Plasmids and inhibitors
Coding sequence of SARS-CoV-2 Mpro (GenBank: MT291835.2) was cloned into pcDNA3.1( +) mammalian expression plasmid using BamHI/EcoRI restriction sites to create the SARS-CoV-2 Mpro coding plasmid; thereafter referred to as CoV-2 Mpro. The coding sequence of a dark-to-bright GFP reporter substrate; thereafter referred to as PR-Sub, was also cloned into pcDNA3.1( +) plasmid. The PR-Sub was designed to contain a sequence representing the N-terminal autoproteolytic cleavage site of SARS-CoV-2 Mpro (TSAVLQ*SGFRKM); corresponding to the nsp4/nsp5 cleavage site, between the GFP and the Influenza A/M2 protein hydrophobic tail (CNDSSDPLVVAASIIGILHLILWILDRL). For in vitro expression of the protease, the coding sequence of His6-tagged Mpro was cloned into pET11a bacterial expression plasmid using NdeI and BamHI enzymes. The above mentioned expression constructs were obtained using the gene synthesis service of GenScript.
The protease inhibitors darunavir, saquinavir, lopinavir, tipranavir, indinavir sulfate, and atazanavir sulfate were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH. Ritonavir was obtained from Abbott laboratories, nelfinavir from Agouron, and atazanavir from Bristol-Myers Squibb.
A synthetic oligopeptide used in our in vitro enzymatic assay representing the N-terminal autoproteolytic cleavage site of SARS-CoV-2 Mpro (AVLQ*SGFR) was obtained from a peptide synthesis service (BioBasic).
Analysis of transfection efficiency and proteolysis
293 T human embryonic kidney cells (HEK-293 T) (Invitrogen) were maintained in T-75 flask in 15 mL Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), 1% glutamine and 1% penicillin–streptomycin. Cells were transfected at 70% confluency with 5 µg of either PR-Sub, or CoV-2 Mpro plus PR-Sub plasmids using PEI method . After 24 h incubation, GFP fluorescence was analyzed by flow cytometry using FACS Calibur (BD Biosciences).
Inhibition profiling in cell culture
On the day of transfection, HEK-293 T cells were split and transferred into a 48-well plates (30,000 cells/well) containing serial dilutions of the inhibitor ranging from 200 µM to 5 nM in a total volume of 200 μL DMEM/well, supplemented with 10% FBS, 1% glutamine and 1% penicillin–streptomycin. After 3 h incubation at 37 °C, cells were transfected with 300 ng of CoV-2 Mpro and PR-Sub plasmids using lipofectamine LTX reagent (Thermo Fisher Scientific), then the cells were incubated for 24 h. GFP fluorescence was then measured by flow cytometry using FACS Calibur. The results were analyzed by FlowJo Software Version 10 (Becton, Dickinson and Company; 2019). Calculations of IC50 were performed using GraphPad Prism 5.0 (GraphPad Software, Inc).
Cell viability assay
The day before the assay, HEK-293 T cells were split into a 96-well plates (20,000 cells/well) containing serial dilutions of the inhibitor ranging from 200 µM to 100 nM in a total volume of 200 μL DMEM/well, supplemented with 10% FBS, 1% glutamine and 1% penicillin–streptomycin. The next day, the medium was replaced with 100 μL of OPTI-MEM culture media supplemented with 10% FBS, and 10 µL of the 12 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) stock solution was added to the cells. After 4 h incubation at 37 °C, 85 µL of supernatant was removed, and 50 µL of DMSO was added to the cells followed by incubation for 10 min at 37 °C. Absorbance was measured at 540 nm using Synergy H1 Hybrid Multi-Mode Reader (Agilent).
Expression and purification of SARS-CoV-2 Mpro
The heat-shock transformed BL21(DE3) cells containing the pET11a-His6-Mpro plasmid were incubated in 30 ml Luria–Bertani (LB) medium supplemented with ampicillin (100 µg/ml final concentration) at 37 °C for 16 h. The pre-cultured medium was inoculated into 470 ml LB (100 µg/ml ampicillin) and further incubated at 37 °C. Protein expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) (1 mM final concentration) when the OD600 reached 0.6–0.8. After 3 h incubation, cells were pelleted by centrifugation at 4 °C for 20 min at 5,000 × g (Sorvall Lynx 4000, Thermo Fisher Scientific), the cell pellet was resuspended in 10 ml buffer A (20 mM Tris, 150 mM NaCl, 10 mM imidazole, pH 7.5) and lysed by sonication on ice (Branson Sonifier 450). After a repeated centrifugation at 4 °C for 20 min at 10,000 × g, the pellet was discarded and His6-Mpro was purified from the supernatant by Ni-chelate affinity chromatography with the aid of His-Trap Column (GE Healthcare) using Äkta Prime instrument (Amersham Pharmacia Biotech). The column was equilibrated and washed with buffer A, the His6-Mpro protein was eluted under 20 column volume with a linear gradient of imidazole (0—500 mM imidazole) using buffer B (20 mM Tris, 150 mM NaCl, 500 mM imidazole, pH 7.5). Afterwards, the purification buffer was exchanged to buffer C (20 mM Tris, 50 mM NaCl, 2 mM CaCl2 pH 7.5) using Amicon Ultra centrifugal filters (10 K, Merck Millipore) and then the protein was incubated with Factor Xa (10 µg FXa/ mg protein, BCXA-1060, Haematologic Technologies) at 16 °C for 16 h to remove His6 fusion tag. Before the next purification step, the buffer was changed to buffer D (20 mM Tris, 1 mM DTT, pH 8.0) and the protein was further purified by ion-exchange chromatography using HiTrap Q FF column (GE Healthcare) equilibrated with buffer D, and eluted with buffer E (20 mM Tris, 1 M NaCl, 1 mM DTT, pH 8.0) under 20 column volume with a linear gradient. The high-purity fractions of the untagged Mpro were dialyzed against buffer F (20 mM HEPES, 120 mM NaCl, 0.4 mM EDTA, 4 mM DTT, 20% glycerol pH 6.5), and stored at -20 °C in a small-volume aliquots.
In vitro protease assay
The AVLQ*SGFR oligopeptide was dissolved in distilled water and was used as substrate in activity measurements to test the inhibitory potential of the PIs.
The cleavage reactions contained 10 µL reaction buffer (20 mM Tris, 100 mM NaCl, pH 7.8), 4.8 µL oligopeptide substrate (1.37 mM final concentration), and 0.2 µL DMSO (in control samples) or 0.2 µL of the inhibitor (diluted in DMSO). For inhibitor screening, inhibitors were applied in 100 µM final concentration. Reactions were initiated by the addition of 5 µL of Mpro in a final total protein concentration of 0.12 µM, and the mixtures were incubated at 37 °C for 10 min. The reactions were terminated by the addition of 180 µL 1% trifluoroacetic acid (TFA). The cleavage products were detected using high performance liquid chromatography (HPLC), utilizing a 0–100% water-acetonitrile gradient in the presence of TFA using Merck Hitachi instrument. Relative activity was determined at less than 20% substrate hydrolysis. Activity measured in the presence of DMSO was considered to be 100%. While no potent inhibitor of Mpro was available to perform active-site titration, 100% activity was assumed for the enzyme.
Homology modeling of dark-to-bright GFP substrate was performed using Phyre2 web portal . 97% of residues were modelled at > 90% confidence. Structural figures were prepared PyMol Molecular Graphics System (Version 1.3 Schrödinger, LLC).