TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets

Authors: Bestle D et al.

Link to paper: https://www.biorxiv.org/content/10.1101/2020.04.15.042085v1

Journal/ Pre-Print: bioRxiv

Key Words: TMPRSS2, Furin, viral entry, protease inhibitors, SARS-COV-2, COVID-19

Research Highlights 

1. The proteases furin and TMPRSS2 cleave the viral spike protein of SARS-CoV-2 at different sites (at S1/S2 and S2’ respectively).

2. Knockdown of TMPRSS2 reduces viral entry and replication of SARS-CoV-2 in Calu-3 human airway epithelial cells

3. TMPRSS2 and furin do not compensate for each other, which means inhibition of either one will sufficient to stop the virus entry. Pharmacological inhibition of both TMPRSS2 and furin synergistically reduces SARS-CoV-2 replication.

Summary 

This study characterised proteolytic cleavage of the SARS-CoV-2 spike (S) protein, which is required for host membrane fusion and subsequent virus entry. The authors showed that furin cleaves SARS-CoV-2 S protein at the S1/S2 site, and the serine protease TMPRSS2 cleaves at the S2’ site. Morpholino-mediated knockdown of TMPRSS2 in a human airway epithelial cell reduced replication of SARS-CoV-2. Virus replication was also reduced by TMPRSS inhibitors such as MI-432, MI-1900 and the broad-spectrum serine protease inhibitor aprotinin, and the furin inhibitor MI-1851. Combined use of TMPRSS inhibitors with MI-1851 resulted in enhanced antiviral activity relative to single inhibitors.

Impact for SARS-CoV-2/COVID19 research efforts

Understand the virology and/or cell biology of SARS-CoV2/COVID19

Treatment of SARS-CoV-2/COVID19 positive individuals (study suggests new therapeutic anti-viral drug targets).

Study Type

· In vitro study

- FRET substrates with S1/S2 SARS-CoV-2 sequence used in enzyme kinetic assay to test cleavage by human furin

- Cleavage of SARS-CoV-2 S protein produced by HEK293 cells co-transfected with plasmids encoding TMPRSS2 detected by SDS-PAGE and Western blot

- Virus replication in Calu-3 cells treated with protease inhibitors or TMPRSS2-targeting morpholino, measured by TCID titration and immunostaining using serum against SARS-CoV

Strengths and limitations of the paper

Novelty: The authors have experimentally proven, for the first time, that Furin found to be involved in the activation of coronavirus spike protein (S1/S2 site). Although, previously, furine like cleavage site was predicted by (Coutard et al. (2020) and Walls et al. (2020). Provides evidence for combined TMPRSS2 and furin inhibition in treatment of COVID-19.

Standing in the field: As other papers have also found, show that TMPRSS2 and furin are involved in SARS-CoV-2 S cleavage

Appropriate statistics: No statistical tests used. To make the efficacy trustworthy the study need to be powered up, as it not clear the number of replicates.

Viral model used: SARS-CoV-2 isolate Munich 929

Translatability: Yes, could be, there is great potential in the idea, but safe and target specific inhibitor will be required.

Main limitations: First, as we know the unavailability of TMPRSS2 crystal structure in protein data bank (PDB). So, non-specific, broad range serine-protease inhibitors has been used in this study. Second, although the local application of aprotinin (the biological inhibitor) in the form small particle aerosol has been approved in Russia to treat the mild to moderate respiratory influenza infection. We do not know the side effects if injected intravenously. Thirdly, even being an in vitro study, it doesn’t demonstrate the improvement or rescue of pathophysiological condition or cellular response e.g. anti-inflammatory etc. No validation of results in primary human cells.