Screening Of Antiviral Inhibitors Based On The Activity Of Zika Virus NS2B-NS3 Protease

Zika virus?ZIKV?is a single-strand positive-strand RNA virus transmitted mainly by mosquitoes.The virus can cross the blood-brain barrier,causing some neurological complications,congenital Zika virus syndrome and irreversible neurological damage.With the rapid spread of the virus in the world,it has attracted the attention of the international health organization.However,there are no specific available drugs for zika virus,so it is urgent to seek anti-zika drugs.The main function of ZIKV NS3 protease is to cleave the polyprotein formed by the virus into independent functional proteins,which are necessary for viral replication.Therefore,it is a potential antiviral drug target.In this study,antiviral inhibitor screening based on ZIKV NS2B-NS3 protease activity was conducted.The activity of the protease was detected in vitro by using fluorescence resonance energy transfer method?FRET?,and a high-throughput screening method was established accordingly.The compounds stored in our laboratory were screened to obtain some compounds with significant inhibitory effect on the protease.One compound with prominent inhibitory effect was tested for antiviral study in cell and animals.The specific experimental results are as follows:1.Soluble expression of ZIKV NS2B-NS3 protease and high-throughput screening of its compound libraryIn this study,the expression vector,competent strain and induction temperature and time were explored to obtain the optimal soluble expression conditions,and a large amount of soluble ZIKV NS2B-NS3 protease was purified by Ni²⁺affinity chromatography.Then,In this study,the activity of NS2B-NS3 protease was detected by FRET method,and S/B=13.60 and Z'=0.51were calculated,which met the requirements of high-throughput screening and could be used to screen compound libraries.A library of more than 4,000 small molecule compounds preserved in the laboratory was screened.Through the primary screening and rescreening,the false positive compounds caused by inherent fluorescence and the error-loadings were eliminated,and ten small molecule inhibitors with inhibition rates higher than 80%were finally obtained.The above ten compounds were tested by in vitro enzyme activity inhibition on DENV,JEV and WNV NS3 proteases,among which eight compounds have good inhibitory effects on DENV and JEV proteases,while only three compounds had specific inhibitory effect on WNV protease.On the basis of this experiment,we selected a compound cs-004/04003052 for further study.The following results are obtained:2.Enzyme kinetics study of compound CS-004/04003052 and its Specificity in inhibiting ZIKV proteaseThe reaction rate-substrate concentration double reciprocal curve was obtained by diluting the compound at different concentrations and measuring the reaction rate of the protease under different substrates concentration.According to the Michaelis equation,the inhibition constant K_i value of the compound was 0.1346±0.0243?M/L.Intersection on X axis of lines indicated that the compound and the substrate were competitively inhibited.The enzyme binding constant K_d value of the compound was determined by Microscale thermophoresis Test?MST?test data to be 12.262±5.549?M.Then,Compound CS-004/04003052 was tested for inhibition of enzyme activity on ZIKV,DENV,JEV NS3 protease and PEDV NSP5 protease in vitro,respectively.It was found that the compound CS-004/04003052 showed high specificity only for ZIKV NS3protease.3.Detection of antiviral effects in cellCompound CS-004/04003052 was diluted to 200?M,100?M,50?M,25?M,12.5?M,6.25?M,3.125?M,and 1.59?M,respectively,and added to Vero,BHK-21,Hela cells,after 48 h,add Cell Titer-Glo Luminescent Cell Viability Assay reagent,then add the volume of reagent,read and save the data,analyze the data,and calculate the CC50.Combined with the measured IC50,the compound is still less toxic to the three cells at higher concentrations.The plaque assay showed that the compound could inhibit the titer of the virus;the results of indirect immunofluorescence assay and Western blot showed that the compound can inhibit the protein expression of the virus.Meanwhile,we also explored whether the compound had inhibitory effect on the replication of Japanese encephalitis virus in cell.The results of plaque assay and Western blot test indicated that the compound had no inhibitory effect on the replication of JEV in cell.4.Antiviral effect in animalsCompound CS-004/04003052 was injected into AG129 mice via the tail vein to study the anti-Zika virus effect in vivo.The results showed that the compound can delay the death time of mice infected with Zika virus.The results of HE section staining showed that the pathological symptoms in the drug-used group were much slower than those in the virus group in the brain,testis,ovary and spleen.Meanwhile,real-time PCR and plaque assay were performed to confirm the results.The virus titer in the internal drug group was significantly lower than that in the control group.Comprehensive analysis showed that compound CS-004/04003052 inhibited Zika virus at the living level of animals.