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. 2020 Oct:182:104927.
doi: 10.1016/j.antiviral.2020.104927. Epub 2020 Sep 7.

Structural-based virtual screening and in vitro assays for small molecules inhibiting the feline coronavirus 3CL protease as a surrogate platform for coronaviruses

Sirin Theerawatanasirikul  1 , Chih Jung Kuo  2 , Nanthawan Phecharat  3 , Jullada Chootip  3 , Chalermpol Lekcharoensuk  4 , Porntippa Lekcharoensuk  5
Affiliations

Structural-based virtual screening and in vitro assays for small molecules inhibiting the feline coronavirus 3CL protease as a surrogate platform for coronaviruses

Sirin Theerawatanasirikul et al. Antiviral Res. 2020 Oct.

Abstract

Feline infectious peritonitis (FIP) which is caused by feline infectious peritonitis virus (FIPV), a variant of feline coronavirus (FCoV), is a member of family Coronaviridae, together with severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. So far, neither effective vaccines nor approved antiviral therapeutics are currently available for the treatment of FIPV infection. Both human and animal CoVs shares similar functional proteins, particularly the 3CL protease (3CLpro), which plays the pivotal role on viral replication. We investigated the potential drug-liked compounds and their inhibitory interaction on the 3CLpro active sites of CoVs by the structural-bases virtual screening. Fluorescence resonance energy transfer (FRET) assay revealed that three out of twenty-eight compounds could hamper FIPV 3CLpro activities with IC50 of 3.57 ± 0.36 μM to 25.90 ± 1.40 μM, and Ki values of 2.04 ± 0.08 to 15.21 ± 1.76 μM, respectively. Evaluation of antiviral activity using cell-based assay showed that NSC629301 and NSC71097 could strongly inhibit the cytopathic effect and also reduced replication of FIPV in CRFK cells in all examined conditions with the low range of EC50 (6.11 ± 1.90 to 7.75 ± 0.48 μM and 1.99 ± 0.30 to 4.03 ± 0.60 μM, respectively), less than those of ribavirin and lopinavir. Analysis of FIPV 3CLpro-ligand interaction demonstrated that the selected compounds reacted to the crucial residues (His41 and Cys144) of catalytic dyad. Our investigations provide a fundamental knowledge for the further development of antiviral agents and increase the number of anti-CoV agent pools for feline coronavirus and other related CoVs.

Keywords: Antiviral activity; CoVs surrogate; Feline infectious peritonitis virus (FIP); Small molecules; Virtual screening.

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Figures

Fig. 1
Fig. 1
Heatmap displaying the binding affinities of the best 28 top-ranked compounds (y-axis) and protease active sites in the PDB structures of various coronaviruses (x-axis) (a). The values were normalized by transforming the binding energies to z-score prior to creating a heatmap and clustering by using the average linkage. The shed color schemes were assigned based on the transformed data, which ranged from strong (purple), moderate (white) to weak (red) binding affinities, respectively. The structural interactions for all contacts of CoVs-3CLpro docking complexes were analyzed with the best 11 top-ranked compounds of FIPV 3CLpro (b, upper panel) by Heatmapper (http://www.heatmapper.ca/). Each solid bar presents the number of small molecules making contacts (y-axis) to each amino acid residue (x-axis). The residues within the binding pocket of FIPV 3CLpro that interact directly with NSC282187, NSC629301, and NSC71097 are marked with asterisks. The alignment of the contacting amino acid residues of CoVs-3CLpro with the selected compounds were compared and the purple gradient colors present the amino acid similarity among CoVs (b, lower panel).
Fig. 2
Fig. 2
FRET-based protease assay. Enzyme kinetics of the three hits against FIPV 3CLpro activities using dose-response curves (a) with respect to the substrate peptide (Dabcyl-KTSAVLQSGFRKME-Edans). The Lineweaver–Burk plots of the determined Ki values of NSC282187, NSC629301, and NSC71097, respectively, (b) using different concentrations of the substrates. The IC50s of FIPV 3CLpro, PEDV 3CLpro, SARS-CoV 3CLpro and SARS-CoV-2 3CLpro were shown as means and SDs.
Fig. 3
Fig. 3
Quantitative analysis of FIPV-infected CRFK cells treated with the various concentrations of the hit compounds (a–b). Quantification of FIPV nucleic acids by RT-qPCR was presented as a percent of viral reduction (%) by the treatment conditions (upper panel). Intracytoplasmic staining pattern (lower panel) generated by IPMA reflected the FIPV antigens in the infected cells which was used for calculating EC50 as described in the text.
Fig. 4
Fig. 4
The FIPV 3CLpro–compound and SARs-CoV-2 3CLpro– compound interactions in the binding pocket of NSC629301 (a and c; cyan) and NSC71097 (b and d; purple) are presented in the upper panel. The interactions are presented as dashed lines for alkyl/π-alkyl bonds (pink), π-donor (dark blue), hydrogen bonds (green), π-sulfur (yellow), π-anion (gray), π- π stack (black) and hydrophobic interaction (red circles and ellipses) in the lower panel. The visualizations are generated using UCSF Chimera version 1.10.2, LigPlot software version v.2.0 and Discovery Studio Visualizer 2017 v.12.0.
Supplementary Fig. 1
Supplementary Fig. 1
The phylogenic tree demonstrates the amino acid sequence relations among 3CLpro of CoVs based on amino acid comparison. The 3CLpro of FIPV was closely related to that of TGEV and PEDV and also shared superclustering with the betacoronaviruses (SARS-CoV, SARS-CoV-2 and MERS-CoV) (a). The analysis of CoVs-3CLpro alignment by T-coffee and visualized by Jalview (v2.11.0) revealed the conserved amino acid residues (blue highlight). The two conserved amino acid residues, His41 and Cys144/145/148, which play an important role on the 3CLpro function, are indicated in the alignments (b, red arrows). The 3CLpro structures with focusing on the substrate binding pocket of CoVs are superimposed to demonstrate the conserved 3D structures of the catalytic dyads (c). Analysis of complexes between CoVs-3CLpro with the two best compounds revealed that both compounds are completely buried within the binding pockets. The NSC629301 and NSC71097 are presented in cyan and dark purple, respectively. For clarity, the complexes between a single CoV-3CLpro and each small molecule are shown in (d).
Supplementary Fig. 2
Supplementary Fig. 2
Antiviral activities of two candidate compounds against TGEV replication in SK6 cells with the various concentrations of compounds at 0.01, 0.1, 1, 5, 10, 50, and 100 μM, respectively. The IPMA detects TGEV antigens in the cytoplasm of the SK6-infected cells.
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