Synergistic Block of SARS-CoV-2 Infection by Combined Drug Inhibition of the Host Entry Factors PIKfyve Kinase and TMPRSS2 Protease

Abstract

Repurposing FDA-approved inhibitors able to prevent infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could provide a rapid path to establish new therapeutic options to mitigate the effects of coronavirus disease 2019 (COVID-19). Proteolytic cleavages of the spike S protein of SARS-CoV-2, mediated by the host cell proteases cathepsin and TMPRSS2, alone or in combination, are key early activation steps required for efficient infection. The PIKfyve kinase inhibitor apilimod interferes with late endosomal viral traffic, and through an ill-defined mechanism prevents in vitro infection through late endosomes mediated by cathepsin. Similarly, inhibition of TMPRSS2 protease activity by camostat mesylate or nafamostat mesylate prevents infection mediated by the TMPRSS2-dependent and cathepsin-independent pathway. Here, we combined the use of apilimod with camostat mesylate or nafamostat mesylate and found an unexpected ∼5-10-fold increase in their effectiveness to prevent SARS-CoV-2 infection in different cell types. Comparable synergism was observed using both, a chimeric vesicular stomatitis virus (VSV) containing S of SARS-CoV-2 (VSV-SARS-CoV-2) and SARS-CoV-2 virus. The substantial ∼5-fold or more decrease of half maximal effective concentrations (EC 50 values) suggests a plausible treatment strategy based on the combined use of these inhibitors. IMPORTANCE Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the coronavirus disease 2019 (COVID-2019) global pandemic. There are ongoing efforts to uncover effective antiviral agents that could mitigate the severity of the disease by controlling the ensuing viral replication. Promising candidates include small molecules that inhibit the enzymatic activities of host proteins, thus preventing SARS-CoV-2 entry and infection. They include Apilimod, an inhibitor of PIKfyve kinase and camostat mesylate and nafamostat mesylate, inhibitors of TMPRSS2 protease. Our research is significant for having uncovered an unexpected synergism in the effective inhibitory activity of apilimod used together with camostat mesylate or with nafamostat mesylate.

SARS-CoV-2 spreads through cell-to-cell transmission

“…S4 ), and moreover, their effects on SARS-CoV-2 are weaker than their effects on SARS-CoV. We also noticed that the cell-free infection of SARS-CoV-2 in Vero-ACE2-TMPRRS2 cells was still sensitive to treatment by endosomal inhibitors, similar to a recent report ( 80 ); we reason that this could be associated with the levels of TMPPRS2 expression on the plasma membrane of target cells used for infection, as well as viral stocks that contain furin-defective mutants generated from Vero cells. These observations collectively suggest a less dominant role for the endosomal entry pathway in cell-to-cell transmission of SARS-CoV-2.…”

Increased B Cell Selection Stringency In Germinal Centers Can Explain Improved COVID-19 Vaccine Efficacies With Low Dose Prime or Delayed Boost

“…Thus, incorporating diverse antigens and inter-clonal competition are expected not to affect our qualitative inferences regarding SARS-CoV-2 vaccines. Understanding vaccine efficacies quantitatively may require accurate description of antibody diversity given the multiple virus entry pathways accessible to SARS-CoV-2 and the potential synergy associated with blocking the pathways simultaneously ( 79 , 80 ).…”

Drug Combinations as a First Line of Defense against Coronaviruses and Other Emerging Viruses

“…Synergistic drug pairs that do not involve a polymerase inhibitor have also been identified in Vero E6 cells, including nelfinavir plus amodiaquine ( 71 ), arbidol plus nitazoxanide ( 73 ), nitazoxanide plus emetine ( 73 ), and nelfinavir plus cepharanthine ( 78 ). Additional non-polymerase-targeted pairs found synergistic in Calu3 cells were interferon alpha plus camostat ( 86 ), interferon alpha plus nafamostat ( 96 ), and apilimod plus camostat ( 103 ). Interferon alpha plus nafamostat reduced viral loads in hamsters to a greater extent than the individual component drugs ( 96 ).…”