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

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.