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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,†
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Science Translational Medicine 03 Aug 2020:
eabc5332
DOI: 10.1126/scitranslmed.abc5332

Abstract
Pathogenic coronaviruses are a major threat to global public health, as exemplified by Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and the newly emerged SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus 3C-like protease (3CLpro), an enzyme essential for viral replication. The optimized compounds were effective against several human coronaviruses including MERS-CoV, SARS-CoV and SARS-CoV-2 in an enzyme assay and in cell-based assays using Huh-7 and Vero E6 cell lines. Two selected compounds showed antiviral effects against SARS-CoV-2 in cultured primary human airway epithelial cells. In a mouse model of MERS-CoV infection, administration of a lead compound one day after virus infection increased survival from 0 to 100% and reduced lung viral titers and lung histopathology. These results suggest that this series of compounds has the potential to be developed further as antiviral drugs against human coronaviruses.
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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
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Abstract
Pathogenic coronaviruses are a major threat to global public health, as exemplified by Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and the newly emerged SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus 3C-like protease (3CLpro), an enzyme essential for viral replication. The optimized compounds were effective against several human coronaviruses including MERS-CoV, SARS-CoV and SARS-CoV-2 in an enzyme assay and in cell-based assays using Huh-7 and Vero E6 cell lines. Two selected compounds showed antiviral effects against SARS-CoV-2 in cultured primary human airway epithelial cells. In a mouse model of MERS-CoV infection, administration of a lead compound one day after virus infection increased survival from 0 to 100% and reduced lung viral titers and lung histopathology. These results suggest that this series of compounds has the potential to be developed further as antiviral drugs against human coronaviruses.

INTRODUCTION
Coronaviruses are a large group of viruses that can cause a wide variety of diseases in humans and animals (1). Human coronaviruses generally cause the common cold, a mild upper respiratory illness. However, global outbreaks of new human coronavirus infections with severe respiratory disease have periodically emerged from animal reservoirs, including Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Middle East Respiratory Syndrome coronavirus (MERS-CoV) and, most recently, SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). SARS-CoV-2 emerged in China in December 2019 and subsequently rapidly spread throughout the world. Genetic analysis of SARS-CoV-2 revealed that it is closely related to SARS-like beta-coronaviruses of bat origin, bat-SL-CoVZC45 and bat-SL-CoVZXC21 (2). Despite the periodic emergence of new coronaviruses capable of infecting humans, there are currently no licensed vaccines or antiviral drugs against any coronaviruses, underscoring the urgent need for the development of preventive and therapeutic measures against pathogenic coronaviruses.

The coronavirus genome contains two overlapping open reading frames (ORF1a and ORF1b) at the 5' end terminal, which encode polyproteins pp1a and pp1ab. The polyproteins are processed by a 3C-like protease (3CLpro or Main protease, MPro) (11 cleavage sites) and a papain-like protease (PLpro) (3 cleavage sites), resulting in sixteen mature nonstructural proteins, including an RNA-dependent RNA polymerase (RdRp). Both 3CLpro and PLpro are essential for viral replication, making them attractive targets for drug development (3–7). Coronavirus 3CLpro is a cysteine protease that has two N-terminal domains containing two ß-barrel chymotrypsin-like folds (8–10). The active site of 3CLpro is located in the cleft between the two domains and is characterized by a catalytic Cys-His dyad.

We have developed broad-spectrum inhibitors of an array of viruses, including coronaviruses and noroviruses (11–18) that use 3CLpro for viral replication and picornaviruses that use 3C protease (19). We have shown efficacy of the coronavirus 3CLpro inhibitor, GC376 (currently in clinical development) in animal models of coronavirus infection (20, 21). Specifically, administration of GC376 to cats with feline infectious peritonitis (FIP), a coronavirus-induced systemic disease that is 100% fatal, reversed the progression of FIP and resulted in clinical remission (20, 21). We have recently reported the results of exploratory in vitro studies using a dipeptidyl series of MERS-CoV 3CLpro inhibitors that embody a piperidine moiety as a new design element, as well as pertinent structural and biochemical studies (17). Here, we report the development of 3CLpro inhibitors against multiple coronaviruses, including SARS-CoV-2, and demonstrate in vivo efficacy against MERS-CoV in a mouse model.

RESULTS
3CLpro inhibitors show activity against multiple coronaviruses in enzyme and cell-based assays
The synthesis scheme for compound series 6a-k and 7a-k is shown in Fig. 1 and described in the supplementary materials and methods. The activity of compounds 6a-k and 7a-k against the 3CLpro enzymes of MERS-CoV, SARS-CoV and SARS-CoV-2 was evaluated in a fluorescence resonance energy transfer (FRET) enzyme assay (Table 1, Table S1). Several compounds in this series (6a, 7a, 6c, 7c, 6e, 7e, 6h, 7h, 6j and 7j) were also tested in cell-based assays (Table 2). Table 1 and Table S1 show 50% inhibitory concentration (IC50) values in a FRET enzyme assay for select compounds (6a, 7a, 6c, 7c, 6e, 7e, 6h, 7h, 6j and 7j) and GC376. 50% effective concentration (EC50) values and 50% cytotoxic concentration (CC50) values for select compounds and GC376 were measured in cell culture assays (Table 2). Cell culture assays included Huh7 cells infected with MERS-CoV, Vero E6 cells infected with SARS-CoV-2, CRFK cells infected with feline infectious peritonitis virus (FIPV) and CCL1 cells infected with mouse hepatitis virus (MHV) (Table 2). Inhibitors with a P2 leucine (Leu) residue were more potent than those with a cyclohexylalanine against MERS-CoV 3CLpro (compounds 6h and 7h versus 6i and 7i), with submicromolar IC50 values (Table 1, Table S1). The compounds tested against MERS-CoV in cell culture (7a, 6c, 7e, 7h and 6j) also displayed submicromolar EC50 values. Among these compounds, 6j showed the most potent antiviral activity against MERS-CoV with an EC50 value of 0.04 µM. GC376 with a P2 Leu residue and a non-fluorinated benzyl cap, exhibited 20-fold lower potency against MERS-CoV in cell culture compared to compound 6j (Table 2).

Fig. 1 Synthesis scheme for compound series 6a-k and 7a-k.
Stepwise compound synthesis with intermediate compounds is shown for 3CLpro inhibitors of the 6a-k and 7a-k series. The alcohol inputs were reacted with (L) leucine isocyanate methyl ester or (L) cyclohexylalanine isocyanate methyl ester to yield products 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) 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. a Amino-acid methyl ester isocyanate/TEA/CH3CN/reflux/2 hours; b1M LiOH/THF/RT/ 3 hours; cEDCI/HOBT/glutamine surrogate/DIPEA/DMF/RT/24 hours; d2M LiBH4/THF/methanol/RT/ 12 hours; e Dess-Matin periodinane/DCM/15°C-18°C/ 3 hours; fNaHSO3/ethyl acetated/ethanol/H2O/44-55°C. Full details are provided in the Supplementary Materials and Methods.

Table 1 Structures of 3CLpro inhibitors and their IC50 values in the FRET enzyme assay.
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The compounds were also effective against SARS-CoV-2 with EC50 values ranging from 0.15 to 0.9 µM in Vero E6 cells (Table 2). These compounds were also found to be potent against FIPV and MHV, with EC50 values ranging from 0.07 to 0.22 µM. In the FRET enzyme assay, these compounds were active against the 3CLpro of SARS-CoV and SARS-CoV-2 (Table 1). The IC50 values of these compounds against SARS-CoV-2 3CLpro ranged from 0.17 to 0.82 µM. Among these compounds, 6e showed the most potent antiviral activity against SARS-CoV-2 in the enzyme assay (IC50, 0.17 µM) and cell-based assay (IC50, 0.15 µM) (Tables 1 and 2). Notably, GC376 also exhibited activity against the 3CLpro of SARS-CoV-2 with an IC50 value of 0.62 µM in the enzyme assay (Table 1).

The antiviral effects of compounds 6j and 6e against SARS-CoV-2 were confirmed in cultured primary human airway epithelial cells from 3 donors, who were infected with SARS-CoV-2. In the absence of a 3CLpro inhibitor, viral titers in the infected cultured primary human airway epithelial cells reached 107.3 (donor 1), 107.1 (donor 2), 108.4 (donor 3) plaque forming units (pfu) per ml of culture medium. In the presence of compound concentrations that were approximately 2-3 fold higher than the EC50 values obtained in cultured cells (2 µM, 6j or 0.5 µM, 6e), viral titers were reduced to 106.4 (donor 1), 106.1 (donor 2), 106.3 (donor 3) pfu/ml for compound 6j, or 106.1 (donor 1), 106.5 (donor 2), 108.1 (donor 3) pfu/ml for compound 6e (Table 3). Although there was some variation in viral replication among infected cells from the three donors (especially donor 3), the antiviral effects of both 6j and 6e were evident at the tested concentrations. For infected cells from donors 1 and 2, both compounds inhibited viral replication approximately 10-fold at the tested concentrations. For infected cells from donor 3, 6e inhibited virus replication approximately 50%, whereas 6j inhibited virus replication 100-fold at the tested concentrations.

Co-crystal structures for 3CLpro of MERS-CoV, SARS-CoV and SARS-CoV-2 with 3CLpro inhibitors
We determined multiple high-resolution cocrystal structures of compounds 6b, 6d, 6 g, 6h, 7i or 7j with the 3CLpro of MERS-CoV (Fig. 2A-F, Fig. S1-S3). These inhibitors bound to the active site of MERS-CoV 3CLpro demonstrating that the vicinity of the S4 pocket is encompassed by an array of primarily hydrophobic residues, including Phe188, Val193, Ala171, and Leu170 (Fig. 2C, 2F and Fig. S3). Hydrophobic and hydrogen-bonding functionalities were incorporated into the 3CLpro inhibitors to capture additional interactions, and the position of the cyclohexyl moiety was also examined using appropriate congeners. The bisulfite adducts reverted to the corresponding aldehydes, which subsequently reacted with Cys148 to form nearly identical covalent complexes with a tetrahedral arrangement at the newly-formed stereocenter (Fig. 2A-F and Fig. S1-S3). The backbone of compound 6h (Table 1 and Fig. 2A-C) engaged in H-bond interactions with amino acid residues Gln192, Gln167 and Glu169. Three additional side chain H-bonds between the ?-lactam ring and His166, Phe143 and Glu169 also were clearly evident (Fig. 2B). Furthermore, the side chain of the P2 Leu was ensconced in the hydrophobic S2 pocket (Fig. 2C). The extra methylene group in compound 7j, which was converted to an aldehyde and thus became identical to 6j, resulted in re-orientation of the difluorocyclohexyl group and the formation of three H-bonds between Gln195 and Ala171 and the fluorine atoms, with concomitant loss of one of the Gln192 hydrogen bonds and the displacement of Phe143 (Fig. 2E). The substitution of the P2 Leu with P2 cyclohexylalanine (compound 7i, Table S1) resulted in the loss of an H-bond with Gln192, but otherwise adopted the same interactions as observed for compound 6h (Fig. S2A). The electron density, hydrogen bond interactions and electrostatic surface representations for MERS-CoV 3CLpro in complex with compounds 6b, 6 g and 6d are shown in Fig. S1-S3.

Fig. 2 X-ray cocrystal structures of compounds with coronavirus 3CL proteases.
Shown are cocrystal structures of the MERS-CoV 3CLpro with compound 6h (A, B and C) and MERS-CoV 3CLpro with compound 7j (D, E, F). Shown are cocrystal structures of the SARS-CoV 3CLpro with compound 6h (G, H and I) or 7j (J, K and L). Panels A, D, G, J show Fo-Fc omit maps (green mesh) contoured at 3s. Panels B, E, H, K show hydrogen bond interactions (dashed lines) between the inhibitor and the 3CLprotease. Panels C, F, I, L show electrostatic surface representation of the binding pocket occupied by the inhibitor. Neighboring residues are colored yellow (nonpolar), cyan (polar) and white (weakly polar).

Next, compound 7j was cocrystallized with SARS-CoV or SARS-CoV-2 3CLpro and the structures were compared to that for MERS-CoV 3CLpro and 7j. In the SARS-CoV 3CLpro-7j complex (Fig. 2G-I), the backbone of compound 7j formed direct H-bonds with Cys145, His163, His164, Glu166, and Gln189. Compound 7j also formed an additional H-bond with His41 and a water-mediated contact with Gly143 (Fig. 2H). However, there was a loss of the three H-bonds between Gln195 and Ala171 and the fluorine atoms, compared to the cocrystal structure of 7j with MERS-CoV 3CLpro. Notably, the electron density map was consistent with both possible enantiomers at the new stereocenter formed by covalent attachment of the S? atom of Cys145 in the cocrystal structure of SARS-CoV 3CLpro with 7j. The electron density map for compound 7j in complex with SARS-CoV-2 3CLpro was most consistent with a single enantiomer, although it adopted a similar binding mode and hydrogen bond interactions as observed in the SARS-CoV 3CLpro-7j cocrystal structure (Fig. 2J-L). Superposition of compound 7j with MERS-CoV 3CLpro, SARS-CoV 3CLpro and SARS-CoV-2 3CLpro (Fig. S4) revealed a very similar binding mode for 7j among all three viral proteases.

3CLpro inhibitor treatment increases survival and reduces lung viral load in infected hDPP4-KI mice
The most potent compound of the series, 6j, was identified in a cell-based assay and had an EC50 value of 0.04 µM against MERS-CoV (Fig. 3A). We determined the efficacy of compounds 6j and 6h in transgenic hDPP4-KI mice expressing human dipeptidylpeptidase 4, a model of MERS-CoV infection. First, hDPP4-KI mice were infected with the mouse-adapted MERS-CoV (MERSMA -CoV) virus strain and then were treated with compounds 6h, 6j (50 mg/kg/day, once a day) or vehicle as a control starting one day post virus infection (1 dpi) and continuing until 10 dpi. All mice treated with vehicle control died by 8 dpi (Fig. 3B). In contrast, 40% of mice treated with compound 6h survived, and all mice treated with compound 6j were alive at the end of the study (15 dpi) (Fig. 3B). The survival of mice treated with compound 6j or 6h was increased compared to the vehicle control (p<0.05), and the 6j-treated mice had an improved survival rate compared to 6h-treated mice (p<0.05). All mice treated with compound 6j rapidly recovered from body weight loss starting at 3 dpi (Fig. 3C). The mice that survived after 6h treatment continued to lose body weight until 6 dpi, but then started to gain weight from 9 dpi (Fig. 3C).


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Fig. 3 Effects of treating hDPP4-KI mice infected with MERSMA-CoV with compounds 6j or 6h.
(A) Shown is a dose-dependent curve for inhibition of MERS-CoV in cell culture by compound 6j. Serial dilutions of compound 6j were added to confluent Huh-7 cells, which were immediately infected with MERS-CoV at a multiplicity of infection (MOI) of 0.01. After incubation of the cells at 37°C for 48 hours, viral titers were determined using a plaque forming assay and EC50 values were determined with GraphPad Prism software. (B, C) hDPP4-KI mice infected with MERSMA-CoV (n=6) were treated with compounds 6j or 6h starting at 1 day post-infection (dpi) for up to 10 days and survival (B) and body weight (C) were monitored for 15 days. Control mice received vehicle only. (D, E) hDPP4-KI mice infected with MERSMA-CoV were treated with compound 6j (n=5) starting at 1 dpi, 2 dpi or 3 dpi, and survival (D) and body weight (E) were monitored for 15 days. Untreated mice and vehicle-treated mice (n=4) were included as controls. The mice were infected with MERSMA-CoV at 0 dpi. The data points represent the mean and the standard error of the mean for one experiment. The analysis of survival curves in groups was performed using a Log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test.

After we observed that treatment with compound 6j resulted in the survival of MERSMA-CoV-infected hDPP4-KI mice, we conducted another study by delaying treatment initiation until 3 dpi. Similar to the first study, no untreated mice or mice given vehicle (control) survived, and there was no statistical difference between these two groups (0% survival) (Fig. 3D). When 6j treatment was started on 1 dpi, four of the five mice survived (80% survival), and there was a statistically significant increased survival in mice treated starting at 1 dpi compared to untreated or vehicle control-treated mice (P<0.05, Fig. 3D). When 6j treatment was delayed by one additional day (2 dpi), the survival of mice treated with 6j decreased to 40%, but this was still higher than 0% survival for untreated or vehicle control-treated mice. However, there was no statistical difference between the 6j treatment group starting at 2 dpi and the untreated or vehicle control-treated groups (Fig. 3D). Treatment with 6j starting at 3 dpi also failed to improve the survival of mice compared to the untreated or vehicle control-treated groups (Fig. 3D). All mice lost body weight following virus infection, but surviving mice treated with 6j regained the lost weight by 15 dpi (Fig. 3E). Recovery of body weight was faster in mice treated with 6j starting at 1 dpi than at 2 dpi (Fig. 3E). These results show that survival of mice markedly increased when 6j was given at 1 dpi. The antiviral effect of 6j in this mouse model was greater than that of 6h with better survival rates and faster recovery of body weight in the 6j-treated group (Fig. 3D, E).

Treating infected hDPP4-KI mice with compound 6j reduces lung viral titers
The lung pathology caused by MERSMA-CoV infection of hDPP4-KI mice resembles that seen in severe cases of human MERS-CoV infection, with diffuse alveolar damage, pulmonary edema, hyaline membrane formation, and infiltration of lymphocytes into the alveolar septa (22). A group of hDPP4-KI mice were infected with MERSMA-CoV and treated with compound 6j or vehicle as a control starting at 1 dpi. Mouse lungs were collected for determination of virus load at 3 and 5 dpi, and for histopathology at 6 dpi. Lung virus titers decreased in the 6j-treated mice compared to control mice at both 3 and 5 dpi (P<0.01) (Fig. 4A). Edema in the lungs of the treated mice was reduced compared to control mice (P<0.01) (Fig. 4B). Scores for hyaline membrane formation were reduced in 6j-treated mice but were not statistically different from control mice. MERSMA-CoV-infected hDPP4-KI mice treated with vehicle showed patches in lung tissue variably composed of cellular inflammation, vascular congestion, and atelectasis (Fig. 4C and E). The airways of these animals were generally intact, with only scattered, uncommon sloughed cells (Fig. 4C and E). In some lungs from these mice, lymphatic vessels were filled with degenerative cells and cellular debris (Fig. 4C and E). Alveolar edema was detected in some lung tissue sections (Fig. 4C and E). In contrast, there were few observed lesions in the lungs of MERSMA-CoV-infected hDPP4-KI mice treated with compound 6j starting at 1 dpi (Fig. 4D and F).


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Fig. 4 Lung virus titers and histopathology of 6j-treated hDPP4-KI mice infected with MERSMA-CoV.
hDPP4-KI mice were infected with MERSMA-CoV at 0 days post infection (dpi) and then were treated with vehicle as a control or compound 6j starting at 1 dpi until euthanasia (n=4 or 5 per group). (A) Lungs were collected and virus titers measured at 3 and 5 dpi. Lungs were examined for edema and for hyaline membrane formation (B) and lung sections were stained with hematoxylin/eosin stain for histopathology at 6 dpi (C-F). (B) Tissues were scored for edema and hyaline membrane formation using the scale: 0, none; 1, rare (<5 alveoli); 2, <33% of lung fields; 3, 34-66% lung fields, and 4, >66% lung fields (30). (C-F) Representative histopathology images are shown for vehicle control (C, E) and compound 6j treatment (D, F) at 40X (C, D) or 100X (E, F). Asterisks indicate p<0.01 by multiple t tests.

DISCUSSION
There are currently no approved vaccines or small molecule therapeutics for the treatment of MERS-CoV, SARS-CoV or SARS-CoV-2 infection. However, numerous preventive and therapeutic options are under development (3–7). The most clinically-advanced antiviral compound with a broad-spectrum of activity is remdesivir (GS-5734). This nucleoside analog originally was developed as an antiviral drug against Ebola virus and has been shown to be effective against both MERS-CoV and SARS-CoV in cell culture assays and in animal models of coronavirus infection (23–26). Prophylactic treatment or early therapeutic treatment of infected mice with remdesivir reduced MERS-CoV or SARS-CoV-mediated weight loss and decreased lung virus titers and lung injury scores compared to vehicle-treated animals (23, 26). Remdesvir also showed potent activity against SARS-CoV-2 in cell culture assays and animal models (27), and was recently issued emergency use authorization by the US Food and Drug Administration as an investigational antiviral drug for COVID-19. Another nucleoside analog, EIDD-2801, which is a broad-spectrum inhibitor against multiple viruses including influenza viruses, was also shown to be effective against MERS-CoV and SARS-CoV in mouse models (28).

Our group has been engaged in the discovery of broad-spectrum inhibitors targeting the 3CLpro of multiple human and animal coronaviruses. We initially generated dipeptidyl and tripeptidyl series of compounds (29) and observed that the dipeptidyl compound series had superior pharmacokinetic (PK) profiles compared to the tripeptidyl compound series (20). A representative compound of the dipeptidyl series is GC376, which is currently in clinical development for FIP in cats and COVID-19. The PK characteristics of multiple dipeptidyl compounds similar to compound 6j, including GC376, were examined through intraperitoneal or subcutaneous administration in animals, including mice. It was determined that their Cmax values were > 100-fold of the EC50 for the target virus, and that the elimination half-life (T1/2) was 3-5 hours. The in vivo efficacy of GC376 against mice infected with MHV or murine norovirus has been demonstrated (11, 15).

Inspection of previously-obtained crystal structures of the dipeptidyl compounds in a complex with coronavirus 3CLpro (17, 19) revealed the potential to achieve enhanced binding interactions with the S4 subsite by introducing diverse functionalities at the cap position in the inhibitors. In the current study, a new dipeptidyl series focusing on the design of structural variants in the cap substructure were synthesized and evaluated for their activity against coronavirus 3CLpro. The EC50 of GC376 against MERS-CoV was determined to be ~1 µM. One of our goals was to generate compounds with near or below 0.1 µM potency against MERS-CoV and other target coronaviruses. All synthesized compounds displayed varying degrees of inhibitory activity against multiple coronaviruses in the FRET enzyme assay and cell-based assays. Among these compounds, 6e showed the most potent antiviral activity against SARS-CoV-2 3CLpro in a FRET enzyme assay (IC50 0.17 µM) and cell-based assays (EC50 0.15 µM), and 6j showed the most potent antiviral activity against MERS-CoV with an EC50 value of 0.04 µM (Table 2).

It was previously demonstrated that optimal potency is attained when the P1 and P2 residues are a glutamine surrogate and Leu, respectively, and that replacement of the P2 Leu with a cyclohexylalanine is inimical to potency (17-18). This is clearly evident when comparing the relative potencies of 6h and 7h versus 6i and 7i (Table 1 and Table S1). Furthermore, compounds with Leu at the P2 position showed higher CC50 values compared to those with cyclohexylalanine at P2 (Table S1). X-ray crystallography confirmed the mechanism of action of the inhibitors, which involves formation of a covalent bond between the active site cysteine and the carbonyl carbon of the aldehyde. X-ray crystallography also identified the structural determinants associated with binding, accounting for the observed differences in potency. The high-resolution cocrystal structures of 3CLpro inhibitors 7j and 7i with MERS-CoV 3CLpro also confirmed that the difference in activity arose from the loss of a H-bond with Gln192 and the loss of two additional H-bonds from the displacement of Gln167 and Phe143 with 7i (Fig. S2A). The nature of the interaction of 7j with the S4 subsite is unique among the compounds examined and provides strong support for our approach, vis-a-vis our focus on the cap position for enhancing binding affinity and potency. Compound 7j was cocrystallized with MERS-CoV, SARS-CoV, and SARS-CoV-2 3CLpro. Superposition of compound 7j with these 3CLpro enzymes revealed a similar binding mode amongst all three proteases. However, a key difference lies with the different conformation adopted by the difluorocyclohexyl ring in the MERS-CoV 3CLpro S4 subsite, enabling it to engage in additional H-bond binding interactions (Fig. 2E and Fig S4). Compound 7j had moderately lower potency against SARS-CoV 3CLpro and SARS-CoV-2 3CLpro compared to MERS-CoV 3CLpro in the FRET enzyme assay, suggesting that moieties forming H-bonds that were accommodated at the S4 subsite had an important impact on potency. Notably, the barrier to the development of drug resistance increases when an inhibitor engages in H-bond interactions with the backbone of the 3CLpro.

We used a robust mouse model of MERS-CoV infection (30-32) to evaluate the efficacy of compounds 6j and 6h. hDPP4-KI mice expressing human dipeptidylpeptidase 4 were infected with a mouse-adapted MERS-CoV virus (MERSMA-CoV). The infected mice develop fatal lung disease with severe inflammation and weight loss (32). Furthermore, the lung pathology caused by MERSMA-CoV infection of the hDPP4-KI mice closely resembles that of severe human MERS-CoV infection and is characterized by diffuse alveolar damage, pulmonary edema, hyaline membrane formation, and infiltration of lymphocytes into the alveolar septa (22). In the current study, we demonstrated that survival rates in this mouse model were higher with 6j treatment compared to 6h treatment (Fig. 3B). Interestingly, compounds 6j and 6h share a near identical structure except for the extra methylene group present in compound 6j. Compound 6h showed potent anti-3CLpro activity, whereas the antiviral activity of compound 6h in cell culture was lower than that of compound 6j (Tables 1,2), which may have been the reason for its diminished therapeutic efficacy in the mouse model (Fig. 3B).

Our findings indicate that therapeutic treatment of infected mice with compound 6j was associated with a reduction in lung viral load and lung pathology (Fig. 3). Moreover, treatment of mice with 6j at 1 dpi resulted in the survival of infected mice, whereas delaying treatment initiation until 3 dpi resulted in decreased survival. Overall, mouse survival was markedly increased only when 6j was given to mice at 1 dpi (p<0.05; Fig. 3D, E). Treatment with compound 6j starting at 2 dpi resulted in moderately increased survival of infected mice, but this was not statistically significant (p> 0.05). These results emphasize the importance of early therapeutic intervention in attaining a positive clinical outcome.

Earlier studies from our group showed that GC376 can cure fatal feline coronavirus disease in cats (20, 21), demonstrating that a specific coronavirus protease inhibitor can be effective therapeutically against coronavirus disease in a natural host. The MERS-CoV mouse model used here provides proof-of-principle regarding the therapeutic potential of our protease inhibitors for treating severe human respiratory coronavirus disease. Limitations of the current study include differences in host receptor usage, mortality and transmissibility between MERS-CoV and SARS-CoV-2. Thus, further evaluation of our protease inhibitors in mice, hamsters or nonhuman primates experimentally infected with SARS-CoV-2 will be crucial to assess these inhibitors as potential therapeutic options for COVID-19.

Our study showed that these compounds were broadly active against the 3CLpro of several coronaviruses, with compound 6j displaying the highest activity against MERS-CoV and compound 6e displaying the highest activity against SARS-CoV-2. Clinical efficacy is influenced by many factors, including drug bioavailability, PK, metabolism and the chemical stability of a compound. This poses a major challenge with respect to reliably predicting whether the difference in potency against different coronaviruses in assays in vitro can be translated to differences in clinical efficacy. Therefore, further research is needed to establish whether one protease inhibitor can be an effective therapeutic for both MERS-CoV and SARS-CoV-2 infections in humans. We have demonstrated that the dipeptidyl compound series can serve as a platform suitable for the structure-guided design of one or more inhibitors against highly virulent human coronaviruses. We have generated potent inhibitors of the 3CLpro of several coronaviruses, including SARS-CoV-2, and tested their efficacy in cultured cells and primary human airway epithelial cells. Furthermore, we have demonstrated proof-of-concept therapeutic efficacy for one 3CLpro inhibitor 6j in hDPP4-KI mice infected with MERSMA-CoV. Our study lays the foundation for advancing this compound series further along the drug development pipeline.