24 25 Cyclophilins play a key role in the lifecycle of coronaviruses. Alisporivir (Debio 025) is 26 a non-immunosuppressive analogue of cyclosporin A with potent cyclophilin inhibition 27 properties. Alisporivir reduced SARS-CoV-2 RNA production in a dose-dependent manner in 28 VeroE6 cell line, with an EC 50 of 0.46±0.04 µM. Alisporivir inhibited a post-entry step of the 29 SARS-CoV-2 lifecycle. These results justify that a proof-of-concept Phase 2 trial be rapidly 30 conducted with alisporivir in patients with SARS-CoV-2 infection. 31 32 33 on June 9, 2020 by guest http://aac.asm.org/ Downloaded from 101 SARS-CoV-2 life cycle through mechanisms that remain to be unraveled. These results justify 102 that a proof-of-concept Phase 2 trial be rapidly conducted to assess the antiviral properties 103 and the effect of alisporivir on COVID-19 clinical outcomes in infected patients. 104 on June 9, 2020 by guest http://aac.asm.org/ Downloaded from 6Alisporivir has been shown to be well tolerated when administered as a 105 monotherapy 12 . Preclinical pharmacology data indicate that, after oral administration, 106 alisporivir is widely distributed in the whole body, including the lungs, and that its EC 90 107 against SARS-CoV-2 in VeroE6 cells is clinically achievable in patients. In addition, because 108 alisporivir inhibits all cellular cyclophilins, it also blocks mitochondrial cyclophilin-D, a key 109 regulator of mitochondrial permeability transition pore (mPTP) opening, a mechanism 110

Small Extracellular Vesicles (sEVs) are 50–200 nm in diameter vesicles delimited by a lipid bilayer, formed within the endosomal network or derived from the plasma membrane. They are secreted in various biological fluids, including airway nasal mucus. The goal of this work was to understand the role of sEVs present in the mucus (mu‐sEVs) produced by human nasal epithelial cells (HNECs) in SARS‐CoV‐2 infection. We show that uninfected HNECs produce mu‐sEVs containing SARS‐CoV‐2 receptor ACE2 and activated protease TMPRSS2. mu‐sEVs cleave prefusion viral Spike proteins at the S1/S2 boundary, resulting in higher proportions of prefusion S proteins exposing their receptor binding domain in an ‘open’ conformation, thereby facilitating receptor binding at the cell surface. We show that the role of nasal mu‐sEVs is to complete prefusion Spike priming performed by intracellular furin during viral egress from infected cells. This effect is mediated by vesicular TMPRSS2 activity, rendering SARS‐CoV‐2 virions prone to entry into target cells using the ‘early’, TMPRSS2‐dependent pathway instead of the ‘late’, cathepsin‐dependent route. These results indicate that prefusion Spike priming by mu‐sEVs in the nasal cavity plays a role in viral tropism. They also show that nasal mucus does not protect from SARS‐CoV‐2 infection, but instead facilitates it.

The 2019 global coronavirus (COVID-19) pandemic has brought the world to a grinding halt, highlighting the urgent need for therapeutic and preventive solutions to slow the spread of emerging viruses. The objective of this study was to assess the anti-SARS-CoV-2 effectiveness of 8 FDA-approved cationic amphiphilic drugs (CADs). SARS-CoV-2-infected Vero cells, Calu-3 cells and primary Human Nasal Epithelial Cells (HNEC) were used to investigate the effects of CADs and revealed their antiviral mode of action. Among the CADs tested, desloratadine, a commonly used antiallergic, well-tolerated with no major side effects, potently reduced the production of SARS-CoV-2 RNA in Vero-E6 cells. Interestingly, desloratadine was also effective against HCoV-229E and HCoV-OC43 showing that it possessed broad-spectrum anti-coronavirus activity. Investigation of its mode of action revealed that it targeted an early step of virus lifecycle and blocked SARS-CoV-2 entry through the endosomal pathway. Finally, the ex vivo kinetic of the antiviral effect of desloratadine was evaluated on primary Human Nasal Epithelial Cells (HNEC), showing a significant delay of viral RNA production with a maximal reduction reached after 72 h of treatment. Thus, this treatment could provide a substantial contribution to prophylaxis and systemic therapy of COVID-19 or other coronaviruses infections and requires further studies.