| H5N1 highly pathogenic avian influenza virus,belongs to Orthomyxoviridae,influenza virus genus,is a subtype of influenza A virus,which can cause zoonotic diseases.It is characterized by strong infectiousness,spreading rapidly and high pathogenicity.The H5N1 avian influenza virus was first detected in domestic geese in Guangdong,China in1996,subsequently erupted around the word,such as China,Thailand,Vietnam,Indonesia,Canada and Nigeria,which have caused serious economic losses to poultry industry in many countries.The fatality of sick chickens is as high as 100%.After human infection with H5N1 avian influenza virus,the mortality is as high as 60%,which poses a serious threat to human health.At present,H5N1 highly pathogenic avian influenza is mainly controlled by vaccination,but due to the constant occurrence of H5 virus mutation,vaccines are unable to provide timely protection,and lacking therapeutic antibodies for human against H5 subtype avian influenza virus.Therefore,it must rely on drugs to control.Currently,resistant strains of neuraminidase?NA?inhibitors?oseltamivir and zanamivir?that are effective drugs recommended by the WHO for the H5N1 avian influenza,have emerged.Therefore,it is an important issue to strengthen the study of new drugs to treat H5N1 avian influenza virus,especially the new targets and clear mechanisms of drugs or lead compounds.In recent years,the research of HA protein inhibitors for the prevention and treatment of H5N1 virus infection has attracted much attention.HA protein plays an important role in virus binding,fusion and invasion of cells,and it has obvious advantages if it can block the initial entry phase of viral life cycle and interrupt the replication process.Previous studies had found that the diosgenin play an important role in the entry of H5N1 influenza virus into the cell.In this study,several compounds were designed and synthesized on the basis of the active compound XC-27,and the activity of the anti H5N1 avian influenza virus was further studied,and the mechanism was discussed.This paper used MTT assay and plaque assay found the compound XC-27-1,XC-27-2and UA-Nu-ph-5 could protect MDCK cells at an early stage and inhibit H5N1 avian influenza virus entry.By constructing a high-throughput screening system for wild-type H5N1 pseudovirions with three plasmids,we found that the compounds XC-27-1 and XC-27-2 have a greater inhibitory activities to H5N1 pseudovirus,the IC500 values were12.76±7.01?M and 18.47±1.82?M,respectively.Further neuraminidase assays showed that the two compounds weakly inhibited the NA at a high concentration of 400?M.The two compounds do not inhibit H5-type hemagglutinin-mediated hemagglutination.The results showed that the two compounds could selectively act on HA protein,but the binding site was not located at the sialic acid receptor-binding domain?RBD?.To further elucidate the binding of compounds to the H5N1 avian influenza virus HA protein,we constructed the structural model of HA receptor based on the PDB database of HA protein crystal structure and the HA gene sequence of wild-type parental strain,and predicted the compounds possible binding mode and the docking of amino acid residues by molecular docking method,found that I48,L329,T331,W367,T387,I391,V394,T395 are possible related residues for molecular docking.Then,the site-directed mutagenesis results showed that inhibitory activity of XC-27-1 and XC-27-2 on H5N1 subtype virus was significantly decreased after mutation of amino acid residues I391 and T395,and the IC50 increased more than 5 times,indicating that the binding may be located in the HA stem and globular head adjacent to the region,I391,T395 amino acid residues as the main binding site.Furthermore,the binding ability between the compounds and the HA protein of H5N1subtype virus were studied by Bio-Layer Interferometry?BLI?.The results showed that the compounds could specifically bind to HA protein,and the binding affinity constants?KD?were 2.57×10-4?3.67×10-4,respectively. |
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