key: cord-283739-p7b4mtbl
authors: Theerawatanasirikul, Sirin; Kuo, Chih Jung; Phecharat, Nanthawan; Chootip, Jullada; Lekcharoensuk, Chalermpol; Lekcharoensuk, Porntippa
title: Structural-based virtual screening and in vitro assays for small molecules inhibiting the feline coronavirus 3CL protease as a surrogate platform for coronaviruses
date: 2020-09-07
journal: Antiviral Res
DOI: 10.1016/j.antiviral.2020.104927
sha: 
doc_id: 283739
cord_uid: p7b4mtbl

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 (3CL(pro)), which plays the pivotal role on viral replication. We investigated the potential drug-liked compounds and their inhibitory interaction on the 3CL(pro) 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 3CL(pro) activities with IC(50) 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 EC(50) (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 3CL(pro)-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.

Feline infectious peritonitis (FIP), which is caused by feline infectious peritonitis virus 44 (FIPV), is a life threatening, immunopathogenic disease affecting multiple organs in cats 45 (Pedersen, 2014a) . and 1% antibiotics-antimycotics (Invitrogen TM , Carlsbad, USA) at 37°C with 5%CO 2 for 48 175 h. The cytopathic effect (CPE) was observed daily under an inverted microscope (Olympus 176 CKX41, Tokyo, Japan). After the fifth passages of viral propagation, the virus stock was 177 quantitated as decribed previously (Lekcharoensuk, et al., 2012) presented as means and standard deviation, and qPCR data were analyzed and reported as the 256 percentage of viral reduction for each compound. Selectivity Index (SI) was determined as the 257 ratio of CC 50 to EC 50 for each compound. 258

In this study, the structure-based virtual screening was performed in which the 261 compounds were placed within the binding pocket of the 3CL pro due to high similarity of 262 amino acid residues among various CoVs. Then, the most favorable binding energies between 263 the compounds and the catalytic dyads within the binding pocket of CoVs-3CL pro were 264 analized. The binding affinity scores of the 28 top-ranked compounds and CoVs-3CL pro 265 structure were ranged from -8.7 to -5. annotate the active sites of all CoVs, which their 3D structures were highly conserved 282 especially at His41 and Cys144 numbering based on the positions on the FIPV 3CL pro analysis of other CoVs-3CL pro in respect to the FIPV 3CL pro sequence exhibits highly 285 conserved amino acid residues at Leu27, Pro39, Val42, Phe139, Cys144, His162, Glu165, 286

Leu166 and Asp186) in the catalytic dyad which strongly interact with the candidate 287 compounds (Supplementary Figure 1d) . These results indicates that FIPV 3CL pro has a 288 potential use as a surrogate platform to screen the candidate compounds that could inhibit the 289 CoVs-3CL pro activities. 290 291

According to the FIPV 3CL pro structurral based study, we determined if the candidate 293 compounds that could bind to the active site and inhibited the protease activity still actively 294

impeded the viral growth in cell culture. The candidate compounds possessing the inhibitory 295 effect on protease activity had IC 50 values of 3.57 ± 0.36 μM (NSC282187), 16.14 ± 2.76 μM 296 (NSC629301), and 25.90 ± 5.93 μM (NSC71097) as shown in Table 1 . The Ki values of these 297 three compounds were 2.04 ± 0.08, 9.39 ± 0.53, and 15.21 ± 1.76 μM, respectively ( Figure 2) . 298

In addition, the protease inhibition assay of PEDV 3CL pro and SARs-CoV 3CL pro was also 299 performed and the inhibition results are corresponding that of FIPV 3CL pro (Figure 2c ). 300 301

After screening using in silico and in vitro protease assay, the three selected 303 compounds were further examined for their cytotoxicity. The results showed that compound 304 NSC282187 had no effect on the CRFK cells even though at the concentration as high as 305 510.70 μM, whereas the CC 50 values of NSC629301 and NSC71097 were 28.13 ± 5.19 μM 306 and 68.47 ± 4.82 μM, respectively (Table 1) .

prophylaxis experiment was designed to allow the entry of small compounds into the host 310 cells, and also determined that the compounds remaining in the supernatant still have a 311 sufficient viral inhibition potency. We found that compounds NSC629301 and NSC71097 312 had inhibitory effect on FIPV with EC 50 = 6.11 ± 1.90 μM and 1.99 ± 0.30 μM, respectively. 313

Although, the protease inhibitory assay showed that NSC282187 could strongly inhibit FIPV 314 3CL pro activity with very low Ki value (Figure 2 (Figure 3) . In this study, 1% DMSO was 333 used instead of the compound in the mock-treated FIPV infected CRFK cells as a non-334 inhibitor control, which was not influent on FIPV infectivity (data not shown). 335

We also studied the interaction of NSC629301 and NSC71097 with FIPV 3CL pro 337 binding pocket using molecular docking simulation and protein ligand interaction analysis 338 ( Figure 4) . The results showed that the molecule of NSC629301 folded and fit well within the 339 active site of the protease (Figures 4a) . Two nitrogen atoms of a benzonitrile group formed 340 hydrogen bonds with Thr47 and His41 residues. The compound buried perfectly in the 341 binding pocket and formed hydrophobic interaction with hydrophobic residues (amino acid 342 residue: Leu27, Val42, and Leu164), and Asn25, Thr47, His163, and Cys144, respectively. In 343 addition, Cys144 residue can form non-covalent bonds, such as π-alkyl and π-donor 344 interactions with benzofuran and nitrile functional groups, respectively. For NSC71097, the 345 compound favorably located into the active site of FIPV 3CL pro (Figure 4b) . Particularly, 346

His41 residue in the catalytic dyad links with oxygen atom by hydrogen bond, and also form 347 π-π stack bond to the 4-chlorophenol functional group of this compound. Most hydrophobic 348 bonds were well defined which involve amino acid residues: Leu27, Thr47, Cys144, Leu164, 349

Asp186, Gln187 and Pro188, respectively. In addition, Pro188 can form amide-π stack with 350 4-chlorophenol functional group and form alkyl bond to the chloride atom of this functional 351 group. Cys144 and His41, the active residues of FIPV 3CL pro , formed the π-alkyl interaction 352 and hydrogen bond to the compounds, respectively. The Absorption, Distribution, 353

Metabolism, Excretion and Toxicity (ADME/T) properties of the three compounds are shown 354 in Supplementary table 1. All three compounds -NSC282187, NSC629301, and NSC71097-355 did not violate the druglikeness of Lipinski's rule of five when analyzed using SwissADME 356 tract. Moreover, compounds NSC282187 and NSC71097 might be able to permeate blood-358 brain barrier as shown in Supplementary Table 1 

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The interactions are presented as dashed lines for alkyl/π-alkyl 640 bonds (pink), π-donor (dark blue), hydrogen bonds (green) and hydrophobic interaction (red 641 circles and ellipses) in the right panel. The visualizations are generated using UCSF Chimera 642 version 1.10.2 and LigPlot software version 2.0. of TGEV and PEDV and also shared superclustering with the 647 betacoronaviruses (SARS-CoV, SARS-CoV-2 and MERS-CoV) (a). The analysis of CoVs-648 3CL pro alignment by T-coffee and visualized by Jalview

Analysis of complexes between CoVs-3CL pro with the two best compounds 654 revealed that both compounds are completely buried within the binding pockets. The 655 NSC629301 and NSC71097 are presented in cyan and dark purple