An important SARS-CoV-2 enzyme is responsible for low in vivo antiviral efficacy of hydroxychloroquine

A research group from the Scripps Research Institute in Jupiter, US, has shown that the hydroxychloroquine drug interferes with only one of two somewhat redundant pathways that activate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S-protein) to mediate the infectious process. Their paper is currently available on the bioRxiv* preprint server.


Figure 1. SARS-CoV-1 and SARS-CoV-2 fusion can be activated by either or both of two pathways.

The coronavirus disease (COVID-19) represents a highly pathogenic viral infection caused by SARS-CoV-2 and an ongoing global health concern. Very quickly after the start of the pandemic, the concept of drug repurposing came into the scientific spotlight.

Chloroquine and hydroxychloroquine were among the first candidates, as they have shown the propensity to prevent viral infection in cell-cultures. Nonetheless, human clinical trials did not find a significant improvement in treated COVID-19 patients. Such a pronounced difference between in vitro and in vivo trials still intrigues the researchers.

Maybe the answer lies in viral pathophysiology. After SARS-CoV-2 binds to the cell receptor ACE2 (angiotensin-converting enzyme 2), a lysosomal endopeptidase enzyme known as cathepsin L can cleave its S-protein, activating membrane fusion for subsequent cell entry.

Likewise, the plasma membrane-associated protease TMPRSS2 can also cleave the aforementioned S-protein and activate viral entry at the cell surface. The same process is observed in SARS-CoV-1, which is the cause of the original SARS outbreak in 2002 and 2003.

Furthermore, human cells that express both ACE2 and TMPRSS2 are present in multiple tissues, which include lungs, nasal mucosa, buccal mucosa, ileum, colon, and myocardial epithelial cells.

In this study, the researchers from the Department of Immunology and Microbiology at the Scripps Research Institute in Jupiter, US, evaluated the infectivity of SARS-CoV-1 and SARS-CoV-2 S-protein on cells in the presence and absence of TMPRSS2.

Cell systems and pseudoviruses

To determine the precise proteases implicated in the SARS-CoV-2-S proteolysis activation, the researchers interrogated a panel of cysteine protease inhibitors in the HEK293T-ACE2 cell system.

Considering the latter, the HEK293T cell line expressing human ACE2 has been created by transduction with produced vesicular stomatitis virus (VSV) G protein-pseudotyped murine leukemia viruses (MLV).

Moreover, pseudoviruses were enriched with a variant spike or envelope proteins. In other words, such pseudovirions carried SARS-CoV-1 or SARS-CoV-2 S-proteins, and 48 hours after the infection, cells they have entered were lysed in wells and subjected to specific luminescence assays.

Finally, to confirm whether the activity of TMPRSS2 masks the antiviral effect of hydroxychloroquine on SARS-CoV-1 and SARS-CoV-2, these researchers also explored if the suppression of TMPRSS2 can rescue the antiviral efficiency of the drug.

SARS-CoV-2 more sensitive to TMPRSS2 than SARS-CoV-1

"In this study we present multiple lines of evidence that SARS-CoV-2 is more sensitive to the presence of TMPRSS2 than is SARS-CoV-1", say study authors. "This difference can largely be explained by the presence of a furin cleavage site in the SARS-CoV-2 S-protein", they add.

The authors have also shown that the TMPRSS2 expression overcomes the antiviral effect of hydroxychloroquine, providing a mechanistic explanation for its poor therapeutic efficacy against SARS-CoV-2 – despite somewhat encouraging cell culture results.

The data makes clear that, in the absence of TMPRSS2, SARS-CoV-2 utilizes cathepsin L much less efficiently in comparison to SARS-CoV-1. This difference may be explained by the relative instability of the wild-type SARS-CoV-2 S-protein.

Treatment implications

"The greater dependence of SARS-CoV-2 on TMPRSS2 has an immediate implication for the treatment of COVID-19", study authors emphasize the importance of their findings in this recent bioRxiv paper.

More specifically, this implies that inhibitors of endosomal acidification may have less impact on SARS-CoV-2 in the presence of TMPRSS2. TMPRSS2 aids in bypassing the hydroxychloroquine-mediated inhibition of infection with SARS-2 pseudovirus.

As a majority of physiologically relevant target cells in the human body express TMPRSS2 (including ciliated nasal epithelial and cells type II lung cells), the potent inhibition of SARS-CoV-2 by hydroxychloroquine in Vero E6 cells (where TMPRSS2 is largely absent) overestimated its potency by 10- to 40-fold.

However, our results suggest that some efficacy of hydroxychloroquine could be restored if TMPRSS2 is inhibited by camostat (i.e., a serine protease inhibitor), an observation directly relevant to clinical trials",

In any case, this study shows that the inhibition of both TMPRSS2 and cathepsin L may be required to completely block viral entry into cells that express both proteases. Further studies will be needed to corroborate these findings.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Ou, T. et al. (2020). Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2. bioRxiv.

Dr. Tomislav Meštrović

Written by

Dr. Tomislav Meštrović

Dr. Tomislav Meštrović is a medical doctor (MD) with a Ph.D. in biomedical and health sciences, specialist in the field of clinical microbiology, and an Assistant Professor at Croatia's youngest university - University North. In addition to his interest in clinical, research and lecturing activities, his immense passion for medical writing and scientific communication goes back to his student days. He enjoys contributing back to the community. In his spare time, Tomislav is a movie buff and an avid traveler.


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