COBICISTAT FOR PREVENTION AND/OR TREATMENT OF CORONAVIRUS INFECTIONS

Abstract

The present invention relates to cobicistat and its derivatives or prodrugs for use in the prophylaxis and/or treatment of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, severe acute respiratory syndrome coronavirus (SARS-CoV) infection and/or Middle East respiratory syndrome coronavirus (MERS-CoV) infection. The present invention further relates to methods of prevention and/or treatment of SARS-CoV-2 infection.

Claims

1. A composition comprising cobicistat, or a derivative or prodrug thereof, for use in the prophylaxis and/or treatment of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, severe acute respiratory syndrome coronavirus (SARS-CoV) infection and/or Middle East respiratory syndrome coronavirus (MERS-CoV) infection, wherein said derivative or prodrug is ritonavir or desoxy-ritonavir, and wherein said composition comprises a further drug selected from remdesivir, chloroquine, hydroxychloroquine, molnupiravir, tipranavir, nelfinavir, lopinavir, atazanavir, plitidepsin, favipiravir, an anti-inflammatory glucocorticoid, januskinase (JAK) inhibitor, a palmitoyl protein thioesterase 1 (PPT1) inhibitor, and a monoclonal antibody targeting viral replication or host inflammation.

2-5. (canceled)

6. The composition according to claim 1, wherein said further drug is selected from dexamethasone, prednisone, methylprednisolone, hydrocortisone, baricitinib, ruxolitinib, upadacitinib, GNS561, and tocilizumab.

7. The composition according to claim 1, wherein the further drug is remdesivir.

8. The composition according to claim 1, wherein the further drug is at least one of remdesivir, tipranavir, chloroquine, hydroxychloroquine, molnupiravir, favipiravir, nelfinavir, lopinavir, atazanavir, plitidepsin, dexamethasone, baricitinib, and GNS561.

9. The composition according to claim 6, further comprising one or more further drug, selected from tipranavir, nelfinavir, lopinavir, and atazanavir.

10. The composition according to claim 1, comprising cobicistat, or a derivative or prodrug thereof, and chloroquine in combination with one or more further drugs selected from tipranavir, nelfinavir, lopinavir, and atazanavir.

11. The composition according to claim 1, wherein cobicistat is present in a therapeutically effective amount, which is higher than the dosage used for HIV-1 treatment.

12-16. (canceled)

17. A method of prevention and/or treatment of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, severe acute respiratory syndrome coronavirus (SARS-CoV) infection and/or Middle East respiratory syndrome coronavirus (MERS-CoV) infection, comprising the step of: administering, to a subject in need of such prevention and/or treatment, a therapeutically effective amount of cobicistat or a derivative or prodrug thereof wherein said derivative or prodrug is ritonavir or desoxy-ritonavir.

18. The method of claim 17, wherein the therapeutically amount is higher than the dosage used for HIV-1 treatment.

19. The method of claim 17, wherein administration of cobicistat is oral, at a daily dosage in the range from 300 to 1,000 mg, or wherein administration of cobicistat is intranasal and/or via inhalation and the administration is via a dry powder inhaler, or via a nebulizer or a soft mist spray dispenser.

20. (canceled)

21. The method of claim 19, wherein administration is intranasal and/or inhalation and the amount administered is in the range from 2 to 15 μM per day.

22. The method according to claim 17, wherein prophylaxis comprises pre- and post-exposure prophylaxis to SARS-CoV-2 infection.

23. The method according to claim 17, wherein said cobicistat, or derivative or prodrug thereof, is administered in combination with one or more further drug.

24. The method according to claim 23, wherein the one or more further drug is selected from remdesivir, chloroquine, hydroxychloroquine, molnupiravir, and favipiravir.

25. The method according to claim 23, wherein the one or more further drug is selected from tipranavir, nelfinavir, lopinavir, and atazanavir.

26. The method according to claim 23, wherein the one or more further drug is selected from plitidepsin, dexamethasone, prednisone, methylprednisolone, hydrocortisone, baricitinib, ruxolitinib, upadacitinib, GNS561, and tocilizumab.

27. The method according to claim 23, wherein the further drug is remdesivir.

28. The method according to claim 23, wherein the one or more further drug is selected from remdesivir, tipranavir, chloroquine, hydroxychloroquine, molnupiravir, favipiravir, nelfinavir, lopinavir, atazanavir, plitidepsin, dexamethasone, baricitinib, and GNS561.

29. The method according to claim 23, wherein cobicistat is administered in combination with remdesivir and further in combination with one or more further drug, selected from tipranavir, nelfinavir, lopinavir, and atazanavir.

30. The method according to claim 23, wherein cobicistat is administered in combination with chloroquine in combination with one or more further drug selected from tipranavir, nelfinavir, lopinavir, and atazanavir.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0126] FIG. 1 Cobicistat is an inhibitor of SARS-CoV-2 replication.

[0127] A-C) In-silico docking (A,B) and molecular dynamics (C-E) analysis of the putative mode and energy of binding of cobicistat to SARS-CoV-2 3CL.sub.pro.

[0128] A) Docking pose showing the ligand interaction of cobicistat to the active site of 3CL.sub.pro and the formation of hydrogen bonds to ASN142, GLY143 and GLN189 of 3CL.sub.pro.

[0129] B) Overlay of crystal structures of SARS-Cov-2 3CL.sub.pro showing the amino acids important for the binding of cobicistat to the active site of the enzyme. Residues of the catalytic dyad (Cys145 and His41) of 3CL.sub.pro were among the highest contributors to non covalent binding to cobicistat. The source and list of structures used are detailed in Example 1.

[0130] C) Schematic representation of time course experiments evaluating in vitro inhibition of SARS-CoV-2 replication by cobicistat.

[0131] D,E) Effect of various concentrations of cobicistat, added according to the scheme of (D) on intracellular and supernatant SARS-CoV-2 RNA content in Calu-3 cells. Viral RNA content was measured by qPCR using the 2019-nCoV_N1 primer set (Center of Disease Control). Fold change values in intracellular RNA (D) were calculated by the delta-delta CT method, using the Tata-binding protein (TBP) gene as housekeeper control. Expression levels in supernatant (E) were quantified using an in vitro transcribed standard curve generated as described in Example 1. Data are expressed as mean with SD and were analyzed by two-way ANOVA followed by Dunnet's post-test (N=3 independent experiments). *P<0.05; **P<0.01; ***P<0.001.

[0132] FIG. 2. Cobicistat decreases replication of SARS-CoV-2 and rescues viability of infected cells in multiple in vitro models.

[0133] A,B) Effect of serial dilutions of cobicistat on SARS-CoV-2 RNA concentration in supernatants (A) and on the viability of infected and uninfected cell lines of lung (Calu-3), gut (T84) and kidney (Vero E6) origin (A,B). Cells were infected with SARS-CoV-2 at two different MOIs (0.05 and 0.5) and left untreated or treated with cobicistat two hours post-infection. Forty-eight hours post-infection supernatants were collected and viral RNA was assayed by qPCR while cellular viability was measured by MTT assay (A) or by crystal violet staining (B). Inhibition of viral replication was calculated as described in Example 1 while viability data were normalized to the uninfected or to the untreated control. Half maximal inhibitory (IC50) concentration values were calculated by nonlinear regression. Each point in panel A represents a mean of 3 independent experiments. Pictures in panel B are derived from infections at MOI 0.5 (Calu-3 and T84 cells) or MOI 0.05 (Vero E6 cells).

[0134] C) Comparison between the IC50 and CC50 values of cobicistat determined in vitro and the peak plasma levels detectable in mice and in after administration of a single dose of the drug. Determination of in vitro CC50 values is based on the data shown in (D).

[0135] D) Uninfected cell lines of lung (Calu-3), gut (T84) and kidney (Vero E6) origin were left untreated or treated with serial dilutions of cobicistat. Forty-eight hours post-treatment cellular viability was measured by MTT assay. Data, expressed as mean±SD of three independent experiments, were normalized to the untreated control and CC50 values were calculated by nonlinear regression.

[0136] FIG. 3. Cobicistat decreases SARS-CoV-2 S-protein content and syncytia formation.

[0137] A,B). Screening of putative inhibitors of the enzymatic activity of 3CL.sub.pro. The activity of 3 CL.sub.pro was measured by FRET assay and normalized over the untreated condition (A). Apart from cobicistat, compounds tested included HIV-1 protease inhibitors [nelfinavir, tipranavir] and compounds previously administered in clinical trials as SARS-CoV-2 therapeutics [darunavir, lopinavir], as well as two positive controls known to inhibit 3CL.sub.pro activity [MG132 and GC376]. EC50 values were calculated by nonlinear regression (B).

[0138] C) Effect of cobicistat on the expression of S- and N-proteins in SARS-CoV-2 infected Vero E6 cells. Cells were infected at 0.5 MOI and left untreated or treated, two hours post-infection, with various concentrations of cobicistat, of the RdRP inhibitor remdesivir, or the 3CL.sub.pro inhibitor GC376. Cells were harvested 24 hours post-treatment and subjected to protein extraction and subsequent analysis by Western Blot. Expression of S- and N-proteins, and expression of the housekeeping protein actin-(3, were detected using primary monoclonal antibodies followed by incubation with fluorescent-conjugated secondary antibodies and detection on a LI-COR Odyssey® CLx instrument. Data are representative of three independent experiments.

[0139] D,E) Effect of cobicistat on S-protein-mediated syncytia formation. Vero E6 cells were transfected with the SARS-CoV-2 S-protein and left untreated or treated with various concentrations of cobicistat or with sera isolated from convalescent SARS-CoV-2 patients (1:100 dilution). Syncytia formation was examined 24 hours post-transfection by immunofluorescence (IF) staining for DAPI and S-protein (D) and quantified as the number of cells forming syncytia (E). Data were analyzed using the nonparametric Kruskal-Wallis test followed by Dunn's post-test. Horizontal lines represent mean values. **P<0.01; ****P<0.0001. Scale bar=50 μM.

[0140] FIG. 4. Expression of the metabolic targets of cobicistat in uninfected and SARS-CoV-2 infected cell lines and primary human organoids.

[0141] A,B) The relative expression of CYP3A4/5 and P-gp was analyzed by qPCR in uninfected (A) and SARS-CoV-2 infected or mock infected (B) cells. Infections were carried out at MOI 0.5 for 48 hours. Raw data were used to calculate delta CT values (A), by using the TBP gene as housekeeping control. Fold changes, in infected over mock infected cells, were then calculated using the delta-delta CT method. Data in (B) are expressed as mean±SD (N=3).

[0142] FIG. 5. The combination of cobicistat and remdesivir synergistically inhibits SARS-CoV-2 activity.

[0143] A-F) Synergistic activity of cobicistat and remdesivir in inhibiting replication and cytopathic effects of SARS-CoV-2 in Vero E6 cells. Cells were infected at 0.5 MOI and left untreated or treated with the drugs at the indicated concentrations two hours-post infection. Forty Eight hours post-treatment: cells were fixed for immunofluorescence (IF) staining (A,B), supernatants were collected for qPCR (C-E) or cellular viability was analyzed (F). For IF detection, cells were stained with sera of SARS-CoV-2 patients and with the J2 antibody, which binds to double stranded RNA (Pape et al. 2020). The percentage of infected cells was determined by automatic acquisition of nine images per well (A), as described in Example 1. Scale bar=100 μM. Viral RNA in supernatants was detected by qPCR using an in vitro transcribed standard curve for absolute quantification (C-E) and data, expressed as mean±SD, were transformed as Logic) to restore normality and analyzed by one-way ANOVA, followed by Holm-Sidak's post-test (C). Cellular viability was measured by MTT assay (F).

[0144] Isobologram analysis of synergism (D) (Chou 2010) was performed using the IC90 values for SARS-CoV-2 replication of cobicistat, remdesivir, or their combination, calculated by non-linear regression. Synergism analyses of the inhibition of viral replication (E) or cytopathic effects (F) were performed with the SynergyFinder web-tool using the Zero Interaction Potency (ZIP) model based on inhibition values calculated as described in Example 1.

[0145] G) Effect of the combination of cobicistat and remdesivir on SARS-CoV-2 RNA expression in supernatants of a primary human colon organoid. Treatment with cobicistat/remdesivir was performed two hours post-infection and supernatants were collected forty-eight hours post-treatment. Viral RNA was quantified as described for panel (C).

[0146] For all panels N=3 independent experiments, except for panel E (N=2 independent experiments) and panel G (N=2 replicates from one colon organoid donor). ***P<0.001; **P<0.01; *P<0.05.

[0147] FIG. 6. Synergistic antiviral effect of cobicistat and remdesivir in the Vero E6 and T84 cell lines.

[0148] Effect of combined treatment of cobicistat and remdesivir on the viability of SARS-CoV-2 infected Vero E6 cells (A) and on viral replication (B,C) and inhibition of cytopathic effects (D) in T84 cells. Cells were infected at 0.5 MOI and left untreated or treated with the drugs at the indicated concentrations two hours-post infection. Forty Eight hours post-treatment: cells were fixed for crystal violet (A) or immunofluorescence (IF) (B) staining, supernatants were collected for qPCR (C), or cellular viability was analyzed (D). For IF detection, cells were stained with sera of SARS-CoV-2 patients (B). Viral RNA in supernatants was detected by qPCR using an in vitro transcribed standard curve for absolute quantification and data, expressed as mean±SD, were analyzed by non-parametric Friedman test, followed by Dunn's post-test (C). Scale bar=100 μM. Cellular viability was measured by MTT assay and synergism analysis of the inhibition cytopathic effects was performed with the SynergyFinder web-tool using the Zero Interaction Potency (ZIP) model based on inhibition values calculated as described in the Methods section. For panels C,D N=3 independent experiments. *P<0.05.

[0149] FIG. 7 Synergistic antiviral effect of cobicistat and chloroquine in the Calu-3 and T84 cell lines.

[0150] Effect of combined treatment of cobicistat and remdesivir on the viability of SARS-CoV-2 infected Calu-3 (A) and T84 (B) cells. Cells were infected at 0.5 MOI and left untreated or treated with the drugs at the indicated concentrations two hours-post infection. Forty Eight hours post-treatment cellular viability was analyzed by MTT assay. Synergism analysis of the inhibition cytopathic effects was performed with the SynergyFinder web-tool using the Zero Interaction Potency (ZIP) model.

EXAMPLES

Example 1 Material and Methods

1. Virtual Screening and Molecular Docking

[0151] Identification of potentially active SARS-CoV-2 inhibitors with desirable Absorption, Distribution, Metabolism, Excretion and Toxicity (ADME-Tox) properties, was performed by structure-based virtual screening (SBVS) of Drugbank V. 5.1.5(72) compounds targeting the three-dimensional structure of SARS-CoV-2 3CL.sub.pro. The analysis was focused on the substrate-binding site, which is located between domain I and II of 3CL.sub.pro. The binding site was identified using the publicly available 3D crystal structure [Protein Data Bank (PDB) ID: 6W63]. Structures of the previously described non-covalent protease inhibitor X77 (Andrianov et al., 2020), natively co-crystallized with 3CL.sub.pro were used as a reference for the identification of binding-site coordinates and dimensions for the virtual screening workflow, as well as for the docking validation of positions generated from the screening.

[0152] Protein structure analysis and preparation for docking were performed using the Schrödinger protein preparation wizard (Schrödinger Inc). Missing hydrogen atoms were added, bond orders were corrected and unknown atom types were assigned. Protein side-chain amides were fixed using program default parameters and missing protein side chains were filled-in using the prime tool. All non-amino acid residues, including water molecules, were removed. Further, unrelated ligand molecules were removed and active ligand structures were extracted and isolated in separate files. Finally, the minimization of protein strain energy was achieved through restrained minimization options with default parameters. The centroids of extracted ligands were then used to identify the binding site with coordinates and dimensions extended for 20 Å stored as Glide grid file. Drug screening was performed using the Glide software (Friesner et al., 2004). High throughput virtual screening (HTVS) was performed with the fastest search configurations. After post-docking minimization, the top-scoring tenth percentile of the output docked structures were subjected to the standard precision docking stage (SP). Then, active ligand structures were extracted and isolated in separate files. Finally, the top 10% scoring compounds were selected and retained only if their good scoring states were confirmed by Extra precision docking.

[0153] Remdesivir docking to CYP3A4, CYP3A5 and P-gp structures was performed to assess its capacity as a substrate/inhibitor for these proteins. CYP3A4, CYP3A5 and P-gp structures were collected from Protein Data Bank (PDB), IDs: 5VC0, 5VEU and 6QEE, respectively, and were subjected to the same preparation steps described above. Native inhibitors were used for identification of binding sites; the centroid of the known inhibitor Zosuquidar was used to identify the drug binding pocket of the P-gp protein structure. Further, co-crystallized Ritonavir was used for identification of the drug binding pocket in both CYP3A4/5. Receptor grids were generated for protein structures, for both CYP3A4 and CYP3A5. The heme iron of the Protoporphyrin ring was added as metal coordination constraint, allowing metal-ligand interaction in the subsequent docking steps. Docking was performed using flexible ligand conformer sampling allowing ring sampling with a 2.5 kcal/mol window. Retained poses for the initial docking phase were set to 5000 poses and only 800 best poses per ligand were selected for energy minimization. Finally, post-docking minimization was carried out for 10 poses per ligand with a 0.5 kcal/mol threshold for rejecting minimized poses.

2. Cell Lines and Primary Human Organoids

[0154] The following cell lines were used for infection and/or relative quantification of gene expression: Calu-3 (ATCC HTB-55), Caco-2 (ATCC HTB-37), T84 (ATCC CCL-24) and VeroE6 (ATCC CRL-1586). Primary organoids derived from human colon and ileum were seeded in 2D as described in (Stanifer et al. 2020). Culture conditions and susceptibility to SARS-CoV-2 infection have been previously described (Cortese et al. 2020; Stanifer et al. 2020).

3. Virus Stock Production and Infection

[0155] Viral stocks used for infections were produced by passaging the BavPatl/2020 SARS-CoV-2 strain in Vero E6 cells and the infectious titer was estimated by plaque assay, as previously described. Infection experiments were conducted using 25,000 or 250,000 cells per well in 96 and 12 well plates, respectively. Cell lines were infected at 0.05 or 0.5 MOI in medium with low FCS content (2%). Colon organoids were infected in a 24-well plate using 60000 plaque forming units (PFU) per well. Two hours post-infection cells were washed twice in PBS and resuspended in complete medium.

4. Drug Treatments

[0156] The following compounds were tested to determine their effects on 3CL.sub.pro activity, cytotoxicity or inhibition of SARS-CoV-2 replication: cobicistat (#sc-500831; Santa Cruz Biotechnology), remdesivir (#S78932; Selleckchem Chemicals), tipranavir (#sc-220260; Santa Cruz Biotechnology), nelfinavir mesylate hydrate (#PZ0013, Sigma-Aldrich), darunavir, lopinavir (both obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID), MG-132 (#M8699; Sigma-Aldrich), GC376 (BPS Bioscience), Chloroquine (#C 6628, Sigma Aldrich).

5. RNA Isolation and cDNA Retrotranscription

[0157] RNA extraction was performed on cell lysates or supernatants using the NucleoSpin RNA, Mini kit for RNA purification (Macherey-Nagel, Duren, Germany) according to the manufacturer's instructions. The concentration of RNA extracted from cell lysates was measured using a P-class P 300 NanoPhotometer (Implen GmbH, Munich, Germany).

[0158] Retrotranscription to cDNA was performed with 500 ng of intracellular RNA or 10 μL of RNA from supernatants, using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., USA) following the manufacturer's instructions.

6. SARS-CoV-2 RNA Standard

[0159] For the preparation of a viral RNA standard to use in qPCR for quantification of viral copies in supernatants, SARS-CoV-2 N sequence was reverse transcribed from total RNA isolated from cells infected with the SARS-CoV-2 BavPatl stain using Superscript 3 and specific primers (TTAGGCCTGAGTTGAGTCA, SEQ ID NO. 1). The resulting cDNA was amplified and cloned into the pJET1.2 plasmid. Ten μg of plasmid DNA was linearized by Adel restriction enzyme digestion and DNA was purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany). For in vitro transcription T7 RNA polymerase was used as previously described (Fischl and Bartenschlager 2013). In vitro transcripts were purified by phenol-chloroform extraction and resuspended in RNase-free water. RNA integrity was confirmed by agarose gel electrophoresis.

7. qPCR Analysis

[0160] Gene and/or viral expression were analyzed by SYBR green qPCR using, for each reaction, 10 μL of SsoFast™ EvaGreen® Supermix (Bio-Rad Laboratories, Hercules, Calif., USA), 500 nM of forward and reverse primer (0.1 μL each from 100 μM stock), 8.8 μL water and 1 μL cDNA. The primers used are listed in Table 2. The qPCR reaction was performed on a CFX96/C1000 Touch qPCR system (Bio-Rad Laboratories, Hercules, Calif., USA) using the following PCR program: polymerase activation/DNA denaturation 98° C. for 3 min, followed by 45 cycles of denaturation at 98° C. for 10 s; annealing/extension at 60° C. for 40 s and a final extension step at the end of the program at 65° C. for 30 s. Gene expression data were normalized using the delta-delta CT method [2(−ΔΔC(T)) method] (Livak and Schmittgen 2001), using the Tata-binding protein (TBP) gene as housekeeper control.

TABLE-US-00002 TABLE 2 List of qPCR primers used in the study SEQ Name Sequence Source ID NO. 2019-nCoV_N1- GAC CCC AAA ATC (1)  2 Forward AGC GAA AT 2019-nCoV_N1- TCT GGT TAG TGC  3 Reverse CAG TTG AAT CTG 2019-nCoV_N2- TTA CAA ACA TTG (2)  4 Forward GCC GCA AA 2019-nCoV_N2- GCG CGA CAT TCC  5 Reverse GAA GAA Hum Cyp3A4- TGA TGG CTC TCA  6 Forward TCC CAG AC Cyp3A4- AGC CCC ACA CTT  7 Reverse TTC CAT AC AGM Cyp3A4- TGA TGG ACC TCA  8 Forward TCC CAG AC Hum Cyp3A5- CGA CAA ACA AAA  9 Forward GCA CCG AC Hum Cyp3A5- TTA TTG ACT GGG 10 Reverse CTG CGA G AGM Cyp3A5- CGA CAA ACA AAA 11 Forward GCA CCG AG AGM Cyp3A5- TAA TTG ATT GGG 12 Reverse CCA CGA G P-gp(MDR1)-F CCC ATC ATT GCA Gao et 13 ATA GCA GG al., 2015 P-gp(MDR1)-R TGT TCA AAC TTC 14 TGC TCC TGA TBP-F CCA CTC ACA GAC Stanifer 15 TCT CAC AAC et al., TBP-R CTG CGG TAC AAT 2020 16 CCC AGA ACT Hum = human AGM = african green monkey (1) https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html (2) https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html

8. Western Blot

[0161] For Western blot experiments 0.5×10.sup.6 cells were lysed in a buffer (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 0.1% SDS, and 0.5% sodium deoxycholate supplemented with protease and phosphatase inhibitors (Sigma-Aldrich, Saint Louis, Mich., USA). Lysates were boiled at 95° C. for 10 min and sonicated for 5 min using a Bioruptor® Plus sonication device (Diagenode, Liege, Belgium). Protein lysates were then run on a precast NuPAGEBis-Tris 4-12% (Thermo Fisher Scientific, Waltham, Mass., USA) SDS-PAGE at 100-120 V and transferred onto a nitrocellulose membrane (GE Healthcare, Little Chalfont, UK) for 2.5 h at 25 V using a Trans-Blot device for semi-dry transfer (Bio-Rad Laboratories, Hercules, Calif., USA). Membranes were blocked using the LI-COR Intercept (PBS) Blocking Buffer (LI-COR Biosciences, Lincoln, Nebr., USA) for 1 h at RT and incubated overnight at 4° C. with the following primary antibodies in blocking buffer with 0.2% Tween 20: α-β-actin (1:10,000), (Sigma-Aldrich, Saint Louis, Mich., USA), a-SARS-CoV-2 spike protein [(rabbit; 1:1000) ab252690 Abcam], α-SARS-CoV-2 nucleocapsid [(mouse; 1:1000) AB_2827977, Sino Biological)], sera of SARS-CoV-2 positive individuals (1:200). Sera were collected as described in (Pape et al. 2020), following signing of informed consent by the donors, as well as ethical approval by Heidelberg University Hospital. After primary antibody incubation, membranes were washed three times with 0.1% PBS-Tween and incubated for 1 h with the following fluorescence-conjugated secondary antibodies: IRDye® 800CW Goat anti-Human IgG, IRDye® 800CW anti rabbit, IRDye® 700CW anti mouse (LI-COR Biosciences, Lincoln, Nebr., USA). All secondary antibodies were diluted 1:15000 in blocking buffer+0.2% Tween. After three washes with 0.1% PBS-Tween and one wash in PBS, fluorescence signals were acquired using a LI-COR Odyssey® CLx instrument.

9. Re-Processing of Microarray and RNA-Seq Data

[0162] Microarray gene expression data for CYP3A4/5 and P-gp in different anatomical tissues or cell lines were retrieved from Homo Sapiens Affymetrix Human Genome U133 Plus 2.0 Array dataset. Data were filtered by applying the criteria “Healthy sample status” and “No experimental treatment”. From the initial list, tissues with sample size<25 were filtered out. The anatomy search tool was used to plot Log 2 expression ratios of the tested genes. Gene expression data in cell lines were retrieved, apart from the aforementioned microarray dataset, from the RNAseq “mRNA Gene Level Homo sapiens (ref: Ensembl 75)” dataset. The cell line condition filter was used to refine the analysis and include exclusively cell lines susceptible to SARS-CoV-2 infection (i.e. T84, Caco2, Calu-3 and A-549).

10. Cell Viability

[0163] Cell viability was evaluated by (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT) assay and by crystal violet staining as previously described (Shytaj et al. 2020; Feoktistova, Geserick, and Leverkus 2016). Briefly, the MTT assay was conducted using the CellTiter 96® Non-Radioactive Cell Proliferation Assay (MTT) (Promega; Madison, Wis., USA). Cells were plated in a 96-well plate at a concentration of 3×10.sup.6 cells/mL in 100 μl of medium. The MTT solution (15 μl) was added to each well and, after 2-4 h, the reaction was stopped by the addition of 100 μl of 10% SDS. Absorbance values were acquired using an Infinite 200 PRO (Tecan, Männedorf, Switzerland) multimode plate reader at 570 nm wavelength.

[0164] For the crystal violet staining, cells were fixed in 6% formaldehyde and incubated with 0.1% crystal violet for 15 mins. Unbound staining was then washed with H.sub.2O and cells were imaged using a Nikon Eclipse Ts2-FL microscope.

11. 3CL.SUB.pro .FRET Assay

[0165] The activity of 3CL.sub.pro was measured by FRET assay (BPS Bioscience, San Diego, Calif., USA) according to the manufacturer's instructions and as previously described (Zhang et al. 2020). Briefly, serial dilutions of test compounds and known 3CLpro were incubated in a 384 well plate with the 3CL.sub.pro and its appropriate buffer, containing 0.5 M DTT. Wells without drugs or without 3CL.sub.pro were used as positive control of 3CL.sub.pro activity and blank control, respectively. After a 30 min incubation, the 3CL.sub.pro substrate was added to each well and the plate was stored for 4 hours in the dark. The fluorescence signal was acquired on an Infinite 200 PRO (Tecan, Männedorf, Switzerland) using an excitation wavelength of 360 nm and a detection wavelength of 460 nm. All Three separate experiments were conducted, with each experiment performed in duplicate. Relative 3CL.sub.pro was expressed as percentage of the positive control after subtraction of the blank.

12. Immunofluorescence and Syncytia Formation Assay

[0166] Cells were seeded on iBIDI glass bottom 96 well plate and infected with SARS-CoV-2 strain BavPatl/2020 for 24-48 h at MOI 0.5. Cells were rinsed in PBS and fixed with 6% PFA, followed by permeabilization with 0.5% Triton X100 (Sigma) in PBS for 15 minutes. Cells were then subjected to a standard immunofluorescence staining protocol. Briefly, cells were blocked in 2% milk (Roth) in PBS and incubated with primary antibodies in PBS (anti ds-RNA mouse monoclonal J2 antibody (Scicons) 1:2000 and patient serum 1:250). Cells were washed twice in PBS 0.02% tween and incubated with secondary antibody in PBS (1:1000 anti-mouse 568, Goat anti-human IgG-AlexaFluor 488 (Invitrogen, Thermofisher Scientific) for immunoglobulins detection in human serum and goat anti-mouse IgG-AlexaFluor 568 (Invitrogen, Thermofisher Scientific) for dsRNA detection). Nuclei were counterstained with Hoechst 33342 (Thermofisher Scientific, 0.002 μg/ml in PBS) for 5 minutes, washed twice with PBS and stored at +4° C. until imaging.

[0167] For syncytia formation assay, Vero E6 cells (0.2×10.sup.6 cells/well) were seeded on cover slips in a 12 well plate 24 h prior transfection. Cells were transfected using TransIT-2020 or TransIT-LT1 (Mirus) with 0.75 μg of pCDNA3.1(+)-SARS-CoV-2-S and 100 μl Opti-MEM per well. 2 h post transfection, cells were treated with cobicistat (final concentration of 1 μM, 5 μM and 10 μM), serum of patients (1:500 or 1:100) or DMSO (same concentration as in 10 μM cobicistat). 24 h post transfection, cells were washed twice with PBS and fixed in 4% PFA for 20 min at room temperature. After another washing step, cells were permeabilized in 0.5% Triton for 5 min at room temperature, washed and blocked in 3% lipid-free BSA in PBS-0.1% Tween-20 for 1 h at room temperature. After washing, cells were stained with the primary rabbit polyclonal anti-SARS-CoV-2 spike glycoprotein antibody (1:1000, Abcam) for 1 h at room temperature or overnight at 4° C. After washing, cells were incubated with the secondary Alexa Fluor 488 goat anti-rabbit IgG antibody (1:500, Life Technologies) for 1 h at room temperature. After washing, cells were incubated with DAPI (1:1000, Sigma-Aldrich) for 1 min followed by washing with PBS and deionized water. Images were acquired with Nikon Eclipse Ts2-FL Inverted Microscope. Syncytia with three or more nuclei surrounded by the antibody staining were used for the quantification. The edges of the antibody staining were overdrawn with the polygon selection tool in ImageJ.

13. Microscopy and Image Analysis

[0168] Cells were imaged using motorized Nikon Ti2 widefield microscope or with Nikon/Andor (CSU W1) spinning disc using a Plan Apo lambda 20×/0.75 air objective and a back-illuminated EM-CCD camera (Andor iXon DU-888). JOBS module was used for automatic acquisition of 9 images per well. Images were acquired in 3 channels using the following excitation/emission settings: Ex 377/50, Em 447/60 (Hoechst); Ex 482/35, Em 536/40 (AlexaFluor 488); Ex 562/40, Em 624/40 (AlexaFluor 568). When spinning disc was used the excitation was performed with 405 nm, 488 nm and 561 nm lasers.

[0169] Quantification of infected cells (expressed as percentage of total cells imaged per well) was performed using a custom-made macro in ImageJ. After camera offset subtraction and local background subtraction using the rolling ball algorithm, nuclei were segmented using automated local thresholding based on the Niblack method. Region of interest (represented by the ring (5 pixel wide) around the nucleus) was determined for each individual cell. Median signal intensity was measured in the region of interest in Alexa488 (serum) and Alexa568 (dsRNA) channels. Threshold for calling infected cells was manually determined for each individual experiment using the data from mock transfected cells. The same image analysis procedure and threshold was used for all wells within one experiment.

14. Statistical Analysis

[0170] Data normality assumptions were tested by D'Agostino & Pearson normality test (for >3). Multiple group comparisons were conducted by non-parametric Kruksal Wallis test, followed by Dunn's post-test, or by Two-Way ANOVA followed by Dunnet's post-test. Half maximal inhibitory (IC50) and cytotoxic (CC50) concentrations of the compounds tested were estimated by nonlinear regression using relative inhibition values calculated according to the formula: % inhibition=100*(1−(X−mock infected)/(infected untreated−mock infected)), where X is each given treatment condition. Data analysis was conducted using GraphPad Prism v6 (GraphPad Software, San Diego, Calif., USA). Synergy scores were calculated using the SynergyFinder web-tool (Ianevski et al. 2020) using the Zero Interaction Potency (ZIP) model (Yadav et al. 2015).

[0171] The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

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