The novel emerged SARS-CoV-2 has rapidly spread around the world causing acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. For SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study, we show that S can be cleaved by the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2' site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 human airway epithelial cells through antisense-mediated knockdown of TMPRSS2 expression. Furthermore, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851 in human airway cells. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Combining various TMPRSS2 inhibitors with furin inhibitor MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. Therefore, this approach has considerable therapeutic potential for treatment of COVID-19.

https://www.life-science-alliance.org/content/lsa/3/9/e202000786.full.pdf

TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells.

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity.

Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 mM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat's safety, make it a likely candidate drug to treat COVID-19.

https://www.mdpi.com/1999-4915/12/6/629/pdf

The Anticoagulant Nafamostat Potently Inhibits SARS-CoV-2 S Protein-Mediated Fusion in a Cell Fusion Assay System and Viral Infection In Vitro in a Cell-Type-Dependent Manner.

Antiviral therapy is urgently needed to combat the coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The protease inhibitor camostat mesylate inhibits SARS-CoV-2 infection of lung cells by blocking the virus-activating host cell protease TMPRSS2. Camostat mesylate has been approved for treatment of pancreatitis in Japan and is currently being repurposed for COVID-19 treatment. However, potential mechanisms of viral resistance as well as camostat mesylate metabolization and antiviral activity of metabolites are unclear. Here, we show that SARS-CoV-2 can employ TMPRSS2-related host cell proteases for activation and that several of them are expressed in viral target cells. However, entry mediated by these proteases was blocked by camostat mesylate. The camostat metabolite GBPA inhibited the activity of recombinant TMPRSS2 with reduced efficiency as compared to camostat mesylate and was rapidly generated in the presence of serum. Importantly, the infection experiments in which camostat mesylate was identified as a SARS-CoV-2 inhibitor involved preincubation of target cells with camostat mesylate in the presence of serum for 2 h and thus allowed conversion of camostat mesylate into GBPA. Indeed, when the antiviral activities of GBPA and camostat mesylate were compared in this setting, no major differences were identified. Our results indicate that use of TMPRSS2-related proteases for entry into target cells will not render SARS-CoV-2 camostat mesylate resistant. Moreover, the present and previous findings suggest that the peak concentrations of GBPA established after the clinically approved camostat mesylate dose (600 mg/day) will result in antiviral activity.

https://biorxiv.org/content/10.1101/2020.08.05.237651v1.full.pdf

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity

Summary In a short period, many research publications that report sets of experimentally validated drugs as potential COVID-19 therapies have emerged. To organize this accumulating knowledge, we developed the COVID-19 Drug and Gene Set Library ( https://amp.pharm.mssm.edu/covid19/ ), a collection of drug and gene sets related to COVID-19 research from multiple sources. The platform enables users to view, download, analyze, visualize, and contribute drug and gene sets related to COVID-19 research. To evaluate the content of the library, we compared the results from six in vitro drug screens for COVID-19 repurposing candidates. Surprisingly, we observe low overlap across screens while highlighting overlapping candidates that should receive more attention as potential therapeutics for COVID-19. Overall, the COVID-19 Drug and Gene Set Library can be used to identify community consensus, make researchers and clinicians aware of new potential therapies, enable machine-learning applications, and facilitate the research community to work together toward a cure.

The COVID-19 Drug and Gene Set Library.

The most rapid path to discovering treatment options for the novel coronavirus SARS-CoV-2 is to find existing medications that are active against the virus We have focused on identifying repurposing candidates for the transmembrane serine protease family member I(TMPRSS2), which is critical for entry of coronaviruses into cells Using known 3D structures of close homologs, we created seven homology models We also identified a set of serine protease inhibitor drugs, generated several conformations of each, and docked them into our models We used three known chemical (non-drug) inhibitors and one validated inhibitor of TMPRSS2 in MERS as benchmark compounds and found six compounds with predicted high binding affinity in the range of the known inhibitors We also showed that a previously published weak inhibitor, Camostat, had a significantly lower binding score than our six compounds All six compounds are anticoagulants with significant and potentially dangerous clinical effects and side effects Nonetheless, if these compounds significantly inhibit SARS-CoV-2 infection, they could represent a potentially useful clinical tool

/pdf/homology-modeling-of-tmprss2-yields-candidate-drugs-that-may-1w2r1utqjh.pdf

Liu Tianyun

Papers

Homology Modeling of TMPRSS2 Yields Candidate Drugs That May Inhibit Entry of SARS-CoV-2 into Human Cells