The SARS-CoV-2 Cytopathic Effect Is Blocked by Lysosome Alkalizing Small Molecules

Gorshkov, Kirill; Chen, Catherine Z.; Bostwick, Robert; Rasmussen, Lynn; Tran, Bruce Nguyen; Cheng, Yu-Shan; Xu, Miao; Pradhan, Manisha; Henderson, Mark; Zhu, Wei; Oh, Eunkeu; Susumu, Kimihiro; Wolak, Mason; Shamim, Khalida; Huang, Wenwei; Hu, Xin; Shen, Min; Klumpp-Thomas, Carleen; Itkin, Zina; Shinn, Paul; Carlos de la Torre, Juan; Simeonov, Anton; Michael, Sam G.; Hall, Matthew D.; Lo, Donald C.; Zheng, Wei

[Image: see text] Understanding the SARS-CoV-2 virus’ pathways of infection, virus–host–protein interactions, and mechanisms of virus-induced cytopathic effects will greatly aid in the discovery and design of new therapeutics to treat COVID-19. Chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including alkalizing lysosomes and blocking autophagy as well as exhibiting dose-limiting toxicities in patients. Therefore, we evaluated additional lysosomotropic compounds to identify an alternative lysosome-based drug repurposing opportunity. We found that six of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero E6 cells with half-maximal effective concentration (EC(50)) values ranging from 2.0 to 13 μM and selectivity indices (SIs; SI = CC(50)/EC(50)) ranging from 1.5- to >10-fold. The compounds (1) blocked lysosome functioning and autophagy, (2) prevented pseudotyped particle entry, (3) increased lysosomal pH, and (4) reduced (ROC-325) viral titers in the EpiAirway 3D tissue model. Consistent with these findings, the siRNA knockdown of ATP6V0D1 blocked the HCoV-NL63 cytopathic effect in LLC-MK2 cells. Moreover, an analysis of SARS-CoV-2 infected Vero E6 cell lysate revealed significant dysregulation of autophagy and lysosomal function, suggesting a contribution of the lysosome to the life cycle of SARS-CoV-2. Our findings suggest the lysosome as a potential host cell target to combat SARS-CoV-2 infections and inhibitors of lysosomal function could become an important component of drug combination therapies aimed at improving treatment and outcomes for COVID-19.