The Coronavirus Disease 2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome Related Coronavirus 2 (SARS-CoV-2) is a global health emergency. As only very limited therapeutic options are clinically available, there is an urgent need for the rapid development of safe, effective, and globally available pharmaceuticals that inhibit SARS-CoV-2 entry and ameliorate COVID-19 severity. In this study, we explored the use of small compounds acting on the homeostasis of the endolysosomal host-pathogen interface, to fight SARS-CoV-2 infection. We find that fluoxetine, a widely used antidepressant and a functional inhibitor of acid sphingomyelinase (FIASMA), efficiently inhibited the entry and propagation of SARS-CoV-2 in the cell culture model without cytotoxic effects and also exerted potent antiviral activity against two currently circulating influenza A virus subtypes, an effect which was also observed upon treatment with the FIASMAs amiodarone and imipramine. Mechanistically, fluoxetine induced both impaired endolysosomal acidification and the accumulation of cholesterol within the endosomes. As the FIASMA group consists of a large number of small compounds that are well-tolerated and widely used for a broad range of clinical applications, exploring these licensed pharmaceuticals may offer a variety of promising antivirals for host-directed therapy to counteract enveloped viruses, including SARS-CoV-2.

Influenza A virus (IAV) is a common pathogen of respiratory disease. The IAV-induced seasonal epidemics and the sporadic pandemics are associated with high morbidity and mortality. Therefore, effective protection and therapy for IAV infections is an important challenge in countering this public health threat. Because vaccinations only protect against known circulating strains, and the currently available antivirals pose the risk of resistance formation, drugs targeting host cell factors needed for viral replication offer a promising therapeutic approach. In this study, we describe the use of the antifungal therapeutics posaconazole and itraconazole in the therapy of IAV. We show that both drugs efficiently inhibit the propagation of IAV in the cell culture model without being cytotoxic. The mode of action is probably based on several targets and includes both a priming of the interferon response and the induced imbalance of cellular cholesterol. The antiviral effect of itraconazole could be confirmed in the mouse model, where the administration of itraconazole led to a drastic reduction in mortality and a significant increase in the survival rate. Thus, our data indicate a promising therapeutic potential of at least itraconazole in influenza therapy.

The Corona Virus Disease 2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome Related Coronavirus 2 (SARS-CoV-2) is a global health emergency. As only very limited therapeutic options are clinically available, there is an urgent need for the rapid development of safe, effective, and globally available pharmaceuticals that inhibit SARS-CoV-2 entry and ameliorate COVID-19. In this study, we explored the use of small compounds acting on the homeostasis of the endolysosomal host-pathogen interface, to fight SARS-CoV-2 infection. We find that fluoxetine, a widely used antidepressant and a functional inhibitor of acid sphingomyelinase (FIASMA), efficiently inhibits the entry and propagation of SARS-CoV-2 in the cell culture model without cytotoxic effects. Mechanistically, fluoxetine induced both impaired endolysosomal acidification and the accumulation of cholesterol within the endosomes. As the FIASMA group consists of a large number of small compounds that are well-tolerated and widely used for a broad range of clinical applications, exploring these licensed pharmaceuticals may offer a variety of promising antivirals for host-directed therapy to counteract SARS-CoV-2 and COVID 19.SignificanceOnly very limited therapeutic options are clinically available for treatment of Corona Virus Disease 2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome Related Coronavirus 2 (SARS-CoV-2), and the development of safe, effective, and globally available pharmaceuticals are urgently required. We report that the widely used antidepressant fluoxetine efficiently inhibits the early entry and propagation of SARS-CoV-2 in the cell culture model, and may offer a promising approach for host-directed therapy to counteract SARS-CoV-2 and COVID 19.

The Sonic hedgehog (Shh) pathway controls embryonic development and tissue homeostasis after birth. Long-lasting questions about this pathway are how dual-lipidated, firmly plasma membrane-associated Shh ligand is released from producing cells to signal to distant target cells, and how the resistance-nodulation-division transporter Dispatched (Disp) regulates this process. Here we show that Disp inactivation in Shh expressing cells impairs proteolytic Shh release from its lipidated terminal peptides, a process called ectodomain shedding. We also show reduced cholesterol export from Disp-deficient cells, that these cells contain increased cholesterol amounts in the plasma membrane, and that Shh shedding from Disp-deficient cells is restored by pharmacological membrane cholesterol extraction and by overexpressed transgenic Disp or structurally related Patched (Ptc, a putative cholesterol transporter). These data suggest that Disp can regulate Shh function via controlled cell surface shedding and that membrane cholesterol-related molecular mechanisms shared by Disp and Ptc exercise such sheddase control.

Staphylococcus aureus is one of the first and most prevalent pathogens cultured from the airways of cystic fibrosis (CF) patients, which can persist there for extended periods. Airway infections in CF patients are characterized by a strong inflammatory response of highly recruited neutrophils. One killing mechanism of neutrophils is the formation of neutrophil extracellular traps (NETs), which capture and eradicate bacteria by extracellular fibers of neutrophil chromatin decorated with antimicrobial granule proteins. S. aureus secretes nuclease, which can degrade NETs. We hypothesized, that S. aureus adapts to the airways of CF patients during persistent infection by escaping from NET-mediated killing via an increase of nuclease activity. Sputum samples of CF patients with chronic S. aureus infection were visualized by confocal microscopy after immuno-fluorescence staining for NET-specific markers, S. aureus bacteria and overall DNA structures. Nuclease activity was analyzed in sequential isogenic long persisting S. aureus isolates, as confirmed by whole genome sequencing, from an individual CF patient using a FRET-based nuclease activity assay. Additionally, some of these isolates were selected and analyzed by qRT-PCR to determine the expression of nuc1 and regulators of interest. NET-killing assays were performed with clinical S. aureus isolates to evaluate killing and bacterial survival depending on nuclease activity. To confirm the role of nuclease during NET-mediated killing, a clinical isolate with low nuclease activity was transformed with a nuclease expression vector (pCM28nuc). Furthermore, two sputa from an individual CF patient were subjected to RNA-sequence analysis to evaluate the activity of nuclease in vivo. In sputa, S. aureus was associated to extracellular DNA structures. Nuclease activity in clinical S. aureus isolates increased in a time-and phenotype-dependent manner. In the clinical isolates, the expression of nuc1 was inversely correlated to the activity of agr and was independent of saeS. NET-mediated killing was significantly higher in S. aureus isolates with low compared to isolates with high nuclease activity. Importantly, transformation of the clinical isolate with low nuclease activity with pCM28nuc conferred protection against NET-mediated killing confirming the beneficial role of nuclease for protection against NETs. Also, nuclease expression in in vivo sputa was high, which underlines the important role of nuclease within the highly inflamed CF airways. In conclusion, our data show that S. aureus adapts to the neutrophil-rich environment of CF airways with increasing nuclease expression most likely to avoid NET-killing during long-term persistence.