key: cord-1036255-679qfp2s authors: De Meyer, Sandra; Bojkova, Denisa; Cinati, Jindrich; Van Damme, Ellen; Buyck, Christophe; Van Loock, Marnix; Woodfall, Brian; Ciesek, Sandra title: Lack of Antiviral Activity of Darunavir against SARS-CoV-2 date: 2020-04-08 journal: nan DOI: 10.1101/2020.04.03.20052548 sha: a68fdcd0f4e55fda6918c216bc3a5b37ef45289e doc_id: 1036255 cord_uid: 679qfp2s Given the high need and the absence of specific antivirals for treatment of COVID-19 (the disease caused by severe acute respiratory syndrome-associated coronavirus-2 [SARS-CoV-2]), human immunodeficiency virus (HIV) protease inhibitors are being considered as therapeutic alternatives. Prezcobix/Rezolsta is a fixed-dose combination of 800 mg of the HIV protease inhibitor darunavir (DRV) and 150 mg cobicistat, a CYP3A4 inhibitor, which is indicated in combination with other antiretroviral agents for the treatment of HIV infection. There are currently no definitive data on the safety and efficacy of DRV/cobicistat for treatment of COVID-19. The in vitro antiviral activity of darunavir against a clinical isolate from a patient infected with SARS-CoV-2 was assessed. DRV showed no activity against SARS-CoV-2 at clinically relevant concentrations (EC50 >100 μM). Remdesivir, used as a positive control, showed potent antiviral activity (EC50 = 0.38 μM). Overall, the data do not support the use of DRV for treatment of COVID-19. In December 2019, the severe acute respiratory syndrome-associated coronavirus disease-2 2019 (SARS-CoV-2; COVID-19) emerged in Wuhan, Hubei Province, China (1) . The virus was subsequently identified as a coronavirus (CoV), in addition to SARS-CoV-1 and Middle East respiratory syndrome CoV (MERS-CoV) that passed from animals to humans where it can cause severe respiratory illness (2) . As of March 2020, COVID-19 has spread around the world with the WHO declaring a global pandemic (3) . Given the extent of the COVID-19 pandemic, there is an urgent need to identify potential treatments for the disease as well as to develop a vaccine. As no specific antivirals for treatment of COVID-19 are available, one avenue of clinical interest is the use of human immunodeficiency virus (HIV) protease inhibitors (PIs) as a therapeutic intervention. The potential for HIV PIs as a treatment for COVID-19 is mainly based on limited virologic and clinical data on the HIV protease inhibitor lopinavir with low-dose ritonavir (as a pharmacoenhancer; LPV/r) in patients infected with severe acute respiratory syndrome related to a coronavirus (SARS-CoV) (4) . After demonstrating the in vitro antiviral activity of LPV against SARS-CoV-1, the clinical response of patients with SARS to a combination of LPV/r and ribavirin was examined. Patients treated with LPV/r had lower rates of adverse clinical outcomes at day 21 following the onset of symptoms compared with historical controls (4). However, recent data in hospitalized adults with severe confirmed COVID-19 treated with LPV/r in addition to a standard care of ventilation, oxygen, vasopressor support, antibiotics and renalreplacement therapy showed that there was no significant improvement in time to clinical improvement or mortality at day 28 compared with the standard care (5). The HIV PI darunavir with cobicistat as a pharmacoenhancer (DRV/c, 800/150 mg given orally once daily with food) in combination with other antiretroviral agents is approved for both treatment-naïve and -experienced patients with HIV-1 infection (6-7). The efficacy and safety All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.03.20052548 doi: medRxiv preprint profile of boosted-DRV combination therapy is well-established in the HIV setting, based on phase III clinical studies as well as real-world evidence (8) (9) (10) (11) . To date, no clear clinical evidence supports the use of DRV (boosted with either ritonavir or cobicistat) in viral diseases other than HIV. In this paper, the antiviral activity of DRV against SARS-CoV-2 was investigated in an in vitro model at clinically relevant concentrations. When DRV/c is taken at the indicated once-daily dose, the median trough plasma concentration of DRV was 3.4 µM (1875 ng/mL). (6) . This cell culture assay was shown to be suitable for antiviral assays. Productive viral infection takes place in this model with the number of SARS-CoV RNA molecules increasing continuously after infection, indicating that the virus undergoes full replicatory cycles (12) . Remdesivir (GS-5734), a nucleotide analog initially developed for Ebola virus disease, has shown to inhibit SARS-CoV-2 replication in vitro with an EC 50 equal to 0,770 µM (13) and was therefore used as a positive control. All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.03.20052548 doi: medRxiv preprint Human colon carcinoma cell line (Caco-2) cells (obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) were cultured in Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and containing penicillin (100 IU/mL) and streptomycin (100 μ g/mL) in a 5% CO 2 atmosphere at 37°C. All culture reagents were purchased from Sigma (Hamburg, Germany). SARS-CoV-2 was isolated from human samples and cultured in Caco-2 cells, as previously described (3). After one passage in Caco-2 cells, viral stocks were stored at -80°C prior to use. Confluent layers of Caco-2 cells were cultured at 37°C in a 5% CO 2 atmosphere for 72 hours on author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.03.20052548 doi: medRxiv preprint the MTT method was done for two of the three experiments. Data were analyzed by a fourparameter curve-fitting from a dose-response curve using GraphPad Prism (version 7.00) to calculate the EC 50 (concentration of the compound that inhibited 50% of the infection) based on visual CPE scoring or based on the MTT method. To assess the effects of the compounds on Caco-2 cell viability, cell viability was measured in confluent cell layers treated with a range of compound concentrations in absence of virus using the Rotitest Vital (Roth) according to manufacturer's instructions, as previously described (12) . All assays were performed three times independently in triplicate. From this the CC 50 (cytotoxic concentration of the compound that reduced cell viability to 50%) was calculated from a doseresponse curve in GraphPad Prism (version 7.00) using four-parameter curve-fitting. The selectivity index for each of the compounds was determined as the ratio of the CC 50 to the EC 50 . In general, in silico docking can be a useful approach for identifying subsets of molecules for in vitro studies and can be used to explain in vitro observations on a structural level. The coordinates of the crystal structure of the main SARS-CoV-2 protease were retrieved from the PDB database (https://www.rcsb.org/structure/6lu7). author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.03.20052548 doi: medRxiv preprint 'quickprep' settings to prepare the protein complex. The original ligand was then removed. General docking settings were then altered to have 50 initial placements. All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.03.20052548 doi: medRxiv preprint Remdesivir showed strong antiviral activity against SARS-CoV-2 with an EC50 of 0.11 µM based on visual scoring of inhibition of CPE (Figure 1a) author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.03.20052548 doi: medRxiv preprint Current efforts to manage the COVID-19 pandemic have largely focused on improved hygiene, quarantine of infected individuals, social distancing to limit transmission and development of a vaccine (15) . Despite the expedited efforts to develop a vaccine and collaborative efforts to screen compounds in discovery and development across the broader pharmaceutical industry for activity against COVID-19, patients are in immediate need of therapeutic interventions (12, 16) . Current data on the therapeutic effect of HIV protease inhibitors in patients with COVID-19 are far from comprehensive. This study demonstrated that DRV showed no in vitro antiviral activity against SARS-CoV-2 at clinically relevant concentrations. Furthermore, structural analyses using protease structures are consistent with these data. DRV binds to the active site of the HIV virus' dimeric aspartic protease (14) . The crystal structure of this protease is well-elucidated and has been shown to have an extensive hydrogen-bonding network with DRV, allowing for the potent in vitro activity (EC 50 values = 1.2 to 8.5 nM) of this protease inhibitor against HIV (6-7). By contrast, the SARS-CoV-2 main protease is a cysteine protease (Protein Data Bank-code 6LU7) and while several docking poses have been found for DRV in in silico models, unlike in HIV, these poses showed little interaction with the SARS-CoV-2 main protease active site. Several publications describe in silico docking experiments on the main coronavirus protease that specifically focus on or include DRV (17) (18) (19) (20) (21) . Although these studies suggest DRV as a candidate for further investigation, such promising docking results could not be reproduced in our in silico docking studies. Such discrepancies can often result from in silico docking, which is primarily a useful approach for identifying subsets of molecules for in vitro activity testing. No in vitro antiviral activity of DRV against SARS-CoV-2 was found in the experiments reported here. All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04. 03.20052548 doi: medRxiv preprint In this study, remdesivir demonstrated activity against SARS-CoV-2 with an EC 50 of 0.38 μ M, which is in line with the earlier reported remdesivir EC 50 of 0.77 µM, indicating that the in vitro antiviral assay used is appropriate to assess antiviral activity against SARS-CoV-2 (12) . In conclusion, the lack of in vitro antiviral activity of DRV against SARS-CoV-2 does not support the use of DRV for treatment of COVID-19. Hence, the use of DRV (boosted with either ritonavir or cobicistat) should remain solely for treatment of patients with HIV infection. All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.03.20052548 doi: medRxiv preprint A novel coronavirus from patients with pneumonia in China Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding Evidence of SARS-CoV-2 infection in returning travelers from Wuhan, China Role of lopinavir/ritonavir in the treatment of SARS: Initial virological and clinical findings A trial of lopinavir-ritonavir in adults hospitalized with severe Covid-19 Prezcobix prescribing information Rezolsta Summary of product characteristics Cobicistat-boosted darunavir in HIV-1-infected adults: week 48 results of a Phase IIIb, open-label single-arm trial All rights reserved. No reuse allowed without permission author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the Final 192-week efficacy and safety of once-daily darunavir/ritonavir compared with lopinavir/ritonavir in HIV-1-infected treatment-naïve patients in the ARTEMIS trial Week 48 analysis of once-daily vs. twice-daily darunavir/ritonavir in treatment-experienced HIV-1-infected patients Profile of once-daily darunavir/cobicistat fixed-dose combination for the treatment of HIV SARS-CoV-2 infected host cell proteomics reveal potential therapy targets Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro Structural and thermodynamic basis for the binding of TMC114, a next-generation human immunodeficiency virus type 1 protease inhibitor Therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in Wuhan, China. F1000Res Covid-19 -The search for effective therapy Potential therapeutic agents for COVID-19 based on the analysis of protease and RNA All rights reserved author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the Available at Shanghai Institute of Materia Medica Insight derived from molecular docking and molecular dynamics simulations into the binding interactions between HIV-1 protease inhibitors and SARS-CoV-2 3CLpro Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Identification of FDA approved drugs targeting COVID-19 virus by structure-based drug repositioning Medical writing supporting for the development of this manuscript was provided by Patrick Hoggard of Zoetic Science, an Ashfield company, part of UDG Healthcare plc; this support was funded by Janssen Pharmaceuticals. We thank Lena Stegmann for technical support by antiviral assays. All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.03.20052548 doi: medRxiv preprint author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is the All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.03.20052548 doi: medRxiv preprint