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

Severe acute respiratory coronavirus 2 (SARS-CoV-2) is a newly identified virus that has resulted in over 2.5 million deaths globally and over 116 million cases globally in March, 2021. 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 shown in vitro activity against Ebola viruses and demonstrated activity against SARS-CoV-2 in vivo. Most notably, the RNA polymerase targeting remdesivir demonstrated activity in vitro and efficacy in the early stage of the disease in humans. Testing other small-molecule drugs that are active against Ebola viruses (EBOVs) would appear 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 viruses in vitro in HeLa cells and mouse-adapted EBOV in mice in 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 IC50 values of 180 nM and IC50 198 nM, respectively. We used microscale thermophoresis to test the binding of these molecules to the spike protein, and tilorone and pyronaridine bind to the spike receptor binding domain protein with K d values of 339 and 647 nM, respectively. Human Cmax for pyronaridine and quinacrine is greater than the IC50 observed in A549-ACE2 cells. We also provide novel insights into the mechanism of these compounds which is likely lysosomotropic.