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

Abstract

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.

A Drug Repurposing Approach for Antimalarials Interfering with SARS-CoV-2 Spike Protein Receptor Binding Domain (RBD) and Human Angiotensin-Converting Enzyme 2 (ACE2)

“…Mefloquine, quinine and pyronaridine display antiviral activity against SARS-CoV-2 in vitro [21,26]. The activity of pyronaridine against SARS-CoV-2 has also been evaluated by measuring its interaction with the RBD by microscale thermophoresis [36,37].…”

Atovaquone and Berberine Chloride Reduce SARS-CoV-2 Replication In Vitro

“…The absence of a SARS-CoV-2 antiviral compound is not for a lack of trying. At the onset of the pandemic, many groups tested countless compounds in the hope of developing SARS-CoV-2 antivirals [1][2][3][4][5]. These studies identified potential candidates, several of which are or were in clinical trials.…”

Recent advances in developing small-molecule inhibitors against SARS-CoV-2

“…10 ( 126 ) Tamoxifen citrate In vitro 8.98 37.96 Preclinical 245 Fig. 10 ( 127 ) Imatinib mesylate In vitro 5.32 >30.86 Preclinical 245 In vivo (humanized mice carrying hPSC-derived lung xenografts) 2.15 ‒ Preclinical 248 Fig. 10 ( 128 ) Bruceine A In vitro 0.011 31.4 Preclinical 247 Fig.…”

“…10 ( 158 ) Pyronaridine In vitro 0.198 ‒ Preclinical 249 Fig. 10 ( 159 ) Mycophenolic acid In vivo (humanized mice carrying hPSC-derived lung xenografts) 0.9 ‒ Preclinical 248 Fig. 10 ( 160 ) Quinacrine dihydrochloride In vivo (humanized mice carrying hPSC-derived lung xenografts) 0.84 ‒ Preclinical 27 …”

“…Puhl et al 248 found that some drugs shown to have in vitro activity against Ebola also showed activity against SARS-CoV-2. Therefore, they tested three small-molecule drugs active against Ebola virus in various cell lines (VeroE6, Vero76, Caco-2, Calu-3, A549-ACE2, HUH-7 and monocytes) infected with SARS-CoV-2 248 . The compilation of these results suggests that there is considerable variability in antiviral activity observed in different cell lines.…”