key: cord-350903-nwagvvc5 authors: Softic, Laurent; Brillet, Rozenn; Berry, François; Ahnou, Nazim; Nevers, Quentin; Morin-Dewaele, Margot; Hamadat, Sabah; Bruscella, Patrice; Fourati, Slim; Pawlotsky, Jean-Michel; Ahmed-Belkacem, Abdelhakim title: Inhibition of SARS-CoV-2 Infection by the Cyclophilin Inhibitor Alisporivir (Debio 025) date: 2020-06-23 journal: Antimicrob Agents Chemother DOI: 10.1128/aac.00876-20 sha: doc_id: 350903 cord_uid: nwagvvc5 Cyclophilins play a key role in the life cycle of coronaviruses. Alisporivir (Debio 025) is a nonimmunosuppressive analogue of cyclosporine with potent cyclophilin inhibition properties. Alisporivir reduced SARS-CoV-2 RNA production in a dose-dependent manner in Vero E6 cells, with a 50% effective concentration (EC(50)) of 0.46 ± 0.04 μM. Alisporivir inhibited a postentry step of the SARS-CoV-2 life cycle. These results justify rapidly conducting a proof-of-concept phase 2 trial with alisporivir in patients with SARS-CoV-2 infection. I n December 2019, an outbreak of pneumonia emerged in the Chinese city of Wuhan. A novel coronavirus was identified as the pathogen causing the disease, named COVID-19 (for coronavirus disease 2019). This new virus was called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) because of its genetic proximity to SARS-CoV. At the time of writing, over 3.5 million people have been diagnosed with COVID-19 worldwide, while over 250,000 of them have died from complications of the disease. Currently, there are no vaccines or effective antiviral drugs targeting SARS-CoV-2. A pragmatic approach is to assess whether drugs that are approved for other indications or have reached late clinical developmental stages are effective against SARS-CoV-2 and could be rapidly repurposed for this indication. For instance, chloroquine has been shown to bear potent antiviral properties against SARS-CoV-2 in vitro, and several clinical trials are under way to assess its efficacy in patients with COVID-19. The nucleotide analogues remdesivir and favipiravir, as well as the antiretroviral drug lopinavir in combination with ritonavir, are also under clinical investigation. Cyclophilins are cellular peptidyl-prolyl cis-trans isomerases that catalyze the interconversion of the two energetically preferred conformers of the planar peptide bond preceding an internal proline residue. Cyclophilins play a key role in the life cycle of many coronaviruses, including human coronaviruses 229E (HCoV-229E) and NL-63 (HCoV-NL63), feline infectious peritonitis coronavirus (FPIV), SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) (1-7). Cyclosporine A (CsA), a potent cyclophilin inhibitor, blocks the replication of various coronaviruses in vitro, including HCoV-229E, HCoV-NL63, FPIV, mouse hepatitis virus (MHV), avian infectious bronchitis virus, and SARS-CoV (5, (8) (9) (10) . However, CsA cannot be used in patients with COVID-19 because of its strong immunosuppressive properties. Alisporivir (Debio 025) is a nonimmunosuppressive analogue of CsA that potently inhibits cyclophilins. Alisporivir has been administered to more than 1,800 patients with chronic hepatitis C virus infection in phase 2 and 3 clinical trials, alone or in combination with pegylated interferon alpha and/or ribavirin. In vitro, alisporivir inhibits the replication of HCoV-229E, HCoV-NL63, MHV, SARS-CoV, and MERS-CoV at lowmicromolar concentrations without cytotoxic effect (1, 10, 11) . The goal of this study was to assess the antiviral properties of alisporivir against SARS-CoV-2, with the objective of generating the preclinical proof of concept of antiviral effectiveness required to start a clinical trial in patients with COVID-19. The antiviral effectiveness of increasing concentrations of alisporivir was measured in Vero E6 cells infected with a clinical isolate of SARS-CoV-2 at a multiplicity of infection (MOI) of 0.02 (Fig. 1A) . Dimethyl sulfoxide (DMSO) was used as a negative control, while chloroquine was used as a positive control of antiviral inhibition. The compounds were added at the beginning of infection, and viral RNA was extracted from supernatants at 48 h postinfection and quantified by reverse transcriptase quantitative PCR (RT-qPCR). Alisporivir reduced SARS-CoV-2 RNA production in a dose-dependent manner: the 50% effective concentration (EC 50 ) was 0.46 Ϯ 0.04 M, and the EC 90 was 3.10 Ϯ 1.40 M. The maximum viral RNA reduction was 2 log 10 at 5 M. For comparison, the EC 50 of chloroquine was 0.35 Ϯ 0.02 M (Fig. 1A) . Neither alisporivir nor chloroquine was cytotoxic at the effective concentration, with 50% cytotoxic concentrations (CC 50 s) of Ͼ20 M and therapeutic indexes of Ͼ43 and Ͼ57, respectively. We confirmed the anti-SARS-CoV-2 effectiveness of alisporivir by immunofluorescence. Vero E6 cells were infected at an MOI of 0.4 for 2 h in the presence of increasing concentrations of alisporivir. After virus removal, infected cells were incubated for 24 h in the presence of alisporivir and immunostained with an anti-double-stranded-RNA (dsRNA) antibody. Alisporivir reduced the number of SARS-CoV-2-infected cells in a dose-dependent manner, and complete inhibition was attained at 10 M (Fig. 1B) . Chloroquine also inhibited SARS-CoV-2 in this assay (data not shown). The next experiment was aimed at identifying the step of the SARS-CoV-2 life cycle targeted by alisporivir. Chloroquine, which inhibits endosome-mediated viral entry, was used as a control. Vero E6 cells were infected at an MOI of 0.4 for 2 h in the presence of 5 M alisporivir or chloroquine. After virus removal, cells were incubated for 7 h in the absence of the compounds, fixed, and immunostained with the anti-dsRNA antibody. No infected cells were detected in the presence of 5 M chloroquine, confirming that chloroquine prevents SARS-CoV-2 entry into Vero E6 cells. In contrast, alisporivir did not inhibit SARS-CoV-2 entry into Vero E6 cells (Fig. 1C) . This result was confirmed by a time-of-addition experiment showing that, in contrast to that of chloroquine, the effect of alisporivir was preserved when the compound was added 3 h postinfection. The antiviral effect of alisporivir was abolished when the compound was added 6 h postinfection (Fig. 1D) . These results suggest that alisporivir inhibits a postentry step of the SARS-CoV-2 life cycle. Taken together, our results demonstrate that the nonimmunosuppressive macrocyclic cyclophilin inhibitor alisporivir (Debio 025) exhibits strong, dose-dependent antiviral properties against SARS-CoV-2 in vitro. Alisporivir inhibits a postentry step of the SARS-CoV-2 life cycle through mechanisms that remain to be unraveled. These results justify rapidly conducting a proof-of-concept phase 2 trial to assess the antiviral properties and the effect of alisporivir on COVID-19 clinical outcomes in infected patients. Alisporivir has been shown to be well tolerated when administered as a monotherapy (12) . Preclinical pharmacology data indicate that, after oral administration, alisporivir is widely distributed in the whole body, including the lungs, and that its EC 90 against SARS-CoV-2 in Vero E6 cells is clinically achievable in patients. In addition, because alisporivir inhibits all cellular cyclophilins, it also blocks mitochondrial cyclo- philin D, a key regulator of mitochondrial permeability transition pore (mPTP) opening, a mechanism involved in triggering cell death. Therefore, besides its antiviral properties, alisporivir may also be effective in preventing lung tissue damage. A phase 2, proof-of-concept trial with alisporivir in patients with COVID-19 is planned to start very soon. Human coronavirus NL63 replication is cyclophilin A-dependent and inhibited by non-immunosuppressive cyclosporine A-derivatives including alisporivir Function of HAb18G/CD147 in invasion of host cells by severe acute respiratory syndrome coronavirus Cyclophilins and cyclophilin inhibitors in nidovirus replication Nucleocapsid protein of SARS coronavirus tightly binds to human cyclophilin A The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors Feline coronavirus replication is affected by both cyclophilin A and cyclophilin B Genetic deficiency and polymorphisms of cyclophilin A reveal its essential role for human coronavirus 229E replication Cyclosporin A inhibits the replication of diverse coronaviruses Suppression of feline coronavirus replication in vitro by cyclosporin A Influences of cyclosporin A and non-immunosuppressive derivatives on cellular cyclophilins and viral nucleocapsid protein during human coronavirus 229E replication Alisporivir inhibits MERS-and SARS-coronavirus replication in cell culture, but not SARS-coronavirus infection in a mouse model Alisporivir plus ribavirin, interferon free or in combination with pegylated interferon, for hepatitis C virus genotype 2 or 3 infection This work was funded by the Fondation pour la Recherche Médicale and the Agence Nationale de la Recherche, Call ANR FLASH COVID-19.Alisporivir was kindly provided by Debiopharm, Lausanne, Switzerland. S.F. has served as an advisor and/or speaker for Abbvie; J.-M.P. has served as an advisor and/or speaker for Abbvie, Gilead, Merck, and Siemens Healthcare; A.A.-B. has served as a speaker for Abbvie.