The following compounds were purchased from Sigma; PD-150606 (D5946), Ribavirin (R9644), Mycophenolic acid (M3536), Tanespimycin (A8476), acetylsalicylic acid (A5376), Diclofenac (D689), Rhapontigenin (PHL83903), E64d (E8640). The cathepsin inhibitor CA-074me (205531) was purchased from Merck Millipore. Remdesivir was purchased from MedChemExpress (HY-104077). Drug stocks were prepared as follows: PD-160606 (50mM in DMSO), Ribavirin (50mM in H2O), Mycophenolic acid (100mM in MeOH), Tanespimycin (50mM in DMSO), Acetylsalicylic acid (100mM in EtOH), Diclofenac (10mM in MeOH), Rhapontigenin (10mM in DMSO), CA-074me (20mM in DMSO), E64d (10mM in DMSO), Remdesivir (10mM in DMSO).
Cell culture and virus stock
Caco-2 cells were acquired from APC Cork (Alimentary Pharmabiotic Centre, University College Cork, National University of Ireland, Cork, Ireland), HeLa cells (CCL-2), VeroE6 and Calu-3 cells from ATCC. All cells below passage 35 were used for experiments. Caco-2 cells were propagated in DMEM with GlutaMAX Gibco Cat No 61965-026 and 20% heat inactivated Fetal Bovine Serum (FBS). Vero cell culture and virus particle generation and titration was performed as previously described 19. VeroE6 (African green monkey kidney epithelial cell line) and Calu-3 (human lung epithelial cell line) cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Life Technologies) containing 10% or 20% fetal bovine serum, respectively, 100 U/mL penicillin, 100 μg/mL streptomycin and 1% non-essential amino acids (complete medium). Cell lines used in these experiments tested negative for mycoplasma using the MycoAlert Plus mycoplasma detection kit (Lonza) as per manufacturer's instructions.
Visualization of SARS-CoV2 viral N-protein during infection
Caco-2 cells were seeded on iBIDI glass bottom 8-well chamber slides. At indicated times post-infection, cells were fixed in 4% paraformaldehyde (PFA) for 20 mins at room temperature . Cells were washed and permeabilized in 0.5% Triton-X for 15 mins at room temperature. Mouse monoclonal antibody against SARS-CoV NP (Sino biologicals MM05), were diluted in phosphate-buffered saline (PBS) at 1:1000 dilution and incubated for 1h at room temperature. Cells were washed in 1X PBS three times and incubated with Goat anti-mouse Alexa Fluor 568 secondary antibody and DAPI for 45 mins at room temperature. Cells were washed in 1X PBS three times and maintained in PBS. Cells were imaged by epifluorescence on a Nikon Eclipse Ti-S (Nikon).
2D-TPP infection time course
Two days prior to infection, 1.5x106 Caco-2 cells were seeded in triplicate into plastic 10 cm tissue culture dishes (Greiner Cell State) per condition or in 8 well iBIDI chambers for IF staining. Caco-2 cells were infected with SARS-CoV-2 (BetaCoV/Germany/BavPat1/2020 p.1) at an MOI of 0.5 for 1 hour. Following 1 hour infection, virus supernatants were removed and cells were washed, and fresh media was added to cells At indicated times the 10 cm dish samples were harvested as described below (see thermal proteome profiling) and the iBIDI chambers were fixed and stained as described above (visualization of SARS-CoV-2). The percent of infected cells was determined by creating a nuclei mask in Ilastik (www.ilastik.org). CellProfiler (www.cellprofiler.org) was then used to measure the fluorescence intensity inside each nucleus of the mask. The values were thresholded using mock cells as a background control and the percentage of infected cells was calculated by the ratio of nuclei to positive cells.
Thermal proteome profiling
Thermal proteome profiling was performed as previously described 11 with the following modifications. The infected Caco-2 cells were harvested at 1, 2, 4, 7, 12, 24, and 48 hours post-infection. For harvesting, cells were trypsinized, washed once with PBS, and resuspended in 220 uL PBS. Each 20 uL of the concentrated cells were pipetted into a 96-well PCR plate and the samples from each time point were subjected to a thermal gradient (40.0°C, 42.1°C, 43.8°C, 46.5°C, 50.0°C, 54.0°C, 57.3°C, 60.1°C, 62.0°C, 64.0°C) for 3 min in a thermocycler (MJ Research, PTC-0200 DNA Engine) followed by 3 min at room temperature. The cells were then placed on ice and lysed with 30 μl lysis buffer (0.8% NP‐40, 1 mM MgCl2, 1× protease inhibitor (Roche), 1x phosphatase inhibitor (PhosStop, Sigma Aldrich), 250 U/ml benzonase in PBS) for 1 h, shaking at 4°C and 500rpm. A 0.45um 96-well filter plate (Millipore, ref: MSHVN4550) was pre-wetted with 50µl of 0.8% NP-40 in PBS by centrifugation, an additional 100uL of 0.8% NP-40 in PBS was added to each sample, and the samples were transferred to the pre-wet filter plate. The filter plate was transferred to an extraction plate vacuum manifold for Oasis 96-well plates from Waters (Cat. 186001831) and the sample was filtered for the removal of protein aggregates. To verify the effect of the heat treatment, the soluble protein concentration at each temperature for each experiment was determined using the BCA assay, according to the manufacturer’s instructions (ThermoFisher Scientific). Then, 100 uL of each sample was transferred to a new PCR plate, 10uL of denaturing buffer (20mM TCEP (tris(2-carboxyethyl)phosphine) in 2% SDS) was added, the plate was covered with aluminum foil, boiled for 10 min at 95°C and kept at -20°C until prepared for mass spectrometry.
Mass spectrometry-based proteomics
Proteins were digested according to a modified SP3 protocol 34,35. Briefly, approximately 5 μg of protein was diluted in 20 μl of water and added to the bead suspension (10 μg of beads (Thermo Fischer Scientific—Sera‐Mag Speed Beads, (4515‐2105‐050250 and 6515‐2105‐050250) in 10 μl 15% formic acid and 30 μl ethanol). After a 15 min incubation at room temperature with shaking, beads were washed four times with 70% ethanol. Next, proteins were digested overnight by adding 40 μl of digest solution (5 mM chloroacetamide, 1.25 mM TCEP, 200 ng trypsin, and 200 ng LysC in 100 mM HEPES pH 8). Peptides were then eluted from the beads, dried under vacuum, reconstituted in 10 μl of water, and labeled for 30 min at room temperature with 45 μg of TMTpro (Thermo Fisher Scientific) dissolved in 4 μl of acetonitrile. The reaction was quenched with 4 μl of 5% hydroxylamine for 15 min at room temperature, and experiments belonging to the same mass spectrometry run were combined. Samples were desalted with solid‐phase extraction by loading the samples onto a Waters OASIS HLB μElution Plate (30 μm), washing them twice with 100 μl of 0.05% formic acid, eluting them with 100 μl of 80% acetonitrile and 0.05% formic acid, and drying them under vacuum. Finally, samples were fractionated onto 12 fractions on a reversed‐phase C18 system running under high pH conditions. This consisted of an 85 min gradient (mobile phase A: 20 mM ammonium formate (pH 10) and mobile phase B: acetonitrile) at a 0.1 ml/min starting at 0% B, followed by a linear increase to 35% B from 2 min to 60 min, with a subsequent increase to 85% B from up to 62 min and holding this up to 68 min, which was followed by a linear decrease to 0% B up to 70 min, finishing with a hold at this level until the end of the run. Fractions were collected every two minutes from 12 min to 70 min and every 12th fraction was pooled together.
Samples were analyzed with liquid chromatography coupled to tandem mass spectrometry, as previously described. Briefly, peptides were separated using an UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) equipped with a trapping cartridge (Precolumn; C18 PepMap 100, 5 μm, 300 μm i.d. × 5 mm, 100 Å) and an analytical column (Waters nanoEase HSS C18 T3, 75 μm × 25 cm, 1.8 μm, 100 Å). Solvent A was 0.1% formic acid in LC‐MS grade water and solvent B was 0.1% formic acid in LC‐MS grade acetonitrile. Peptides were loaded onto the trapping cartridge (30 μl/min of 0.05% trifluoroacetic acid in LC-MS grade water for 3 min) and eluted with a constant flow of 0.3 μl/min using a 120 min analysis time (with a 2–30% B elution, followed by an increase to 40% B, and a final wash to 80% B for 2 min before re‐equilibration to initial conditions). The LC system was directly coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) using a Nanospray‐Flex ion source and a Pico‐Tip Emitter 360 μm OD × 20 μm ID; 10 μm tip (New Objective). The mass spectrometer was operated in positive ion mode with a spray voltage of 2.3 kV and capillary temperature of 275°C. Full‐scan MS spectra with a mass range of 375–1,200 m/z were acquired in profile mode using a resolution of 70,000 (maximum fill time of 250 ms or a maximum of 3e6 ions (automatic gain control, AGC)). Fragmentation was triggered for the top 10 peaks with charge 2–4 on the MS scan (data‐dependent acquisition) with a 30‐s dynamic exclusion window (normalized collision energy was 30), and MS/MS spectra were acquired in profile mode with a resolution of 35,000 (maximum fill time of 120 ms or an AGC target of 2e5 ions).
Protein identification and quantification
MS data were processed as previously described 18. Briefly, raw MS files were processed with isobarQuant 18, and the identification of peptides and proteins was performed with Mascot 2.4 (Matrix Science) against the human (Proteome ID: UP000005640) and SARS-CoV-2 (Proteome ID: UP000464024) UniProt FASTA, modified to include known contaminants and the reversed protein sequences (search parameters: trypsin; missed cleavages 3; peptide tolerance 10 ppm; MS/MS tolerance 0.02 Da; fixed modifications were carbamidomethyl on cysteines and TMTpro on lysine; variable modifications included acetylation on protein N‐terminus, oxidation of methionine, and TMTpro on peptide N‐termini).
Abundance and thermal stability score calculation
We calculated abundance and thermal stability scores for every protein at every infection time point by combining the data from the three replicates similarly to previously described 10. Briefly, the overall distribution of signal sum intensities was normalized with vsn to compensate for slight differences in protein amounts from each TMT channel. Then, for every protein, we calculated the ratio of the signal sum intensity of each time point to the signal sum of the uninfected sample at the same temperature. The abundance score of each protein at each time point was calculated as the average log2 fold change at the two lowest temperatures weighted for the number of temperatures in which the protein was identified for each replicate (requiring that there was data for two biological replicates). The thermal stability score of each protein at each time point was then calculated by subtracting the abundance score from the log2 fold changes of all temperatures, and summing the resulting fold changes weighted for the number of temperatures in which the protein was identified for each replicate (requiring that there were at least ten data points to calculate this score). To assess the significance of abundance and thermal stability scores, we used a limma analysis, followed by an FDR analysis, using the fdrtool package (see analysis script at: https://github.com/andrenmateus/TPP-SARSCoV2/). Abundance and thermal stability scores for all time points were separately transformed to z-scores. Proteins with calculated |z-score| >1.96 (corresponding to a global p <0.05 for the effect size) and with q-value <0.05 were considered significantly changed.
All proteins having significant stability changes during infection with SARS-CoV-2 were selected, given the same weight and mapped into a custom made human interactome integrating STRING v 11.0 (Edge Score>0.75) and Opentargets interactome (November 2019) (compilation of Intact, Reactome and Signor). All edges were treated as undirected, redundancies and self-loops removed, and a Personalized Pagerank algorithm was used to propagate the signal. Walktrap clustering was performed in the network regions with the highest page rank score (third quantile) to produce modules of interacting genes with no more than 300 genes. Modules were selected as significant if proteins with stability changes were enriched (fisher test with BF multiple testing p value adjustment) or if the distribution of page rank score was significantly higher (Kolmogorov Simonov test with BF multiple testing p value adjustment). Finally, we selected the significant modules that were enriched in proteins with: SARS-CoV-2 viral interactors 3, phosphosites changing during SARS-CoV-2 infections or kinases whose activity is affected by SARS-CoV-2 infection 2. The resulting 6 modules were further simplified keeping only the nodes that are changing at any level during infection (protein stability, phosphosite dynamics, kinase activity, PPI with viral proteins) and the edges connecting them.
Antiviral activity test
For testing the antiviral activity and cytotoxicity of selected compounds, Calu-3 cells were seeded one day prior to infection at a density of 3 x 104 cells per well of a clear 96-well plate (Corning). Antiviral activity and cytotoxicity were tested in duplicates, and remdesivir was included as positive control in each test run (concentration range: 0.5 nM - 10 µM in 3-fold serial dilutions). For the cytotoxicity test, cells were left untreated or treated with the respective drug in 3-fold dilution steps (for concentration ranges see Figures 5 and S5). For the antiviral activity test, cells were additionally infected with SARS-CoV-2 at an MOI of 1. Plates were incubated at 37°C for 20 - 24 h. Cytotoxicity was measured using a CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer’s instruction. For the antiviral activity test, plates were fixed with 6% formaldehyde, washed with PBS, and applied to immunostaining using a double-strand RNA-specific antibody (Scicons) suitable to detect SARS-CoV-2 infected cells. Cells were permeabilized with 0.2% Triton-X100 in PBS for 15 min at room temperature, washed once with PBS, and blocked in 2% milk in PBS with 0.02% Tween-20 for 1 hour at room temperature. Cells were incubated with the mouse anti-double strand RNA antibody for 1 hour at room temperature, washed three times with PBS, incubated with the secondary antibody (anti-mouse IgG, conjugated with horseradish peroxidase) for 1 hour at room temperature and subsequently washed four times with PBS. Detection was done by adding TMB (3,3',5,5'-Tetramethylbenzidine) substrate (Thermo Fisher Scientific) and plates were analyzed by photometry at 620 nm using a plate reader. The background absorbance was measured at 450 nm. The percentage of cell viability and inhibition was determined by dividing the values obtained from the drug-treated cells by the values from the untreated controls. EC50 values were calculated by non-linear regression sigmoidal dose response analysis using the GraphPad Prism 7 software package.