Background Antivirals are needed to combat the COVID-19 pandemic, which is caused by SARS-CoV-2. The clinically-proven protease inhibitor Camostat mesylate inhibits SARS-CoV-2 infection by blocking the virus-activating host cell protease TMPRSS2. However, antiviral activity of Camostat mesylate metabolites and potential viral resistance have not been analyzed. Moreover, antiviral activity of Camostat mesylate in human lung tissue remains to be demonstrated. Methods We used recombinant TMPRSS2, reporter particles bearing the spike protein of SARS-CoV-2 or authentic SARS-CoV-2 to assess inhibition of TMPRSS2 and viral entry, respectively, by Camostat mesylate and its metabolite GBPA. Findings We show that several TMPRSS2-related proteases activate SARS-CoV-2 and that two, TMPRSS11D and TMPRSS13, are robustly expressed in the upper respiratory tract. However, entry mediated by these proteases was blocked by Camostat mesylate. The Camostat metabolite GBPA inhibited recombinant TMPRSS2 with reduced efficiency as compared to Camostat mesylate. In contrast, both inhibitors exhibited similar antiviral activity and this correlated with the rapid conversion of Camostat mesylate into GBPA in the presence of serum. Finally, Camostat mesylate and GBPA blocked SARS-CoV-2 spread in human lung tissue ex vivo and the related protease inhibitor Nafamostat mesylate exerted augmented antiviral activity. Interpretation Our results suggest that SARS-CoV-2 can use TMPRSS2 and closely related proteases for spread in the upper respiratory tract and that spread in the human lung can be blocked by Camostat mesylate and its metabolite GBPA. Funding NIH, Damon Runyon Foundation, ACS, NYCT, DFG, EU, Berlin Mathematics center MATH+, BMBF, Lower Saxony, Lundbeck Foundation, Novo Nordisk Foundation.

To circumvent time-consuming clinical trials, testing whether existing drugs are effective inhibitors of SARS-CoV-2, has led to the discovery of Remdesivir. We decided to follow this path and screened approved medications "off-label" against SARS-CoV-2. Fluoxetine inhibited SARS-CoV-2 at a concentration of 0.8 µg/ml significantly in these screenings, and the EC50 was determined with 387 ng/ml. Furthermore, Fluoxetine reduced viral infectivity in precision-cut human lung slices showing its activity in relevant human tissue targeted in severe infections. Fluoxetine treatment resulted in a decrease in viral protein expression. Fluoxetine is a racemate consisting of both stereoisomers, while the S-form is the dominant serotonin reuptake inhibitor. We found that both isomers show similar activity on the virus, indicating that the R-form might specifically be used for SARS-CoV-2 treatment. Fluoxetine inhibited neither Rabies virus, human respiratory syncytial virus replication nor the Human Herpesvirus 8 or Herpes simplex virus type 1 gene expression, indicating that it acts virus-specific. Moreover, since it is known that Fluoxetine inhibits cytokine release, we see the role of Fluoxetine in the treatment of SARS-CoV-2 infected patients of risk groups.

The influenza virus hemagglutinin (HA) facilitates viral entry into target cells. Cleavage of HA by host cell proteases is essential for viral infectivity and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease (TTSP) TMPRSS2 has been identified as an HA activator in cell culture and in the infected host. However, it is less clear whether TMPRSS2-related enzymes can also activate HA for spread in target cells. Moreover, the activity of cellular serine protease inhibitors against HA-activating TTSPs is poorly understood. Here, we show that TMPRSS11A, another member of the TTSP family, cleaves and activates the influenza A virus (FLUAV) HA and the Middle East respiratory syndrome coronavirus spike protein (MERS-S). Moreover, we demonstrate that TMPRSS11A is expressed in murine tracheal epithelium, which is a target of FLUAV infection, and in human trachea, suggesting that the protease could support FLUAV spread in patients. Finally, we show that HA activation by the TMPRSS11A-related enzymes HAT and DESC1 but not TMPRSS11A itself is blocked by the cellular serine protease Influenza viruses, members of the family Orthomyxoviridae, are enveloped viruses with a negative sense, segmented RNA genome. Influenza viruses readily adapt to immune pressure and thus constantly circulate in the human population, causing annual influenza epidemics (1,2). Moreover, reassortment of genomic segments between different influenza A viruses can result in the emergence of antigenically new viruses, which may spread pandemically (1,2). The constantly changing nature of influenza viruses also compromises the efficacy of currently available antivirals, since the viruses can rapidly acquire resistance-conferring mutations (3). This may not be the case if host cell factors were targeted, which are required for viral spread but dispensable for cellular survival, and the identification of such factors is in the focus of current research efforts (4).The viral hemagglutinin protein (HA) is incorporated into the viral envelope and facilitates influenza virus entry into target cells (5,6). For this, the surface unit of HA, termed HA1, binds to sialic acids on cell surface receptors and stimulates viral uptake into host cell endosomes. Thereafter, the transmembrane unit HA2 fuses the viral and the endosomal membrane, allowing the delivery of the viral genomic information into the cellular cytoplasm (5,6). HA is synthesized as an inactive precursor protein, HA0, and cleavage of HA0 by host cell proteases primes HA for membrane fusion, which is triggered by endosomal low pH (7,8). The priming of HA, which, for historic reasons, will subsequently be referred to as HA activation, is essential for viral infectivity.As a consequence, the host cell proteases responsible for HA activation are potential targets for antiviral intervention.Evidence is constantly accumulating that the type II transmembrane serine protease TMPRSS2 plays an important role in HA activation. Thus, TMPRSS2 activates H...