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Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2

Tianling Ou, Huihui Mou, Lizhou Zhang, Amrita Ojha, Hyeryun Choe, Michael Farzan
doi: https://doi.org/10.1101/2020.07.22.216150
Tianling Ou
1Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA
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Huihui Mou
1Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA
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Lizhou Zhang
1Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA
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Amrita Ojha
1Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA
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Hyeryun Choe
1Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA
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Michael Farzan
1Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA
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  • For correspondence: mfarzan@scripps.edu
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Abstract

Hydroxychloroquine, used to treat malaria and some autoimmune disorders, potently inhibits viral infection of SARS coronavirus (SARS-CoV-1) and SARS-CoV-2 in cell-culture studies. However, human clinical trials of hydroxychloroquine failed to establish its usefulness as treatment for COVID-19. This compound is known to interfere with endosomal acidification necessary to the proteolytic activity of cathepsins. Following receptor binding and endocytosis, cathepsin L can cleave the SARS-CoV-1 and SARS-CoV-2 spike (S) proteins, thereby activating membrane fusion for cell entry. The plasma membrane-associated protease TMPRSS2 can similarly cleave these S proteins and activate viral entry at the cell surface. Here we show that the SARS-CoV-2 entry process is more dependent than that of SARS-CoV-1 on TMPRSS2 expression. This difference can be reversed when the furin-cleavage site of the SARS-CoV-2 S protein is ablated. We also show that hydroxychloroquine efficiently blocks viral entry mediated by cathepsin L, but not by TMPRSS2, and that a combination of hydroxychloroquine and a clinically-tested TMPRSS2 inhibitor prevents SARS-CoV-2 infection more potently than either drug alone. These studies identify functional differences between SARS-CoV-1 and -2 entry processes, and provide a mechanistic explanation for the limited in vivo utility of hydroxychloroquine as a treatment for COVID-19.

Author Summary The novel pathogenic coronavirus SARS-CoV-2 causes COVID-19 and remains a threat to global public health. Chloroquine and hydroxychloroquine have been shown to prevent viral infection in cell-culture systems, but human clinical trials did not observe a significant improvement in COVID-19 patients treated with these compounds. Here we show that hydroxychloroquine interferes with only one of two somewhat redundant pathways by which the SARS-CoV-2 spike (S) protein is activated to mediate infection. The first pathway is dependent on the endosomal protease cathepsin L and sensitive to hydroxychloroquine, whereas the second pathway is dependent on TMPRSS2, which is unaffected by this compound. We further show that SARS-CoV-2 is more reliant than SARS coronavirus (SARS-CoV-1) on the TMPRSS2 pathway, and that this difference is due to a furin cleavage site present in the SARS-CoV-2 S protein. Finally, we show that combinations of hydroxychloroquine and a clinically tested TMPRSS2 inhibitor work together to effectively inhibit SARS-CoV-2 entry. Thus TMPRSS2 expression on physiologically relevant SARS-CoV-2 target cells may bypass the antiviral activities of hydroxychloroquine, and explain its lack of in vivo efficacy.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted July 22, 2020.
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Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2
Tianling Ou, Huihui Mou, Lizhou Zhang, Amrita Ojha, Hyeryun Choe, Michael Farzan
bioRxiv 2020.07.22.216150; doi: https://doi.org/10.1101/2020.07.22.216150
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Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2
Tianling Ou, Huihui Mou, Lizhou Zhang, Amrita Ojha, Hyeryun Choe, Michael Farzan
bioRxiv 2020.07.22.216150; doi: https://doi.org/10.1101/2020.07.22.216150

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