A cysteine protease inhibitor blocks SARS-CoV-2 infection of human and monkey cells
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Abstract
K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC 50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC 50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 μM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 μM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2, differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.
SIGNIFICANCE
The virus causing COVID-19 is highly infectious and has resulted in a global pandemic. We confirm that a cysteine protease inhibitor, approved by the FDA as a clinical-stage compound, inhibits SARS-CoV-2 infection of several human and monkey cell lines with notable(nanomolar) efficacy. The mechanism of action of this inhibitor is identified as a specific inhibition of host cell cathepsin L. This in turn inhibits host cell processing of the coronaviral spike protein, a step required for cell entry. Neither of the coronaviral proteases are inhibited, and the cleavage site of spike protein processing is different from that reported in other coronaviruses. Hypotheses to explain the differential activity of the inhibitor with different cell types are discussed.
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SciScore for 10.1101/2020.10.23.347534: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Cell cultures and SARS-CoV-2 infection: Evaluation of protease inhibitors in a coronaviral infection assay: Identification of protein targets of K777 using an activity-based probe, 4N-propargyl-K777: 4N-propargyl-K777 was added at concentrations of 1 μM to Vero E6 cells, with and without infection by SARS CoV-2 viral particles at an MoI of 0.1. Vero E6suggested: RRID:CVCL_XD71)Protein extracts from Vero E6, HeLa/ACE2, Calu-3 and … SciScore for 10.1101/2020.10.23.347534: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Cell cultures and SARS-CoV-2 infection: Evaluation of protease inhibitors in a coronaviral infection assay: Identification of protein targets of K777 using an activity-based probe, 4N-propargyl-K777: 4N-propargyl-K777 was added at concentrations of 1 μM to Vero E6 cells, with and without infection by SARS CoV-2 viral particles at an MoI of 0.1. Vero E6suggested: RRID:CVCL_XD71)Protein extracts from Vero E6, HeLa/ACE2, Calu-3 and A549/ACE2 cell lysates (225 ng) were incubated for 30 min with 10 μM of K777 or CA-074 (Cayman #24679) in 50 mM Sodium acetate, pH 5.5 containing 1 mM EDTA, 1 μM pepstatin, 100 μM AEBSF and 5 mM DTT. Calu-3suggested: NoneA549/ACE2suggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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