Repurposing the Ebola and Marburg Virus Inhibitors Tilorone, Quinacrine and Pyronaridine: In vitro Activity Against SARS-CoV-2 and Potential Mechanisms

SARS-CoV-2 is a newly identified virus that has resulted in over 1.3 M deaths globally and over 59 M cases globally to date. Small molecule inhibitors that reverse disease severity have proven difficult to discover. One of the key approaches that has been widely applied in an effort to speed up the translation of drugs is drug repurposing. A few drugs have shownin vitroactivity against Ebola virus and demonstrated activity against SARS-CoV-2in vivo. Most notably the RNA polymerase targeting remdesivir demonstrated activityin vitroand efficacy in the early stage of the disease in humans. Testing other small molecule drugs that are active against Ebola virus would seem a reasonable strategy to evaluate their potential for SARS-CoV-2. We have previously repurposed pyronaridine, tilorone and quinacrine (from malaria, influenza, and antiprotozoal uses, respectively) as inhibitors of Ebola and Marburg virusin vitroin HeLa cells and of mouse adapted Ebola virus in mousein vivo. We have now tested these three drugs in various cell lines (VeroE6, Vero76, Caco-2, Calu-3, A549-ACE2, HUH-7 and monocytes) infected with SARS-CoV-2 as well as other viruses (including MHV and HCoV 229E). The compilation of these results indicated considerable variability in antiviral activity observed across cell lines. We found that tilorone and pyronaridine inhibited the virus replication in A549-ACE2 cells with IC₅₀values of 180 nM and IC₅₀198 nM, respectively. We have also tested them in a pseudovirus assay and used microscale thermophoresis to test the binding of these molecules to the spike protein. They bind to spike RBD protein with K_dvalues of 339 nM and 647 nM, respectively. Human C_maxfor pyronaridine and quinacrine is greater than the IC₅₀hence justifyingin vivoevaluation. We also provide novel insights into their mechanism which is likely lysosomotropic.