments inhibitory effect.The results of virtual screening showed that didanosine,ganciclovir,cytarabine,ceftazidime and ASFV dUTPase in the FDA drug library have high docking scores,the highest being-10.63Kcal/mol.Second,there are hydrogen bonds and hydrophobic interactions between didanosine,ganciclovir,cytarabine,ceftazidime and ASFV dUTPase.The molecular dynamics simulation results of didanosine,ganciclovir,cytarabine,ceftazidime complex with ASFV dUTPase and free ASFV dUTPas showed that didanosine,ganciclovir,cytarabine and ceftazidime can For stable binding to ASFV dUTPase,the binding free energies are-12.3Kcal/mol,-16.5 Kcal/mol,-26.4 Kcal/mol and-31.2 Kcal/mol,respectively.In vitro enzyme activity inhibition experiments showed that didanosine,ganciclovir,and cytarabine inhibited dUTPase activity by 82%,60%,and 42%,respectively,at a concentration of 100 μm.In conclusion,the ASFV dUTPase inhibitors cytarabine,ganciclovir and didanosine were screened from the FDA drug library in this study.At a concentration of 100 μm,they can inhibit the activity of ASFV dUTPase in vitro.Drug development provides new ideas and lays the foundation for the prevention and treatment of ASF.