Targeting the human protein disulfide isomerase A3 as a broad-spectrum antiviral approach against human respiratory viruses

• The cellular PDIA3 enzyme assists folding of N-linked viral envelope glycoproteins • The small molecule PDIA3 inhibitor LOC14 has a broad-spectrum antiviral activity • LOC14 hinders formation of disulfide bridges in the coronavirus spike glycoprotein • LOC14 reduces the release of infectious coronavirus particles from infected cells • Validation of PDIA3 as a promising broad-spectrum antiviral target The recent emergence of new respiratory virus infections in humans with epidemic or pandemic potential has reiterated the urgent need for effective broad-spectrum antivirals (BSAs) as part of a strategic framework for pandemic prevention and preparedness. In this context, the cellular machinery involved in the maturation of viral proteins in the endoplasmic reticulum (ER) can be considered in the search for host-targeting agents (HTAs) with potential BSA activity. Here, we investigated the feasibility of pharmacological targeting of human protein disulfide isomerase A3 (PDIA3) to identify BSAs against respiratory viruses. PDIA3 catalyzes the formation and isomerization of disulfide bonds in the ER pathway specific for folding N-glycosylated proteins; hence, it may be involved in the folding of many envelope glycoproteins. LOC14, a small molecule employed as a prototype PDIA3 inhibitor, was found to exert antiviral activity in the sub-micromolar range against a panel of representative human respiratory viruses, including the coronaviruses hCoV-OC43 and hCoV-229E, clinical isolates of influenza A and B viruses, and respiratory syncytial virus (RSV), even in a primary human airway epithelial cell model. Using hCoV-OC43 as a model respiratory virus with envelope N-linked glycoproteins to confirm the mechanistic mode of action, LOC14 was observed to affect the folding of the spike glycoprotein by reducing the formation of disulfide bonds, thereby hampering the production of infectious viral particles. Overall, these findings validate PDIA3 as a target for the development of HTAs with BSA activity and further support the advancement of LOC14 as a candidate BSA for respiratory virus infections.