Study on the Antiviral Activities and Hemagglutinin-Based Molecular Mechanism of Novel Chlorogenin 3-O-β-Chacotrioside Derivatives Against H5N1 Subtype Viruses.

Wan-Zhen Shi,Gao-Peng Song,Ling-Zhi Jiang,Chang-Wen Ke,Ping Xiong,Sheng Wang

doi:10.3390/v12030304

Abstract

The objective of this study was to investigate the inhibitory effect of chlorogenin 3-O-β-chacotrioside derivatives against H5N1 subtype of the highly pathogenic avian influenza (HPAI) viruses and its molecular mechanism. A series of novel small molecule pentacyclic triterpene derivatives were designed and synthesized and their antiviral activities on HPAI H5N1 viruses were detected. The results displayed that the derivatives UA-Nu-ph-5, XC-27-1 and XC-27-2 strongly inhibited wild-type A/Duck/Guangdong/212/2004 H5N1 viruses with the IC50 values of 15.59 ± 2.4 μM, 16.83 ± 1.45 μM, and 12.45 ± 2.27 μM, respectively, and had the selectivity index (SI) > 3, which was consistent with the efficacy against A/Thailand/kan353/2004 pseudo-typed viruses. Four dealt patterns were compared via PRNT. The prevention dealt pattern showed the strongest inhibitory effects than other patterns, suggesting that these derivatives act on the entry process at the early stages of H5N1 viral infection, providing protection for cells against infection. Further studies through hemagglutinin inhibition (HI) and neuraminidase inhibitory (NAI) assay confirmed that these derivatives inhibited H5N1 virus replication by interfering with the viral hemagglutinin function. The derivatives could recognize specifically HA protein with binding affinity constant KD values of 2.57 × 10−4 M and 3.67 × 10−4 M. In addition, through site-directed mutagenesis combined with a pseudovirion system, we identified that the high-affinity docking sites underlying interaction were closely associated with amino acid residues I391 and T395 of HA. However, the potential binding sites of the derivatives with HA did not locate at HA1 sialic acids receptor binding domain (RBD). Taken together, these study data manifested that chlorogenin 3-O-β-chacotrioside derivatives generated antiviral effect against HPAI H5N1 viruses by targeting the hemagglutinin fusion machinery.

Highlights

Pathogenic H5N1 avian influenza as a zoonotic disease was caused by A/H5N1 subtype virus in the orthomyxoviridae family, which is a severe infectious disease characterized by sudden death and exceptionally high mortality in poultry
This study aimed at elucidating the molecular interaction of these agents and HA binding sites via Plaque Reduction Neutralization Test (PRNT) and hemagglutinin inhibition data, pseudovirion system combined with site-directed mutagenesis studies, and anti-viral against multiple H5N1 subtype strains as well as significant binding affinity
Madin-Darby Canine Kidney (MDCK) cells were obtained from ATCC (Manassas, VA, USA), LinX-A cells were donated by the Scripps Research Institute in San Diego, CA, USA

Summary

Introduction

Pathogenic H5N1 avian influenza as a zoonotic disease was caused by A/H5N1 subtype virus in the orthomyxoviridae family, which is a severe infectious disease characterized by sudden death and exceptionally high mortality in poultry. The hemagglutinin gene of HPAI A/H5 viruses were undergoing continuous evolution, generating emerging re-assorted subtypes and clades. Four clades 2.2.1.2, 2.3.2.1a, 2.3.2.1c, and 2.3.4.4 strains of H5N1 virus have become predominant pandemic strains with the continued evolution of HA gene [4,11,12,13,14,15,16]. In view of a major public health concern, providing protection against severe infection and death caused by H5N1 has become a research focus in the zoonosis field

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