Two low-MW compounds, 3-(aminocarbonyl)-1-phenylpyridinium (compound 1, Specs ID number: AC-907/25005189) and 2, 3-dichloronaphthoquinone (compound 2, Specs ID number: AQ-390/13304011) were identified after the preliminary screening of 1, 590 compounds from the library. The final concentrations of protease and compounds were 100 nmol/L and 1.25 mmol/L, respectively. During the FRET assays, these two compounds exhibited the ability to inhibit over 90% of the protease activity of PEDV 3CLpro. The percentage of inhibition for the 1, 590 compounds is shown in Fig. 1A. The chemical structures of compounds 1 and 2 are illustrated in Fig. 1B.
Figure 1. Screening of and information on compounds 1 and 2: A High-throughput screening for inhibitors targeting PEDV 3CLpro from a library containing 1590 low-MW compounds was conducted. Each dot represents the percentage of inhibition of a compound. Compounds and substrate were used at final concentrations of 1.25 mmol/L and 40 µmol/L, respectively. B The structures and chemical names of compounds 1 and 2.
We then evaluated the inhibitory effects of the compounds at various concentrations against PEDV 3CLpro, with a DMSO control. The IC50 values of compounds 1 and 2 against 3CLpro were determined at a protease concentration of 100 nmol/L (Fig. 2A). The results indicated that both compounds exhibited concentration-dependent inhibitory activities. Compound 1 showed inhibitory activity against 3CLpro at concentrations of 0.39–12.5 µmol/L, and compound 2 exhibited inhibitory activity at 3.125–200 µmol/L. In addition, the IC50 values of compounds 1 and 2 were 0.1877 µmol/L and 6.765 µmol/L, respectively, indicating that compound 1 exhibited stronger inhibitory activity than compound 2.
Figure 2. Dose response for compounds 1 and 2 in suppressing PEDV 3CLpro activity and PEDV replication in Vero cells. Each concentration in the dose–response experiment was assayed in triplicate: A FRET assays were performed to measure the IC50 values of compounds 1 and 2, reflecting their ability to inhibit PEDV 3CLpro activity. B Cell viability analysis of compound 1 (6.25–100 µmol/L) and compound 2 (3.125–50 µmol/L) in Vero cells. C Inhibitory effects of compound 1 (3.125–25 µmol/L) and compound 2 (3.125–25 µmol/L) in PEDV-infected cells at an MOI of 0.1. Cell culture supernatant was harvested for TCID50 assays after incubation with serial dilutions of the test compounds for 16 h. D Dose–response curves determing the IC50 and EC50 values of compounds 1 and 2 by nonlinear regression. Bars represent the SD from triplicate trials (* represents a significant difference between test concentration and control; *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001).
To further study the antiviral activities of compounds 1 and 2 against PEDV, each compound was added to PEDV-infected Vero cells at serial concentrations of 3.125 µmol/L, 6.25 µmol/L, 12.5 µmol/L, and 25 µmol/L (Fig. 2C). We then obtained the TCID50 values of each concentration (with analyses performed in triplicate) and identified the dose-dependent inhibition of each compound against PEDV in Vero cells. Since 3CLpro plays an important role in PEDV replication and is the protein targeted by compounds 1 and 2, we concluded that these two compounds effectively decreased the viral titre at 3.125–25 µmol/L by blocking the enzyme activity of 3CLpro during PEDV replication. Significantly, the EC50 value of compound 1 (28.63 µmol/L) was lower than its CC50 value (73.8 µmol/L) obtained in Vero cells (Fig. 2B), which indicated its ability to suppress CoV replication with little cytotoxicity. Compound 2 showed moderate inhibitory effects against PEDV, with an EC50 value of 38.45 µmol/L; its CC50 value (21.79 µmol/L) was lower than its EC50 value in Vero cells, indicating potential cytotoxicity of compound 2 to Vero cells. However, the EC50 value of compound 1 was 28.63 µmol/L, and cell viability was higher than 80% in the presence of this compound at 25 µmol/L. Hence, 25 µmol/L might be an effective working concentration of compound 1 for inhibiting viral replication in vivo.
We performed an IFA and Western blotting to verify the results mentioned above. The images captured under fluorescence microscope (Fig. 3A) showed that compounds 1 and 2 each inhibited PEDV replication in a dose-dependent manner. There was no typical CPE of PEDV infection observed at a concentration of 25 µmol/L after 16 h of infection, and the amounts of infected cells at 12.5, 6.25 and 3.125 µmol/L significantly decreased compared to the amount with mock treatment (i.e., without compound 1 or 2 added). Additionally, the cytotoxic effect of compound 2 was evident, as the amount of DAPI-stained nuclei with compound 1 present was less than that with compound 2. The supernatant from infected cells in the presence or absence of the test compounds was collected to perform Western blotting (Fig. 3B). PEDV N protein level was decreased by compounds 1 and 2 in a dose-dependent manner, and no N protein was detectable with either compounds at 25 µmol/L or with compound 1 at 12.5 µmol/L. The concentration of N protein was moderately reduced in the presence of compound 2 at 12.5 µmol/L.
Figure 3. Dose response for compounds 1 and 2 in PEDV-infected Vero cells: A Immunofluorescence assay of compounds 1 and 2 against PEDV in Vero cells. PEDV-infected cells were used as a control group. Serial dilutions of the test compounds from 3.125 µmol/L to 25 µM were assessed independently. Nuclei were stained with DAPI (blue fluorescence), and PEDV infection was detected using a monoclonal antibody followed by FITC-tagged goat anti-rabbit IgG (green fluorescence). B Western blot analysis of the effects of compounds 1 and 2 on the production of CoV N protein in PEDV-infected cells. GAPDH was used as an internal control.
Given the presence of 3CLpro in all CoVs, we sought to determine the antiviral effects of compounds 1 and 2 against other members of the Alphacoronavirus genus. We performed similar antiviral assays with feline kidney cells (CRFK cells) against FIPV (Fig. 4). The EC50, CC50 and selectivity index (SI) values are presented in Table 1. In feline kidney cells (CRFK; CCL-94), compound 2 showed a greater capacity to inhibit FIPV replication than compound 1; the EC50 value of compound 2 was 46.03 µmol/L, whereas that of compound 1 was 59.66 µmol/L (Fig. 4A). The percentage inhibition achieved by compound 2 against FIPV at 25 µmol/L was 35%, whereas cell viability remained at 80% (Fig. 4A and 4B). Western blotting and immunofluorescence assays verified the ability of compounds 1 and 2 to suppress α-CoV replication (Figs. 3A and 3B, 4C and 4D).
Figure 4. A Cell viability analyses of compound 1 (6.25–100 µmol/L) and compound 2 (3.125–50 µmol/L) in CRFK cells. B Inhibitory effects of compound 1 (3.125–25 µmol/L) and compound 2 (3.125–25 µmol/L) in FIPV-infected cells at an MOI of 0.1. Cell culture supernatant was harvested for TCID50 assays after incubation with serial dilutions of test compounds for 20 h (* represents a significant difference between test concentration and control; *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001). (C) Immunofluorescence assay of compounds 1 and 2 against FIPV in CRFK cells. FIPV-infected cells were tested as a control group. Serial dilutions of the test compounds from 3.125 µM to 25 µmol/L were assessed independently. Nuclei were stained with DAPI (blue fluorescence), and FIPV infection was detected using a monoclonal antibody followed by FITC-tagged goat anti-rabbit IgG (green fluorescence). Bars represent the SD from triplicate trials. (D) Western blot analysis of the effects of compounds 1 and 2 on the production of CoV N protein in FIPV-infected cells. GAPDH was used as an internal control.
Compound Name PEDV FIPV IC50a (µmol/L) CC50b (µmol/L) EC50c (µmol/L) SId values CC50 (µmol/L) EC50 (µmol/L) SI values 1 3-(aminocarbonyl)-1-phenylpyridinium 0.1877 73.8 28.63 2.58 60.48 59.66 1.01 2 2, 3-dichloronaphthoquinone 6.765 21.79 38.45 0.56 36.7 46.03 0.80 a50% inhibitory concentration against PEDV 3CLpro;
b50% cytotoxic concentration in subcultured cells;
c50% effective concentration of test compound in inhibiting viral replication;
dselectivity index (SI = CC50/EC50). The IC50, CC50 and EC50 values were determined using GraphPad Prism 7
Table 1. Antiviral activities of compounds 1 and 2 against PEDV and FIPV.
To elucidate the inhibitory mechanism of the inhibitors against the protease, PEDV 3CLpro crystals were soaked with various concentrations of compound 1 and compound 2 to obtain the protein-inhibitor complex. Unfortunately, we were unable to obtain complex structures. Thus, the potential binding sites were analyzed by molecular simulations based on the crystal structure of PEDV 3CLpro obtained by our laboratory (PDB code: 4ZUH) and the structure of the protein in complex with the effective 3CLpro inhibitor GC376 (PDB code: 6L70) (Kim et al. 2013; Ye et al. 2016). To simulate the most likely conformation between the ligand and the macromolecule (3CLpro), we selected the lowest energy conformations for further analysis. The simulated molecular docking results showed that compounds 1 and 2 formed extensive hydrogen bonds with the residues of the protease binding pocket (Fig. 5A and 5B).
Figure 5. In silico molecular docking of compounds 1 and 2 with PEDV 3CLpro. A Docked conformation of compound 1 with PEDV 3CLpro. The protease is shown via a charge-potential surface. The possible hydrogen bond interactions are presented as yellow dashed lines, and the bond distances are shown. The S1 and S4 pockets are labelled. B Docked conformation of compound 2 with PEDV 3CLpro. The compounds and interactions between compounds and binding sites are represented as in A. C Sequence alignment of various coronavirus 3CLpros. The amino acid sequence information was retrieved from the NCBI Database, and the sequence alignments are based on one virus strain each (PEDV-YN144: KT021232.1; HCoV-229E: KU291448.1; HCoV-NL63: NC_005831.2; FIPV-WSU-79/1146: AAY32595.1; TGEV-PUR-MAD: NC_038861.1; SARS-CoV-Tor2: NP_828863.1; SARS-CoV-2-Wuhan-Hu-1: YP_009742612.1; MERS-CoV isolate HCoV-EMC/2012: NC_019843.3; IBV-Beaudette: NP_740626.1; PDCoV/USA/Nebraska145/2015: ANI85845.1; SADS-CoV: QJF53984.1). The key residues for 3CLpro activity sites and the compounds' potential binding sites on PEDV are marked with blue arrows at the bottom. The sequences were aligned using ClustalW2, and images were constructed with ESPript3.0.
In the lowest energy conformations, compound 1 formed hydrogen bonds with Glu165, Gln187, Thr189, and Gln191, and compound 2 bound the active site residues Cys144 and Gly142. Because Glu165 consists of the S1 specificity binding pocket and Cys144 is a conserved active site of 3CLpro (Ye et al. 2016), the two compounds may inhibit PEDV replication via blockage of the recognition and binding of 3CLpro and its substrate.
Sequence alignment of the 3CLpros of multiple CoVs, including PEDV, human coronaviruses (HCoV), FIPV, transmissible gastroenteritis coronavirus (TGEV), SARS-CoV, SARS-CoV-2, MERS-CoV, infectious bronchitis virus (IBV), porcine deltacoronavirus (PDCoV) and swine acute diarrhea syndrome coronavirus (SADS-CoV), indicates that the active sites (His 41, Cys144) of CoV 3CLpros and the potential binding sites (Cys144, Glu165, Gln191) of the compounds in PEDV 3CLpro are conserved (Fig. 5C). This conservation explains the antiviral activity of compounds 1 and 2 against swine and feline CoVs.