Morpholinodiazenyl chalcone blocks influenza A virus capsid uncoating by perturbing the clathrin‐mediated vesicular trafficking pathway

Abstract

Influenza A virus (IAV) is a highly contagious respiratory pathogen that significantly threatens global health by causing seasonal epidemics and occasional, unpredictable pandemics. To identify new compounds with therapeutic potential against IAV, we designed and synthesized a series of 4′‐morpholinodiazenyl chalcones using the molecular hybridization method, performed a high‐content screen against IAV, and found that (E)‐1‐{4‐[(E)‐morpholinodiazenyl]phenyl}‐3‐(3,4,5‐trimethoxyphenyl)prop‐2‐en‐1‐one (MC‐22) completely neutralized IAV infection. While MC‐22 allowed IAV to successfully internalize into the cell and fuse at the acidic late endosomes, it prevented viral capsid uncoating and genome release. Since IAV majorly utilizes clathrin‐mediated endocytosis (CME) for cellular entry, we examined whether MC‐22 had any effect on CME, using nonviral cargoes that enter cells via clathrin‐dependent or ‐independent pathways. Although MC‐22 showed no effect on the uptake of choleratoxin B, a cargo that enters cells majorly via the clathrin‐independent pathway, it significantly attenuated the clathrin‐dependent internalization of both epidermal growth factor and transferrin. Cell biological analyses revealed a marked increase in the size of early endosomes upon MC‐22 treatment, indicating an endosomal trafficking/maturation defect. This study reports the identification of MC‐22 as a novel CME‐targeting, highly potent IAV entry inhibitor, which is expected to neutralize a broad spectrum of viruses that enter the host cells via CME.