cells Article Aprotinin Inhibits SARS-CoV-2 Replication Denisa Bojkova 1, Marco Bechtel 1, Katie-May McLaughlin 2, Jake E. McGreig 2 , Kevin Klann 3 , Carla Bellinghausen 4 , Gernot Rohde 4 , Danny Jonigk 5,6, Peter Braubach 5,6, Sandra Ciesek 1,7,8 , Christian Münch 3,9,10 , Mark N. Wass 2,* , Martin Michaelis 2,* and Jindrich Cinatl, Jr. 1,* 1 Institute for Medical Virology, University Hospital, Goethe University, 60596 Frankfurt am Main, Germany; [email protected] (D.B.); [email protected] (M.B.); [email protected] (S.C.) 2 School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK; [email protected] (K.-M.M.); [email protected] (J.E.M.) 3 Faculty of Medicine, Institute of Biochemistry II, Goethe University, 60590 Frankfurt am Main, Germany; [email protected] (K.K.); [email protected] (C.M.) 4 Department of Respiratory Medicine and Allergology, University Hospital, Goethe University, 60590 Frankfurt am Main, Germany; [email protected] (C.B.); [email protected] (G.R.) 5 Institute of Pathology, Hannover Medical School (MHH), 30625 Hannover, Germany; [email protected] (D.J.); [email protected] (P.B.) 6 Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), The German Center for Lung Research (Deutsches Zentrum für Lungenforschung, DZL), Hannover Medical School (MHH), 30625 Hannover, Germany 7 German Center for Infection Research, DZIF, External Partner Site, 60596 Frankfurt am Main, Germany 8 Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Branch Translational Medicine und Pharmacology, 60596 Frankfurt am Main, Germany 9 Frankfurt Cancer Institute, Goethe University, 60596 Frankfurt am Main, Germany 10 Cardio-Pulmonary Institute, Goethe University, 60590 Frankfurt am Main, Germany * Correspondence: [email protected] (M.N.W); [email protected] (M.M.); [email protected] (J.C.J.) Received: 28 June 2020; Accepted: 28 October 2020; Published: 30 October 2020 Abstract: Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is the cause of the current coronavirus disease 19 (COVID-19) pandemic. Protease inhibitors are under consideration as virus entry inhibitors that prevent the cleavage of the coronavirus spike (S) protein by cellular proteases. Herein, we showed that the protease inhibitor aprotinin (but not the protease inhibitor SERPINA1/alpha-1 antitrypsin) inhibited SARS-CoV-2 replication in therapeutically achievable concentrations. An analysis of proteomics and translatome data indicated that SARS-CoV-2 replication is associated with a downregulation of host cell protease inhibitors. Hence, aprotinin may compensate for downregulated host cell proteases during later virus replication cycles. Aprotinin displayed anti-SARS-CoV-2 activity in different cell types (Caco2, Calu-3, and primary bronchial epithelial cell air–liquid interface cultures) and against four virus isolates. In conclusion, therapeutic aprotinin concentrations exert anti-SARS-CoV-2 activity. An approved aprotinin aerosol may have potential for the early local control of SARS-CoV-2 replication and the prevention of COVID-19 progression to a severe, systemic disease. Keywords: severe acute respiratory syndrome coronavirus; severe acute respiratory syndrome coronavirus 2; 2019-nCoV; COVID-19; antiviral; drug discovery; aprotinin Cells 2020, 9, 2377; doi:10.3390/cells9112377 www.mdpi.com/journal/cells Cells 2020, 9, 2377 2 of 13 1. Introduction Severe acute respiratory syndrome virus 2 (SARS-CoV-2), a novel betacoronavirus, causes a respiratory disease and pneumonia called coronavirus disease 19 (COVID-19) and is the cause of a current pandemic responsible for millions of cases and hundreds of thousands of deaths [1–7]. Drugs for the treatment of COVID-19 are urgently needed. Cell entry of coronaviruses is mediated by the interaction of the viral spike (S) protein with their host cell receptors, which differ between different coronaviruses [8]. For example, Middle East respiratory syndrome coronavirus (MERS-CoV) uses dipeptidyl peptidase 4 (DPP4) as a cellular receptor [8]. Host cell entry of SARS-CoV-2 and of the closely related severe acute respiratory syndrome virus (SARS-CoV) is mediated by angiotensin-converting enzyme 2 (ACE2) [8–10]. S binding to ACE2 depends on S cleavage at three sites (S1, S2, and S2’) by host cell proteases, typically by the transmembrane serine protease 2 (TMPRSS2), and can be inhibited by serine protease inhibitors [9,10]. Camostat was the first serine protease inhibitor that was shown to inhibit TMPRSS2 [9]. Subsequently, additional TMPRSS2 inhibitors, including nafamostat and Arbidol derivatives, were demonstrated to interfere with SARS-CoV-2 internalization into host cells [11–13]. Aprotinin is a serine protease inhibitor, which has previously been shown to inhibit TMPRSS2 and has been suggested as a treatment option for influenza viruses and coronaviruses [14,15]. Herein, we investigated the effects of aprotinin against SARS-CoV-2. 2. Materials and Methods 2.1. Drugs SERPINA1/alpha-1 antitrypsin (Prolastin) was obtained from Grifols (Barcelona, Spain). Aprotinin was purchased from Sigma-Aldrich (Darmstadt, Germany)). 2.2. Cell Culture The Caco2 cell line was obtained from DSMZ (Braunschweig, Germany), and Calu-3 from ATCC (Manassas, VA, US). The cells were grown at 37 ◦C in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL of penicillin, and 100 µg/mL of streptomycin. All culture reagents were purchased from Sigma-Aldrich. Cells were regularly authenticated by short tandem repeat (STR) analysis and tested for mycoplasma contamination. Lung tissue for the isolation of primary epithelial cells was provided by the Hannover Medical School, Institute of Pathology (Hannover, Germany). The use of tissue was approved by the ethics committee of the Hannover Medical School (MHH, Hannover, Germany, number 2701–2015) and was in compliance with The Code of Ethics of the World Medical Association. Primary bronchial epithelial cells were isolated from the lung explant tissue of a patient with lung emphysema as described previously [16]. All patients or their next of kin gave written informed consent for the use of their lung tissue for research. Basal cells were expanded in Keratinocyte-SFM medium supplemented with bovine pituitary extract (25 µg/mL), human recombinant epidermal growth factor (0.2 ng/mL, all from Gibco, Schwerte, Germany), isoproterenol (1 nM, Sigma), Antibiotic/Antimycotic Solution (Sigma-Aldrich), and MycoZap Plus PR (Lonza, Cologne, Germany) and cryopreserved until further use. For differentiation, the cells were thawed and passaged once in PneumaCult-Ex Medium (StemCell Technologies, Cologne, Germany) and then seeded on transwell inserts (12-well plate, Sarstedt, Nümbrecht, Germany) at 4 104 cells/insert. Once the cell layers reached confluency, the medium on × the apical side of the transwell was removed, and medium in the basal chamber was replaced with PneumaCult ALI Maintenance Medium (StemCell Technologies), including Antibiotic/Antimycotic Solution (Sigma-Aldrich) and MycoZap Plus PR (Lonza). During a period of four weeks, the medium was changed and the cell layers were washed with PBS every other day. Criteria for successful differentiation were the development of ciliated cells and ciliary movement, an increase in transepithelial electric resistance indicative of the formation of tight junctions, and mucus production. Cells 2020, 9, 2377 3 of 13 2.3. Virus Infection The isolates SARS-CoV-2/1/Human/2020/Frankfurt (SARS-CoV-2/FFM1), SARS-CoV-2/2/Human/ 2020/Frankfurt (SARS-CoV-2/FFM2), SARS-CoV-2/6/Human/2020/Frankfurt (SARS-CoV-2/FFM6), and SARS-CoV-2/7/Human/2020/Frankfurt (SARS-CoV-2/FFM7) were isolated and cultivated in Caco2 cells as previously described [17,18]. Virus titers were determined as TCID50/mL in confluent cells in 96-well microtiter plates [19,20]. 2.4. Antiviral Assay Confluent cell cultures were infected with SARS-CoV-2 in 96-well plates at a multiplicity of infection (MOI) of 0.01 in the absence or presence of the drug. The cytopathogenic effect (CPE) was assessed visually 48 h post-infection [19]. Concentrations that inhibited CPE formation by 50% (IC50) were determined using CalcuSyn (Biosoft, Cambridge, UK). 2.5. Viability Assay Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay modified after Mosman [21], as previously described [22]. Confluent cell cultures in 96-well plates were incubated with the drug for 48 h. Then, 25 µL of MTT solution (2 mg/mL (w/v) in PBS) were added per well, and the plates were incubated at 37 ◦C for an additional 4 h. After this, the cells were lysed using 200 µL of a buffer containing 20% (w/v) sodium dodecylsulfate and 50% (v/v) N,N-dimethylformamide with the pH adjusted to 4.7 at 37 ◦C for 4 h. Absorbance was determined at 570 nm for each well using a 96-well multiscanner (Tecan, Crailsheim, Germany). After subtracting of the background absorption, the results are expressed as percentage viability relative to control cultures that received no drug. Drug concentrations that inhibited cell viability by 50% (CC50) were determined using CalcuSyn (Biosoft). 2.6. Immunostaining for SARS-CoV-2 S Protein Immunostaining was performed as previously described [23], using a monoclonal antibody directed against SARS-CoV-2 S protein (1:1500 dilution, Sino Biological, Eschborn, Germany) 24 h post-infection. 2.7. Caspase 3/7 Activation Caspase 3/7 activation was determined using the Caspase-Glo® 3/7 Assay (Promega, Walldorf, Germany) according to the manufacturer’s instructions. 2.8. qPCR SARS-CoV-2 RNA from the cell culture supernatant samples was isolated using AVL buffer and the QIAamp Viral RNA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Absorbance-based quantification of the RNA yield was performed using the Genesys 10S UV-Vis Spectrophotometer (Thermo Fisher Scientific, Dreieich, Germany). RNA was subjected to OneStep qRT-PCR analysis using the Luna Universal One-Step RT-qPCR Kit (New England Biolabs, Frankfurt am Main, Germany) and a CFX96 Real-Time System, C1000 Touch Thermal Cycler (Bio-Rad, Feldkirchen, Germany).