$COCP #2https://stm.sciencemag.org/content/early/2020/08/03/scitranslmed.abc5332 3C-like protease inhibitors block coronavirus replication in vitro and improve survival in MERS-CoV-infected mice View ORCID ProfileAthri D. Rathnayake1,*, View ORCID ProfileJian Zheng2,*, View ORCID ProfileYunjeong Kim3, View ORCID ProfileKrishani Dinali Perera3, Samantha Mackin2, View ORCID ProfileDavid K Meyerholz4, View ORCID ProfileMaithri M. Kashipathy5, View ORCID ProfileKevin P. Battaile6, View ORCID ProfileScott Lovell5, View ORCID ProfileStanley Perlman2,†, View ORCID ProfileWilliam C. Groutas1,† and View ORCID ProfileKyeong-Ok Chang3,† See all authors and affiliations
Science Translational Medicine 03 Aug 2020: eabc5332 DOI: 10.1126/scitranslmed.abc5332
The goal of this study was to evaluate the efficacy of 3CLpro inhibitors against human coronaviruses, including SARS-CoV-2, in a FRET enzyme assay and cell culture assays, as well as in a mouse model of MERS-CoV infection. Initial antiviral screening was performed with recombinant 3CLpro from SARS-CoV, MERS-CoV and SARS-CoV-2 in the FRET enzyme assay. Antiviral activity was then assessed in cultured Huh-7 cells infected with MERS-CoV and Vero E6 cells infected with SARS-CoV-2. Selected 3CLpro inhibitors were further examined using X-ray co-crystallization with MERS-CoV, SARS-CoV and SARS-CoV-2 3CLpro to elucidate the mechanism of action and identify the structural determinants of potency. Finally, two selected compounds were evaluated for in vivo efficacy in a mouse model of MERS-CoV infection (hDPP4-KI mice expressing human dipeptidylpeptidase 4 infected with a mouse-adapted MERS-CoV). Age- and sex-matched mice were randomly assigned into various groups for virus infection and treatment studies. Microscopic analysis of lung lesions was conducted in a blinded manner; other experiments were not blinded. No mice were excluded from analysis.
In vivo studies were performed in animal biosafety level 3 facilities at the University of Iowa. All experiments were conducted under protocols approved by the Institutional Animal Care and Use Committee at the University of Iowa according to guidelines set by the Association for the Assessment and Accreditation of Laboratory Animal Care and the U.S. Department of Agriculture.
The studies with MERS-CoV and SARS-CoV-2 were performed in biosafety level 3 facilities at the University of Iowa. All experiments were conducted under protocols approved by the Institutional Biosafety Committee at the University of Iowa according to guidelines set by the Biosafety in Microbiological and Biomedical Laboratories, the U.S. Department of Health and Human Services, the U.S. Public Health Service, the U.S. Centers for Disease Control and Prevention, and the National Institutes of Health.
Synthesis of 3CL protease inhibitors Compounds 6a-k and 7a-k were readily synthesized as illustrated in Fig. 1 and are listed in Table 1 and Table S1. Briefly, the alcohol inputs were reacted with (L) leucine isocyanate methyl ester or (L) cyclohexylalanine isocyanate methyl ester to yield dipeptides 2 which were then hydrolyzed to the corresponding acids with lithium hydroxide in aqueous tetrahydrofuran. Subsequent coupling of the acids to glutamine surrogate methyl ester 8 (33, 34) furnished compounds 4. Lithium borohydride reduction yielded alcohols 5 which were then oxidized to the corresponding aldehydes 6 with Dess-Martin periodinane reagent. The bisulfite adducts 7 were generated by treatment with sodium bisulfite in aqueous ethanol and ethyl acetate (35).
Fluorescence resonance energy transfer (FRET) enzyme assay The expression and purification of the 3CLpro of MERS-CoV, SARS-CoV and FIPV was performed by a standard method described previously by our lab (11, 19, 20). We also cloned and expressed the 3CLpro of SARS-CoV-2. The codon-optimized cDNA of full length of 3CLpro of SARS-CoV-2 (GenBank number MN908947.3) fused with sequences encoding 6 histidine at the N-terminal was synthesized by Integrated DNA (Coralville, IA). The synthesized gene was subcloned into the pET-28a(+) vector. The expression and purification of SARS-CoV-2 3CLpro were conducted following a standard procedure described by our lab (19). Briefly, stock solutions of compounds 6a-k and 7a-k were prepared in DMSO and diluted in assay buffer, which was comprised of 20 mM HEPES buffer, pH 8, containing NaCl (200 mM), EDTA (0.4 mM), glycerol (60%), and 6 mM dithiothreitol (DTT). The protease (3CLpro of MERS-CoV, SARS-CoV, SARS-CoV-2 or FIPV) was mixed with serial dilutions of each compound or with DMSO in 25 µL of assay buffer and incubated at 37°C for 30 min (MERS-CoV and FIPV) or at room temperature for 1 hour (SARS-CoV and SARS-CoV-2), followed by the addition of 25 µL of assay buffer containing substrate (FAM-SAVLQ/SG-QXL®520, AnaSpec, Fremont, CA). The substrate was derived from the cleavage sites on the viral polyproteins of SARS-CoV. Fluorescence readings were obtained using an excitation wavelength of 480 nm and an emission wavelength of 520 nm on a fluorescence microplate reader (FLx800; Biotec, Winoosk, VT) 1 hour following the addition of substrate. Relative fluorescence units (RFU) were determined by subtracting background values (substrate-containing well without protease) from the raw fluorescence values, as described previously (19). The dose-dependent FRET inhibition curves were fitted with a variable slope by using GraphPad Prism software (GraphPad, La Jolla, CA) in order to determine the IC50 values of the compounds.
Antiviral cell-based assays Some compounds in 6a-k and 7a-k series were also investigated for their antiviral activity against the replication of MERS-CoV, FIPV or MHV-1 in Huh-7, CRFK or CCL1 cells, respectively (19). Briefly, medium containing DMSO (<0.1%) or each compound (up to 100 µM) was added to confluent cells, which were immediately infected with viruses at an MOI of 0.01. After incubation of the cells at 37°C for 24 hours, viral titers were determined with the TCID50 method (FIPV or MHV) with the CRFK or CLL1 cells or plaque assay with Vero81 cells (MERS-CoV). For SARS-CoV-2, confluent VeroE6 cells were inoculated with ~ 50-100 plaque forming units/well, and medium containing various concentrations of each compound and agar was applied to the cells. After 48-72 hours, plaques in each well were counted. EC50 values were determined by GraphPad Prism software using a variable slope (GraphPad, La Jolla, CA) (19). To confirm that these inhibitors also inhibit SARS-CoV-2 in primary human cells, differentiated human airway epithelial cells from 3 donors were used as previously described (36, 37). Two compounds (6j and 6e) were tested for their antiviral effects against SARS-CoV2. Briefly, airway epithelial cells were washed with PBS and SARS-CoV2 was inoculated at a MOI of 0.1 for 1 hour. After the inoculum was removed, media containing 6j (2 µM) or 6e (0.5 µM) was added. After 48 hours, cells were subjected to a freeze/thaw cycle, and virus titers were determined by plaque assay on Vero E6 cells.
Measurement of cytotoxicity The cytotoxic dose for 50% cell death (CC50) for compounds 6a-k and 7a-k was determined in Huh-7, CRFK or CCL1 cells. Confluent cells grown in 96-well plates were incubated with various concentrations (1 to 100 µM) of each compound for 72 hours. Cell cytotoxicity was measured by a CytoTox 96 nonradioactive cytotoxicity assay kit (Promega, Madison, WI), and the CC50 values were calculated using a variable slope by GraphPad Prism software. The in vitro therapeutic index was calculated by dividing the CC50 by the EC50.
Protein purification, crystallization and data collection in X-ray crystallographic studies MERS-CoV 3CLpro and SARS-CoV 3CLpro were purified as described previously (17, 19). An E. coli codon optimized construct encoding residues Ser 3264 to Phe 3568 of the orf1ab polyprotein (SARS-CoV-2 3CLpro, Genebank QHD43415.1) was cloned into a pET His6 Sumo TEV LIC cloning vector (2S-T, addgene). Expression and initial Ni-column purification was performed as described for MERS-CoV 3CLpro and SARS-CoV 3CLpro. The SUMO fusion elution fractions of SARS-CoV-2 were dialyzed against 20mM Tris pH 8.0, 100 mM NaCl and treated with TEV protease (1:10 w/w) overnight. This mixture was loaded onto 5 mL HisTrap HP column (GE Healthcare) equilibrated with 20 mM Tris pH 8.0, 100 mM NaCl and eluted with 20mM Tris pH 8.0, 100 mM NaCl, 500mM imidazole using an AKTA Pure FPLC. The flow through fractions, containing SARS-CoV-2 3CLpro without the SUMO fusion loaded onto a Superdex 75 10/300 GL size-exclusion column equilibrated with 20 mM Tris pH 8.0, 100 mM NaCl. The fractions were pooled and concentrated to 9.6 mg/mL for crystallization screening. Note that four residues from cloning (SNIG) remain at the N terminus following treatment with TEV protease.
Purified MERS-CoV 3CLpro, SARS-CoV 3CLpro and SARS-CoV-2 3CLpro in 100 mM NaCl, 20mM Tris pH 8.0 were concentrated to 10.6 mg/mL (0.3 mM), 22 mg/mL (0.64 mM) and 9.6 mg/mL (0.28 mM) respectively for crystallization screening. All crystallization experiments were setup using an NT8 drop-setting robot (Formulatrix Inc.) and UVXPO MRC (Molecular Dimensions) sitting drop vapor diffusion plates at 18°C. 100 nL of protein and 100 nL crystallization solution were dispensed and equilibrated against 50 µL of the latter. Stock solutions (100 mM) of compounds 6b, 6d, 6 g, 6h, 7i, and 7j were prepared in DMSO and complexes were generated by mixing 1 µL of the ligand (2 mM) with 49 µL (0.29 mM) of the protease and incubating on ice for 1 hour. Crystals of the MERS-CoV 3CLpro inhibitor complexes were obtained from the following conditions. Compounds 6b, 6d, 6 g and 6h: Proplex screen (Molecular Dimensions) condition E2 (8% (w/v) PEG 8000, 100 mM sodium citrate pH 5.0), compound 7i: Proplex screen (Molecular Dimensions) condition B8 (15% (w/v) PEG 4000, 100 mM sodium citrate pH 5.0, 100 mM magnesium chloride) and compound 7j: Index HT screen (Hampton Research) condition F6 (25% (w/v) PEG 3350, 100 mM Bis-Tris pH 5.5, 200 mM ammonium sulfate). Crystals of the SARS-CoV 3CLpro complex with compound 7j were obtained from the Index HT screen (Hampton Research) condition H8 (15% (w/v) PEG 3350, 100 mM magnesium formate). Crystals of the SARS-CoV-2 3CLpro complex with compound 7j were obtained in 1-2 days from the PACT HT screen (Molecular Dimensions) condition D7 (20% (w/v) PEG 6000, 100 mM Tris pH 8.0, 200 mM NaCl). Samples were transferred to a fresh drop containing 80% crystallant and 20% (v/v) PEG 200 before storing in liquid nitrogen. X-ray diffraction data were collected at the Advanced Photon Source beamline 17-ID using a Dectris Pilatus 6M (MERS-CoV 3CLpro and SARS-CoV 3CLpro) and Dectris Eiger2 X 9M (SARS-CoV-2 3CLpro) pixel array detector.
Solution and refinement of crystal structures Intensities were integrated using XDS (38, 39) using Autoproc (40) and the Laue class analysis and data scaling were performed with Aimless (41). Structure solution was conducted by molecular replacement with Phaser (42) using a previously determined structure of MERS 3CLpro (PDB: 5WKK (17)) and SARS-CoV 3CLpro (PDB: 1Q2W (43)) SARS-CoV-2 3CLpro (PDB: 6LU7 (44)) as the search models. Structure refinement and manual model building were conducted with Phenix (45) and Coot (46), respectively. Disordered side chains were truncated to the point for which electron density could be observed. Structure validation was conducted with MolProbity (47) and figures were prepared using the CCP4MG package (48). Crystallographic data are provided in Table S2.
Therapeutic treatment in a mouse model of MERS-CoV infection The two best compounds (6j and 6h) in the series were examined for their in vivo efficacy using 10-week old male hDPP4-KI mice infected with MERSMA-CoV (30). In the first study, animals were divided into three groups (n=5-6) and were lightly anesthetized with ketamine/xylazine and infected with 50 µl of 750 pfu MERSMA-CoV via intranasal inoculation. Compounds 6j or 6h were formulated in 10% ethanol and 90% PEG400 and given to mice from 1 to 10 dpi at 50 mg/kg/day (once per day) via intraperitoneal administration. The control mice received vehicle. Animals were weighed daily and monitored for 15 days. Animals were euthanized when an animal lost 30% of initial weight or at 15 dpi.
In the next study, treatment with compound 6j was delayed up to 3 dpi to determine the impact of delayed treatment on mouse survival. Animals were divided into five groups (n=5) and compound 6j (50 mg/kg/day, once per day) was administered to mice starting at one, two or three days after virus challenge (1, 2 or 3 dpi, respectively) until 10 dpi. Mice were monitored for weight loss and survival as described above for 15 days post virus challenge. As controls, vehicle (10% ethanol+90% PEG400) was administered equivalently to the experimental compound or animals received no treatment (untreated). The third study was conducted to assess the effects of therapeutic treatment of compound 6j in the lungs. For lung harvest and virus titration, animals were divided into three groups (n=4) of mice and compound 6j (50 mg/kg/day, once per day) or vehicle was administered to mice starting at 1 dpi until euthanasia. Animals were euthanized at 3 or 5 dpi, and lungs were removed aseptically, disassociated with a manual homogenizer in 1x PBS, briefly centrifuged, and supernatants removed. Samples were titered on Vero-81 cells as reported elsewhere (49). For lung histopathology analyses, animals were divided into two groups (n=5) and compound 6j (50 mg/kg/day, once per day) or vehicle was administered to mice starting at 1 dpi for 5 days. Mice were euthanized at 6 dpi, lungs were fixed with 10% formalin, and hematoxylin and eosin stained tissues were examined by a veterinary pathologist using the post-examination method of masking (50). Briefly, tissues were scored in an ordinal manner for edema and hyaline membrane formation using the following scale: 0, none; 1, rare (<5 alveoli); 2, <33% of lung fields; 3, 34-66% lung fields, and 4, >66% lung fields (30).
Statistical analysis The analysis of survival curves in groups was performed using a Log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test using GraphPad Prism Software (San Diego, CA). Log-transformed viral titers in the lungs, and lung edema and hyaline membrane formation in groups of mice were analyzed with multiple t tests using GraphPad Prism Software.
Fig. S1. X-ray cocrystal structures of compounds with coronavirus 3CLpro showing the Fo-Fc omit map.
Fig. S2. X-ray cocrystal structures of compounds with coronavirus 3CLpro showing hydrogen bond interactions.
Fig. S3. X-ray cocrystal structures of compounds with coronavirus 3CLpro showing electrostatic surface representation.
Fig. S4. X-ray cocrystal structures of comparative binding of 7j with the 3CLpro of MERS CoV, SARS-CoV and SARS-CoV-2.
Table S1. Compound IC50 in the FRET enzyme assay and CC50 in a cell culture assay for MERS-CoV 3CLpro
Table S2. Cocrystal structure data for compounds with the 3CLpro of MERS CoV and SARS CoV.
References (51–56)
Data File S1. Individual-level data for all figures and tables.
This is an open-access article distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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