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Authors: Yamamoto et al.

Link to paper: https://doi.org/10.1101/2020.04.22.054981

Journal/ Pre-Print: BioRxiv

Tags: Drug discovery/Drug repurpose, Molecular biology, Therapeutics

Research Highlights 

1. Nafamostat mesylate inhibits TMPRSS2-induced SARS-CoV2 S protein priming and viral envelope fusion with the target cell.

2. Nafamostat mesylate inhibits the infection of human epithelial cell line (Calu-3) with SARS-CoV2 at EC50 of 10 nM, below average blood concentration of this drug when used for disseminated intravascular coagulation (DIC)

Summary 

The capacity of three Japanese drugs used for pancreatitis and/or disseminated intravascular coagulation-DIC (nafamostat mesylate, camostat mesylate and gabexate mesylate) to inhibit SARS-CoV-2 S protein-initiated fusion with the target cell was tested. Cell-based fusion assay for S protein was developed using cells expressing Renilla luciferase (RL)-split reporter proteins. S was expressed in the effector cells, and the corresponding receptor, ACE2, was co-expressed with TMPRSS2 in target cells. Membrane fusion between these cells was quantitated by determining RL activity. Nafamostat mesylate potently inhibited the fusion by targeting TMPRSS2 priming activity while camostat mesylate was 10-fold less active and gabexate inactive.

Impact for SARS-CoV2/COVID19 research efforts

Inhibition of SARS-CoV2/COVID19 transmission as well as

Treatment of SARS-CoV2/COVID19 positive individuals

Study Type

· In vitro study

Strengths and limitations of the paper

Novelty: Nafamostat mesylate inhibits SARS-CoV2 envelope fusion with the target cell by inhibiting TMPRSS2- induced S protein priming.

Standing in the field: Confirms bioinformatical predictions of nafamostat mesylate being a strong inhibitor of TMPRSS2. Confirms previously experimentally shown inhibitory effect of nafamostat mesylate on TMPRSS2, resulting in the inhibition of MERS-CoV (same group) as well as SARS-CoV2 (Hoffman et al.) entry into the human lung cells.

Appropriate statistics: No statistical tests done despite assays done in quadruplicate.

Viral model used: original SARS-CoV2 as well as various cell lines (HEK293FT, H3255, Calu-3) expressing SARS-CoV2 or MERS-CoV protein S (effector cells) and expressing ACE2 or CD26 + TMPRSS2 (target cells) for membrane fusion assays

Translatability: The study warrants clinical trial with nafamostat mesylate for COVID19 treatment. The drug is already used for the treatment of pancreatitis and DIC in Japan, the toxicity and pharmacokinetics are known, and they found that nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an EC50 around 10 nM, which is lower than circulating levels of the drug in patients after i.v. administration. Additionally, inferior camostat mesylate is already in clinical trial for COVID-19.

Main limitations:

· The researchers were aware that an almost identical paper was published couple of days prior to deposition of this paper to BioRxiv yet they haven’t done any in vivo experiments using nafamostat mesylate, which are still lacking

· Cells were pre-treated with nafamostat mesylate before cells were mixed for fusion assay/exposure to virus so no data on the effect of this drug in reducing pathology in cells already positive for virus. Suggests potential for prophylaxis rather than therapeutics.