Screening Of Focused Compounds Library To Identify Novel Inhibitors For Respiratory Syncytial Virus

Human respiratory syncytial virus(hRSV)is the most important pathogen of acute lower respiratory tract infection in infants under one year of age all over the world.RSV also can infect elderly,immunocompromised individuals and patients after organ transplantation,leading to high fatality rate.In 1956,RSV was first discovered in chimpanzee rhinitis secretions and the next year the same virus was isolated from an infant infected with RSV.The virus can lead infected cells to produce unique cell fusion,so called it "respiratory syncytial virus".RSV infects about 30 million children every year,it especially has the greatest impact on developing countries.To this day,there is no efficacious small molecule drug or vaccine is available to treat or prevent disease.So developing an effective antiviral drug and vaccine are the most urgent work for researcher.Cyclopamine(CPM)is a kind of allosteroid alkaloid,which has a good inhibitory effect on various tumors.In our previous study,we found that CPM acts as a potent inhibitor of RSV replication,but CPM can also inhibit the activity of Hedgehog pathway.It has teratogenic effect that largely preclude its development as a pediatric drug for the treatment of RSV infections.In forthcoming study,our research group has carried out a series of modifications on the basis of CPM structure and designed 14 analogues,according to the different positions of the compound and the different function groups,these compounds are divided into four series.S1-1,S1-2,S1-3,S1-4 are first series,S1-5,S1-6,S1-7,S1-8 are the second series,C2-2,C2-3,C2-4 are the third series,S2-4,S2-5,S2-6 are the fourth series,we call these 14 analogues focused compounds library,and then selected an effective novel compound with stronger inhibiting effect on RSV and less inhibiting effect on Hedgehog pathway.In order to find effective analogue,we have established a method to detect the antiviral effect of the compound rapidly based on the detection of luciferase expression,the firefly luciferase gene was inserted between the P-protein and M-protein genomes of RSV-Long,and the influence of 15 compounds on the replication of RSV-Luc was detected by detecting the expression of luciferase,therefore,the expression level of firefly luciferase was used to indicate the level of virus replication,and the antiviral function of compounds were evaluated indirectly.By screening the antiviral effects of 14 analogues,we found 6 analogues of IC50<5?M:Sl-1,S1-2,S1-3,S1-4,S2-4,S2-5.At the same time,we tested the inhibitory effect of 14 analogues on Hedgehog signaling pathway based on the double-report gene detection method,we found 3 analogues of IC50>5?M:S 1-2,S1-3,S2-5.Through the comprehensive evaluation of the inhibition coefficient ICrh,we screened the effective analogue S2-5.In our research,we measured the antiviral effect of CPM with IC50 is 0.08?M,and S2-5 is 2.25?M,after modification,the effective analogue antiviral effect was 28 times of CPM.The IC50 of the anti-hedgehog pathway of CPM is 0.07?M,and S2-5 is more than 20?M,the effective analogue anti-hedgehog pathway effect was 285 times of CPM after modification.The modification greatly reduced the inhibitory effect of the compound on Hedgehog signaling pathway,there is also a certain level of RSV replication.Minigenome assay(minigenome assay)verified CPM and the effective analogue S2-5 inhibit minigenome downstream gene expression,and 151 M2-1 site is resistant to two compounds.These results indirectly confirmed the CPM and S2-5 have similar antiviral molecular mechanism,which are targeted M2-1 protein.To conclude,we designed and screened a potent inhibitor against respiratory syncytial virus analogue and reducd its impact on Hedgehog pathway at the same time.Our study provides the theoretical foundation for the further research of HRSV replication mechanism,and the best candidate drugs for clinical treatment.