Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors

Abstract

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.

Antiviral and Immunomodulatory Effects of Pelargonium sidoides DC. Root Extract EPs® 7630 in SARS-CoV-2-Infected Human Lung Cells

“…To model the two entry pathways, we infected TMPRSS2-negative Vero cells (endosomal entry) and TMPRSS2-positive Calu-3 cells (endosomal entry and direct fusion) with SARS-CoV-2 spike (SARS-CoV-2-S) protein-carrying VSV-based pseudo particles (VSVpp). As controls, we applied niclosamide (10 µM), which blocks SARS-CoV-2 replication and possibly restricts endosomal entry ( Jurgeit et al, 2012 ; Prabhakara et al, 2021 ; Gassen et al, 2021 ), and camostat mesylate (100 µM), a proven TMPRSS2 inhibitor ( Hoffmann et al, 2020a ). SARS-CoV-2-S VSVpp entry was efficiently blocked (>98%) by niclosamide in both cell lines ( Figure 2A , unprocessed data and controls in Supplementary Figure S2 ) whereas, expectedly, camostat inhibited VSVpp entry only in TMPRSS2-positive Calu-3 cells.…”

Ferristatin II Efficiently Inhibits SARS-CoV-2 Replication in Vero Cells

“…Nevertheless, a relatively uniform binding of RBD to the surface of the cells treated with ferristatin II may indicate that some of the SARS-CoV-2 receptors on Vero cells remain functional and bind the virus, but in the absence of TfR1, the virus cannot enter the cell. This receptor is most likely ACE2, since Prabhakara et al demonstrated an increase in the uptake of labeled RBD along with labeled transferrin by cells overexpressing ACE2, suggesting that ACE2 promotes RBD uptake through clathrin-mediated endocytosis, which is exploitedby TfR1 [28]. Furthermore, there is evidence that SARS-CoV-2 can infect ACE2-negative cells using alternative entry pathways [29]; therefore, a deeper analysis of ferristatin II action on various cell types permissive for SARS-CoV-2 infection will shed light on the role of TfR1 in the virus life cycle.…”

The Physical Basis for pH Sensitivity in Biomolecular Structure and Function, With Application to the Spike Protein of SARS-CoV-2

“…For the membrane-enveloped coronavirus family, cell entry may be through fusion directly at the cell membrane or through fusion at an intracellular membrane, subsequent to receptor-mediated endocytosis of the virus (Whittaker et al, 2021). Although there is evidence that altering endosomal pH impedes viral entry to some extent (Prabhakara et al, 2021) for the SARS-CoV-2 coronavirus (causative agent of the COVID-19 pandemic), the precise balance of genome release routes (cell surface or interior) may depend on other factors, such as the priming cleavage of S1 and S2 subunits of the spike (S) protein (Papa et al, 2021). For membrane-enveloped viruses, mechanisms evolve to protect against mistimed low pHinduced membrane fusion events in the acidic pH secretory pathways for newly synthesized viruses (Fields and Kielian, 2015).…”