Chloroquine does not inhibit infection of human lung cells with SARS-CoV-2
Markus Hoffmann, Kirstin Mösbauer, Heike Hofmann-Winkler, Artur Kaul, Hannah Kleine-Weber, Nadine Krüger, Nils C Gassen, Marcel A Müller, Christian Drosten, Stefan Pöhlmann
Nature, doi:10.1038/s41586-020-2575-3
The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been associated with more than 780,000 deaths worldwide (as of 20 August 2020). To develop antiviral interventions quickly, drugs used for the treatment of unrelated diseases are currently being repurposed to treat COVID-19. Chloroquine is an anti-malaria drug that is used for the treatment of COVID-19 as it inhibits the spread of SARS-CoV-2 in the African green monkey kidney-derived cell line Vero 1-3 . Here we show that engineered expression of TMPRSS2, a cellular protease that activates SARS-CoV-2 for entry into lung cells 4 , renders SARS-CoV-2 infection of Vero cells insensitive to chloroquine. Moreover, we report that chloroquine does not block infection with SARS-CoV-2 in the TMPRSS2-expressing human lung cell line Calu-3. These results indicate that chloroquine targets a pathway for viral activation that is not active in lung cells and is unlikely to protect against the spread of SARS-CoV-2 in and between patients. Chloroquine and hydroxychloroquine are used for the treatment of malaria and have been widely used to treat patients with COVID-19. Both of these drugs are currently under investigation in more than 80 registered clinical trials for the treatment of COVID-19 worldwide 2,3 . Chloroquine and hydroxychloroquine inhibit the ability of SARS-CoV-2 to infect Vero cells 1, 5, 6 , providing a rational for using these drugs for the treatment of COVID-19. However, it is unknown whether these drugs inhibit the infection of lung cells and it is poorly understood how they inhibit infection with SARS-CoV-2. Chloroquine and hydroxychloroquine increase the endosomal pH of cells and inhibit viruses that depend on low pH for cell entry 7 . We investigated whether these drugs could also block the cell entry by SARS-CoV-2 and whether entry inhibition accounted for the prevention of infection with SARS-CoV-2. Moreover, we investigated whether entry inhibition is cell-type-dependent, as the virus can use pH-dependent and pH-independent pathways for entry into cells. The spike (S) protein of SARS-CoV-2, which mediates viral entry, is activated by the endosomal-pH-dependent cysteine protease cathepsin L (CTSL) in some cell lines 4 . By contrast, entry into airway epithelial cells, which express low levels of CTSL 8 , depends on the pH-independent, plasma-membrane-resident serine protease TMPRSS2 4 . Notably, the use of CTSL by coronaviruses is restricted to cell lines 8-10 , whereas TMPRSS2 activity is essential for the spread and pathogenesis of the virus in the infected host 11, 12 . We compared the inhibition by chloroquine and hydroxychloroquine of S-mediated entry into Vero (kidney), TMPRSS2-expressing Vero and Calu-3 (lung) cells. Calu-3 cells, as with the airway epithelium, express low amounts of CTSL 8 and SARS-CoV-2 entry into these cells is dependent on TMPRSS2 4 .
Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this paper. Author contributions M.H. and S.P. designed the study. M.H., K.M., H.H.-W., A.K., H.K.-W., N.K., N.C.G. and M.A.M. performed research. M.H., M.A.M., C.D. and S.P. analysed the data. C.D. provided essential reagents. M.H. and S.P. wrote the manuscript. All authors revised the manuscript.
Competing interests The authors declare no competing interests.
Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-2575-3. Correspondence and requests for materials should be addressed to M.H. or S.P. Reprints and permissions information is available at http://www.nature.com/reprints.
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References
Berger Rentsch, Zimmer, A vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type I interferon, PLoS ONE
Boulware, A randomized trial of hydroxychloroquine as postexposure prophylaxis for COVID-19, N. Engl. J. Med
Corman, Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR, Eurosurveill
Ferner, Aronson, Chloroquine and hydroxychloroquine in covid-19, Br. Med. J
Herzog, Drosten, Müller, Plaque assay for human coronavirus NL63 using human colon carcinoma cells, Virol. J
Hoffmann, SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor, Cell
Iwata-Yoshikawa, TMPRSS2 contributes to virus spread and immunopathology in the airways of murine models after coronavirus infection, J. Virol
Kleine-Weber, Mutations in the spike protein of Middle East respiratory syndrome coronavirus transmitted in Korea increase resistance to antibody-mediated neutralization, J. Virol
Klemm, Mitogen-activated protein kinases (MAPKs) regulate IL-6 over-production during concomitant influenza virus and Staphylococcus aureus infection, Sci. Rep
Kupferschmidt, Big studies dim hopes for hydroxychloroquine, Science
Liu, Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro, Cell Discov
Matsuyama, Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells, Proc. Natl Acad. Sci
Park, Proteolytic processing of Middle East respiratory syndrome coronavirus spikes expands virus tropism, Proc. Natl Acad. Sci
Rolain, Colson, Raoult, Recycling of chloroquine and its hydroxyl analogue to face bacterial, fungal and viral infections in the 21st century, Int. J. Antimicrob. Agents
Shirato, Kanou, Kawase, Matsuyama, Clinical isolates of human coronavirus 229E bypass the endosome for cell entry, J. Virol
Shirato, Kawase, Matsuyama, Wild-type human coronaviruses prefer cell-surface TMPRSS2 to endosomal cathepsins for cell entry, Virology
Touret, De Lamballerie, Of chloroquine and COVID-19, Antiviral Res
Wang, Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro, Cell Res
Yao, In vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Clin. Infect. Dis
Zhou, Protease inhibitors targeting coronavirus and filovirus entry, Antiviral Res
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