key: cord-1000123-0c9q1n8b
authors: Lu, Jimmy; Chen, Sizhu Amelia; Khan, Muhammad Bashir; Brassard, Raelynn; Arutyunova, Elena; Lamer, Tess; Vuong, Wayne; Fischer, Conrad; Young, Howard S.; Vederas, John C.; Lemieux, M. Joanne
title: Crystallization of Feline Coronavirus M(pro) With GC376 Reveals Mechanism of Inhibition
date: 2022-02-24
journal: Front Chem
DOI: 10.3389/fchem.2022.852210
sha: 4bbe32cdb0c9d0cd7cb23024bf00a74c3b168a79
doc_id: 1000123
cord_uid: 0c9q1n8b

Coronaviruses infect a variety of hosts in the animal kingdom, and while each virus is taxonomically different, they all infect their host via the same mechanism. The coronavirus main protease (M(pro), also called 3CL(pro)), is an attractive target for drug development due to its essential role in mediating viral replication and transcription. An M(pro) inhibitor, GC376, has been shown to treat feline infectious peritonitis (FIP), a fatal infection in cats caused by internal mutations in the feline enteric coronavirus (FECV). Recently, our lab demonstrated that the feline drug, GC373, and prodrug, GC376, are potent inhibitors of SARS-CoV-2 M(pro) and solved the structures in complex with the drugs; however, no crystal structures of the FIP virus (FIPV) M(pro) with the feline drugs have been published so far. Here, we present crystal structures of FIPV M(pro)-GC373/GC376 complexes, revealing the inhibitors covalently bound to Cys144 in the active site, similar to SARS-CoV-2 M(pro). Additionally, GC376 has a higher affinity for FIPV M(pro) with lower nanomolar K(i) values compared to SARS-CoV and SARS-CoV-2 M(pro). We also show that improved derivatives of GC376 have higher potency for FIPV M(pro). Since GC373 and GC376 represent strong starting points for structure-guided drug design, determining the crystal structures of FIPV M(pro) with these inhibitors are important steps in drug optimization and structure-based broad-spectrum antiviral drug discovery.

Coronaviruses are single-stranded, positive-sense RNA viruses that affect mammals and birds, causing a variety of diseases (Anand et al., 2003) . Containing one of the largest genomes among RNA viruses (~27-31 kb), the family Coronaviridae makes up four genera: Alpha-, Beta-, Gamma-, and Deltacoronavirus (Báez-Santos et al., 2015) . Coronaviruses take over the host's transcriptional machinery by encoding two overlapping polyproteins, pp1a and pp1ab, which are cleaved by coronavirus-encoded proteases-papain-like protease (PL pro ) and main protease (M pro , also called Due to their rapid transmission and lethality, coronaviruses pose a major threat to public health (Chen et al., 2020) . This was seen in previous global coronavirus outbreaks such as the severe acute respiratory syndrome coronavirus (SARS-CoV) outbreak in 2002/ 3, the Middle East respiratory syndrome coronavirus (MERS-CoV) outbreak of 2012, and more recently, the COVID-19 pandemic caused by SARS-CoV-2 in 2019 and onward (de Wit et al., 2016; Hu et al., 2021) . Aside from humans, coronaviruses infect other mammals including felines, ferrets, mink, and pigs (Perera et al., 2018; Stout et al., 2020; Ye et al., 2020) . Feline enteric coronavirus (FECV), an Alphacoronavirus, is commonly found among domestic cats; however, infected cats are usually asymptomatic or experience mild enteritis (Felten and Hartmann, 2019) . Feline infectious peritonitis (FIP) derives from internal mutations in FECV shifting tropism from enterocytes to macrophages resulting in a 100% fatality rate in cats, thus FIP virus (FIPV) is vertically transmitted (Dye & Siddell, 2005; Felten and Hartmann, 2019) . It is worth noting that mutations in FECV M pro have not been associated with increased virulence in FIPV (Pedersen, 2014) . M pro inhibitors which block viral replication have, therefore, been extensively studied against different coronaviruses as a means to develop broad-spectrum antivirals (Hegyi and Ziebuhr, 2002; Kim et al., 2012; Bai et al., 2021) .

Various peptidomimetic inhibitors have been developed against viral M pro (Yin et al., 2007; St. John et al., 2015; Wang et al., 2016) . In 2011, a peptide-based inhibitor of M pro was reported as a promising antiviral drug to combat norovirus infection (Tiew et al., 2011) . That inhibitor has since been modified and the new derivative, GC376, was shown to inhibit FIPV M pro with sub-micromolar IC 50 values (Kim et al., 2012) . GC376, a dipeptidyl aldehyde bisulfite adduct, is a prodrug that converts into the active-form aldehyde, GC373, upon administration, effectively binding the active site of M pro and stopping viral replication (Kim et al., 2013; Kim et al., 2015) . Other studies have demonstrated that GC376 was successful in reversing the progression of experimentally induced FIP as well as naturally occurring FIP in cats, demonstrating that peptide-based inhibitors are effective against coronavirus infections in vivo (Kim et al., 2016; Pedersen et al., 2018) .

With the success GC376 has had in treating FIP in cats, it was then postulated to be an effective inhibitor to treat SARS-CoV-2 infections (Vuong et al., 2020) . We have previously reported that the prodrug GC376 and drug GC373 are potent inhibitors of SARS-CoV and SARS-CoV-2 M pro with K i values in the nanomolar range (Arutyunova et al., 2021) . The crystal structures of SARS-CoV and SARS-CoV-2 M pro in complex with the feline drugs revealed the inhibitor forming a covalent bond with Cys145 as a hemithioacetal in the active site (Vuong et al., 2020) . The varied effectiveness of GC376 against M pro of different coronaviruses suggests structural differences in drug binding (Kim et al., 2012; Arutyunova et al., 2021) . Despite the research invested in the feline drugs with regard to FIP, no crystal structure of FIPV M pro with GC376 or GC373 has been solved to date.

In this study, we solved the crystal structure of FIPV M pro (FIPV WSU-79/1146) in complex with the drug, GC373, and prodrug, GC376, to reveal the architecture of the active site with bound inhibitors. Furthermore, we examined the improved derivatives of GC376 and demonstrated their higher potency toward FIPV M pro . As GC376 and GC373 were successfully used to treat FIP in cats, they are considered strong starting points in drug design to treat COVID-19 in humans. Here, we compare the structural similarities and differences between SARS-CoV-2 M pro and FIPV M pro with the feline drugs for antiviral drug optimization against SARS-CoV-2 and the development of future broad-spectrum antivirals.

Inhibitors GC373, GC376, and their derivatives, as well as the FRET assay peptide substrate, Abz-SVTLQSG-Y(NO2)-R, were synthesized according to methods previously described (Vuong et al., 2020; Vuong et al., 2021) .

The FIPV WSU-79/1146 M pro gene was synthesized (Bio Basic, Canada) and cloned into pET SUMO expression vector (Invitrogen, United States), generating a fusion protein with a His-tagged SUMO domain at the N-terminus. The construct was transformed into BL21 (DE3) Escherichia coli, where protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) once OD 600 reached 0.5-0.6 and then grown for an additional 5 h at 32°C. The cells were harvested by centrifugation (5,000 ×g for 20 min at 4°C), suspended in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mM imidazole, pH 7.8), and lysed using the Emulsiflex C3 High Pressure Homogenizer. Cellular debris was removed by centrifugation at 20,000 ×g for 45 min at 4°C. The isolated supernatant was applied onto a Ni-NTA column (Qiagen, Canada), the resin was washed with 10 column volumes of lysis buffer containing 20 mM imidazole, and the protein was eluted with a step gradient of 100-1000 mM imidazole in lysis buffer. The eluted fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pooled based on purity and dialyzed against 20 mM Tris-HCl, 150 mM NaCl, 1 mM TCEP, pH 7.8, for 2 h at 4°C. The SUMO tag was cleaved off using His-tagged SUMO protease (McLab, United States) and both the N-terminal SUMO tag and SUMO protease were removed by passing the protein sample through a Ni-NTA column. The flow-through containing FIPV M pro was further purified using size exclusion chromatography (Superdex increase 10/300 GL, GE Healthcare), with buffer containing 20 mM Tris-HCl, 150 mM NaCl, 1 mM TCEP, pH 7.8. The fractions containing FIPV M pro were pooled and concentrated using an Amicon Ultra-15 filter with a MWCO of 10 kDa.

Purified FIPV M pro was dialyzed against 5 mM Tris-HCl, 5 mM NaCl, 1 mM TCEP, pH 7.8 buffer at 4°C overnight and concentrated Frontiers in Chemistry | www.frontiersin.org February 2022 | Volume 10 | Article 852210

to 10 mg/ml using an Amicon Ultra-15 filter with a MWCO of 10 kDa. FIPV M pro was incubated with GC373 or GC376 (5× molar excess) at 4°C for 2 h prior to crystallization. The protein was subjected to the PACT and JCSG crystallization screens (Molecular Dimensions, United States). Crystals were observed with sitting drop trays at room temperature. The crystals of FIPV M pro with GC376 were obtained using a protein:buffer ratio of 1:1 with 2.0 M ammonium sulfate, 0.1 M Bis-Tris, pH 5.5. The crystals of FIPV M pro with GC373 were obtained using a 2:1, protein:buffer ratio with 0.2 M calcium chloride dihydrate, 0.1 M MES, 20% (w/v) PEG 6000, pH 6.0. The crystals were frozen in liquid nitrogen using 19% glycerol as a cryoprotectant.

The diffraction data were collected at Canadian Light Source using beamline CMCF-BM (08B1) and PILATUS3 S 6M detector, Saskatchewan, Canada. Several data sets were collected from different crystals and were processed using SCALA and XDS.

The diffraction data set of the GC373 was processed to 2.05 Å, in a monoclinic C2 space group, while the GC376 were processed to 1.93 Å, in an orthorhombic P2 1 2 1 2 1 space group. The structures were determined by molecular replacement using the crystal structure of the apo-FIPV Mpro (PDB entry: 5EU8) as the search model. GC376 and GC373 were manually fit in the density using Coot. The structures were then refined by using the Phenix software. Data statistics, processing, and model refinement are given in Supplementary Table S1 .

A fluorescence resonance energy transfer (FRET)-based cleavage assay was performed using a synthetic peptide fluorescent substrate containing the cleavage site of FIPV M pro [Abz-SVTLQ↓SG-Tyr (NO2)-R] as described previously (Vuong et al., 2020; Vuong et al., 2021) . For K i determination, 50 nM FIPV M pro was preincubated with GC376 in the concentration range of 0.01-0.4 µM for 10 min at 37°C. The enzymatic reactions using 1-500 μM of FRET substrate in activity buffer (25 mM Bis-Tris, 1 mM DTT, pH 7.0) were started with the addition of protease. For IC 50 determination, 100 nM of FIPV M pro was incubated with an inhibitor concentration range of 0.25 nM-100 µM in activity buffer. The reaction was started with 40 µM of FRET substrate. The fluorescence signal of the FRET peptide cleavage product was monitored at an emission wavelength of 420 nm with excitation at 320 nm, using a Cytation 5 Imaging Multi-Mode Reader (BioTek) for 7 min at 37°C. The kinetic data were analyzed using computer-fit calculation (Prism 9.0, GraphPad Software). The slopes of the Lineweaver-Burk plots were plotted versus the concentration of GC376, and the K i was determined from the x-axis intercept as −K i . The experiments were performed in triplicate.

The Overview of Feline Infectious Peritonitis Virus M pro -GC373/376 Complex Structure

Crystal structures of FIPV M pro in complex with the drug GC373 (PDB: 7SNA) and prodrug GC376 (PDB: 7SMV) were solved to 2.05 Å and 1.93 Å, respectively (Supplementary Table S1 ). GC376 being the dipeptidyl aldehyde bisulfite adduct form of the drug converts into the active-form aldehyde GC373, thus Frontiers in Chemistry | www.frontiersin.org February 2022 | Volume 10 | Article 852210 4 making both structures identical. In both structures, FIPV M pro crystallized as a dimer, with each protomer being comprised of three domains ( Figure 1A) , similar to other viral M pro . Domains I and II have a six-stranded antiparallel β-barrel structure, and domain III is a globular cluster of five antiparallel α-helices, connected to domain II by a long loop. The active site of FIPV M pro contains a Cys144-His41 catalytic dyad located in a cleft between domain I and domain II. Domain III regulates the dimerization of the M pro which is required for its catalytic activity. The N-terminal residues (N-finger) of protomer A fits between domains II and III of the protomer A and interacts with residues in domain II of protomer B helping shape the S1 substrate-binding subsite in the active site.

The GC373 inhibitor covalently binds FIPV M pro and is stabilized by hydrogen bonding and hydrophobic interactions in a similar manner to SARS-CoV and SARS-CoV-2 M pro . In both structures, a covalent bond between Cys144 and the aldehyde of the feline drug reveals that the bisulfite leaving group indeed was removed upon binding ( Figure 1B) . Weak H-bonding was observed between the oxyanion of the inhibitor and His41, the general base in the catalytic dyad, which is distinct from SARS-CoV-2 ( Figure 2A ) (Vuong et al., 2020) . For the P1 position of the inhibitor, the Nγ of the lactam ring sits in the S1 pocket and forms a H-bond with the carbonyl oxygen of Phe139 (Supplementary Figure S1A) , a conserved feature in other M pro structures with GC373 (Arutyunova et al., 2021) . The S2 pocket that supports hydrophobic interactions of a Leu moiety is formed with His41, Ile51, and Leu164 (Supplementary Figure S2A) . This differs from the stabilization network found in SARS-CoV-2 M pro for the same feline drug. Meanwhile, the P3 benzyl moiety interacts with the P1 lactam ring by pi stacking, similar to that observed in the SARS-CoV-2 M pro structure (Vuong et al., 2020) .

FIPV and SARS-CoV-2 M pro share 60% sequence similarity, however, the active-site region exhibits even greater conservation (Supplementary Figure S3) . Comparing the overall structures, both bound to GC373, the RMSD was calculated to be 1.16 Å. While the active site cavity of both FIPV and SARS-CoV-2 M pro are composed of identical residues, the structures of M pro in complex with GC373 or GC376, reveal some differences in inhibitor binding. In FIPV M pro , GC373 is stabilized in the active site by H-bonding with His41 ( Figure 2A, Supplementary Figure S1B ). By contrast, SARS-CoV (not shown) and SARS-CoV-2 M pro form a stable acyl-intermediate with the drug through a H-bonding network with the backbones of Cys145, Ser144, and Gly143 residues ( Figure 2B , Supplementary Figure S6 ). Furthermore, the S2 pocket that supports hydrophobic interactions of the drug's Leu moiety is formed with His41, Ile51, and Leu164 in FIPV M pro , but with His41, Met49, and Met165 in SARS-CoV-2 M pro (Supplementary Figure S2B) . In SARS-CoV-2 M pro , Gln189 plays an integral role in stabilizing the dipeptide backbone of the inhibitor (Bai et al., 2021) , however in FIPV M pro , we observe an unstructured loop fit between the S3 and S4 pocket to form hydrophobic interactions, thus further supporting binding of the inhibitor (Supplementary Figures S4, S5) . In FIPV M pro , Ser1 of the N-terminal finger from protomer B forms a weak H-bond (3.8 Å) with the cyclic glutamine analog nitrogen of GC373 in the active site of protomer A, however, this is not seen in SARS-CoV-2 M pro ( Figure 3A) . Furthermore, the side chain hydroxyl group and backbone amide of Ser1 in protomer B form H-bonds with Glu165 and Phe139 in the active site of protomer A. This is comparable to SARS-CoV and SARS-CoV-2 M pro structures where Ser1 of the N-terminal finger (protomer B) forms H-bonds with Glu166 and Phe140 (protomer A) to shape the P1 position ( Figure 3B ). These structural changes led us to examine the inhibitory parameters of GC376 with FIPV M pro for comparison with SARS-CoV and SARS-CoV-2 M pro .

IC 50 and K i values quantitatively reflect the potency and affinity of a drug and are therefore important parameters to consider when undergoing inhibitor design. First, we determined the catalytic parameters of FIPV M pro using our synthetic peptide FRETsubstrate (Supplementary Table S2 ) (Arutyunova et al., 2021) .

Interestingly, feline coronavirus protease exhibited 24 times slower catalytic turnover rate than M pro of SARS-CoV-2 with the same substrate, and a lower K m value. The K i values for GC376 inhibition were determined to be 2.1 nM for FIPV M pro (Figure 4) , lower in comparison to previously determined K i values of SARS-CoV and SARS-CoV-2 M pro , which were 20 and 40 nM, respectively ( Table 1 ) (Vuong et al., 2020; Arutyunova et al., 2021) .

We recently demonstrated that derivatives of GC376 with singly or doubly modified constituents resulted in improved potency with SARS-CoV-2 M pro , having lower IC 50 and EC 50 values . The singly modified compounds contain derivatives that include a cyclopropyl group (1a) in the P2 position where the S2 pocket typically recognizes a Leu residues side chain, and a 3-fluorobenzyl (2c) or 3chlorophenylethyl group (2d) in the P3 position recognized by the S4 pocket ( Table 2 ). The doubly modified compounds all included a cyclopropyl group in the P2 position, as well as a 3fluorobenzyl (2c), 3-chlorophenylethyl (2d), or 4-methoxyindole (2e) group at the P3 position (Table 3) . In order to assess if these inhibitor derivatives also have improved potency with FIPV M pro as they did with SARS-CoV-2 M pro , IC 50 values were calculated and compared. In vitro analysis with purified FIPV M pro revealed that the doubly modified inhibitor had stronger effects on IC 50 values than a singly modified inhibitor, bringing the IC 50 to the double-digit nanomolar range. This is a similar trend as seen with SARS-CoV-2 M pro using the same doubly modified inhibitors. Overall, this suggests that inhibitors targeting FIPV M pro can be improved and warrant further assessment in cellular and animal studies.

The FECV is commonly detected among domestic house cats and causes mild to no symptoms; however, mutations in FECV lead to FIP, a lethal systemic infection in cats. The M pro inhibitor GC373 and its bisulfide aldehyde GC376 have been shown to treat the otherwise fatal infection in experimentally infected FIP cats, as well as naturally acquired FIP cats. Furthermore, GC376 has also been shown to be an effective inhibitor of other viral M pro such as norovirus (PDB: 3UR9), transmissible gastric epidemic virus (PDB: 4F49), MERS-CoV (PDB: 5WTJ), porcine epidemic diarrhea virus (PDB: 6L70), SARS-CoV (PDB: 7LCQ), and more recently, SARS-CoV-2 (PDB: 6WTJ) (Tiew et al., 2011; Kim et al., 2015; Galasiti Kankanamalage et al., 2018; Vuong et al., 2020; Ye et al., 2020; Arutyunova et al., 2021) . Our work here reports the crystal structure of GC376 and GC373 with FIPV M pro , allowing for its comparison with recent structures of viral proteases with these inhibitors and complementing the work done by others in developing the drug for treating FIP in cats.

The overall architecture of GC373 bound to FIPV M pro is similar to other structures where the drug forms a C-shaped structure with pi stacking between the lactam ring and benzyl group in the P1 and P3 positions, respectively. While structures of SARS-CoV-2 M pro co-crystallized with GC376 solved by other groups have shown the drug binding in both R and S hemithioacetal isomer conformations (Ma et al., 2020) , here we only see the R conformation of the drug bound to FIPV M pro . We observe hydrogen binding of the oxyanion of GC373 to the general base His41 in FIPV M pro , similar to MERS-CoV and norovirus M pro (Kim et al., 2012; Galasiti Kankanamalage et al., 2018) . Nonetheless, this binding is in contrast to SARS-CoV, SARS-CoV-2, and PEDV M pro , where the oxyanion is bound by traditional backbone residues of the oxyanion hole (Kim et al., 2012; Lee et al., 2020; Arutyunova et al., 2021) . Overall, this suggests flexibility in the binding between the active site residues and inhibitor, and further highlights the feline drugs' broad specificity.

We have previously shown that the N-terminal tail of M pro plays a role in dimerization and drug stabilization (Arutyunova et al., 2021) . The FIPV M pro -GC373 complex reveals weak hydrogen bonding of the hydroxyl group of Ser1 (protomer B) with the cyclic glutamine analog in GC373, bound to the active site of protomer A, providing additional coordination for the inhibitor. By contrast, no interaction is observed between Ser1 and GC373 in SARS-CoV-2 M pro . This led us to compare the inhibitory parameters of GC376 between the two M pro to determine if these structural differences lead to improved drug binding. We recently showed that GC376 was an effective inhibitor of SARS-CoV M pro and SARS-CoV-2 M pro with K i values of 20 and 40 nM, respectively ( Table 1 ). In comparison, GC376 inhibited the FIPV M pro with a K i of 2.1 nM, 20 times higher in affinity than SARS-CoV-2 M pro . Together, the difference in K i values further reflects structural plasticity among various M pro that results in differences in how the drug binds the active site and thus affecting drug potency.

In order to increase the potency of GC376, our team has recently developed modified derivatives which showed lower IC 50 values for SARS-CoV-2 M pro compared to the parent compound . The modification of P2 was chosen to be lipophilic since our previous crystal structures demonstrate that the S2 pocket responsible for binding the leucine moiety was mostly hydrophobic. The modification of the P3 position allowed for enhanced dipole interactions with the S4 pocket of the enzyme, potentially contributing to the higher affinity (Wang et al., 2016) . Importantly, these derivatives, in particular the ones with double modifications, exhibit lower IC 50 values with FIPV M pro compared to SARS-CoV-2 M pro . Moving forward, enhanced drugs are needed for both FIPV infections as well as other coronavirus-related outbreaks. This crystal structure of FIPV M pro in complex with GC376 will assist us in accelerating development of new derivatives to be used in clinical trials as broad-spectrum antivirals.

The original contributions presented in the study are included in the article/Supplementary Material, and further inquiries can be directed to the corresponding author.

JL and SAC contributed to protein purification. JL, SAC, MK, HY, and MJL contributed to crystallization and structure determination. TL contributed to FRET-substrate synthesis. WV, CF, and JV contributed to inhibitor design. WV and CF contributed to inhibitor synthesis. RB, JL, SAC, and EA contributed to enzyme 

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We thank the staff at CLS beamline CMCF-BM (08B1), in particular Michel Fodje. Part or all of the research described in this article was performed using beamline CMCF-BM at the Canadian Light Source, a national research facility of the University of Saskatchewan, which is supported by the Canada Foundation for Innovation (CFI), the Natural Sciences and Engineering Research Council (NSERC), the National Research Council (NRC), the Canadian Institutes of Health Research (CIHR), the Government of Saskatchewan, and the University of Saskatchewan.

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2022.852210/ full#supplementary-material Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Publisher's Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors, and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.Copyright © 2022 Lu, Chen, Khan, Brassard, Arutyunova, Lamer, Vuong, Fischer, Young, Vederas and Lemieux. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.