Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease

Abstract

A novel coronavirus SARS-CoV-2, also called novel coronavirus 2019 (nCoV-19), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.96% as of May 4, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (M^pro). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC₅₀ values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC₅₀ values ranging from 0.49 to 3.37 μM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known M^pro inhibitors. A complex crystal structure of SARS-CoV-2 M^pro with GC-376, determined at 2.15 Å resolution with three monomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by M^pro. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.

INTRODUCTION

An emerging respiratory disease COVID-19 started to circulate among human in December 2019. Since its first outbreak in China from an unknown origin, it quickly became a global pandemic. As of May 4, 2020, there are 239,740 deaths among 3,442,234 confirmed cases in 215 countries.¹ The etiological pathogen of COVID-19 is a new coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also called novel coronavirus (nCoV-2019). As the name indicates, SARS-CoV-2 is similar to SARS, the virus that causes severe respiratory symptoms in human and killed 774 people among 8098 infected worldwide in 2003.² SARS-CoV-2 shares ~82% of sequence identity as SARS and to a less extent for Middle East respiratory syndrome (MERS) (~50%).^{3, 4} SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus that belongs to the β-lineage of the coronavirus.⁵ The β-lineage also contains two other important human pathogens, the SARS coronavirus and MERS coronavirus. The mortality rate of SARS-CoV-2 is around 6.96% as of May 4, 2020, which is lower than that of SARS (~10%) and MERS (~34%).² However, current data indicate that SARS-CoV-2 is more contagious and has a larger R0 value than SARS and MERS,⁶ resulting in higher overall death tolls than SARS and MERS. The SARS-CoV-2 virus is currently spreading at an alarming speed in Europe and the United States.

There is currently no antiviral or vaccine for SARS-CoV-2. The SARS-CoV-2 viral genome encodes a number of structural proteins (e.g. capsid spike glycoprotein), non-structural proteins (e.g. 3-chymotrypsin-like protease (3CL or main protease), papain-like protease, helicase, and RNA-dependent RNA polymerase), and accessary proteins. Compounds that target anyone of these viral proteins might be potential antiviral drug candidates.^{7, 8} In this study, we focus on the viral 3CL protease, also called the main protease (M^pro), and aim to develop potent M^pro inhibitors as SAR-CoV-2 antivirals. The M^pro plays an essential role in coronavirus replication by digesting the viral polyproteins at more than 11 sites, and it appears like a high profile target for antiviral drug discovery.^9–12 The M^pro has an unique substrate preference for glutamine at the P1 site (Leu-Gln↓(Ser,Ala,Gly)), a feature that is absent in closely related host proteases, suggesting it is feasible to achieve high selectivity by targeting viral M^pro. As such, we developed the Fluorescence Resonance Energy Transfer (FRET)-based enzymatic assay for the SARS-CoV-2 M^pro and applied it to screen a focused library of protease inhibitors. Here we report our findings of several novel hits targeting SARS-CoV-2 M^pro and their mechanism of action. The in vitro antiviral activity of the hits was also evaluated in cell culture using infectious SARS-CoV-2 virus. Overall, our study provides a list of drug candidates for SARS-CoV-2 with a confirmed mechanism of action, and the results might help speed up the drug discovery efforts in combating COVID-19. The compounds identified herein represent one of the most potent and selective SARS-CoV-2 M^pro inhibitors so far with both enzymatic inhibition and cellular antiviral activity.^{9, 11, 12} The X-ray crystal structure of SARS-CoV-2 M^pro with GC-376 showed that the compound can adapt two configurations R and S, offering a molecular explanation of the high-binding affinity of the aldehyde-containing inhibitors. Significantly, the discovery of calpain II and XII inhibitors as potent SARS-CoV-2 antivirals suggest that it might be feasible to design dual inhibitors against the viral M^pro and the host calpains, both of which are important for viral replication.

RESULTS

Establishing the FRET-based assay for the SARS-CoV-2 main protease (M^pro)

The M^pro gene from SARS-CoV-2 strain BetaCoV/Wuhan/WIV04/2019 was inserted into pET-29a(+) vector and expressed in BL21(DE3) E. Coli. with a His-tag in its C-terminus. The M^pro protein was purified with Ni-NTA column to high purity (Fig. 1A). To establish the FRET assay condition, we designed a FRET based substrate using the sequence between viral polypeptide NSP4-NSP5 junction from SARS-CoV-2: Dabcyl-KTSAVLQ/SGFRKME(Edans). We then tested the M^pro proteolytic activity in buffers with different pH. We found that M^pro displays highest activity in pH 6.5 buffer (Fig. 1B), which contains 20 mM HEPES, 120 mM NaCl, 0.4 mM EDTA, and 4 mM DTT and 20% glycerol. As such, all the following proteolytic assay was conducted using this pH 6.5 buffer. Next, we characterized the enzymatic activity of this SARS-CoV-2 M^pro by measuring the K_m and V_max values. When 100 nM M^pro was mixed with various concentration of FRET substrate (0 to 200 μM), the initial velocity was measured and plotted against substrate concentration. Curve fitting with Michaelis-Menton equation gave the best-fit values of K_m and V_max as 32.8 ± 3.5 μM and 29.4 ± 1.1 RFU/s, respectively (Fig. 1C).

Figure 1: SARS-CoV-2 M^pro expression and characterization.

(A) SDS-PAGE of His-tagged-Main protease (M^pro) (lane 1); Lane M, protein ladder; the calculated molecular weight of the His-tagged-M^pro is 34,992 Da. (B) Reaction buffer optimization: 250 nM His-tagged-M^pro was diluted into three reaction buffers with different pH values. (C) Michaelis-Menten plot of 100 nM His-tagged- M^pro with the various concentrations of FRET substrate in pH 6.5 reaction buffer.

Primary screening of a focused protease library against the SARS-CoV-2 M^pro.

With the established FRET assay condition, we screened a collection of protease inhibitors from the Selleckchem bioactive compound library to identify potential SARS-CoV-2 M^pro inhibitors. The protease inhibitors are grouped based on their targets and mechanism of action and include proteasome inhibitors (1-8); HIV protease inhibitors (9-14); γ-secretase inhibitors (15-22); HCV NS3-4A protease inhibitors (23-29); DPP-4 inhibitors (30-35); miscellaneous serine protease inhibitors (36-39); cathepsin and calpain protease inhibitors (40-43); miscellaneous cysteine protease inhibitors (44-48); matrix metalloprotease inhibitors (49-51); and miscellaneous protease inhibitors (52-55). The inhibitors were pre-incubated with 100 nM of M^pro at 30 °C for 30 minutes in the presence of 4 mM 1,4-dithiothreitol (DTT) before the addition of 10 μM FRET substrate. The addition of DTT was to quench non-specific thiol reactive compounds and to ensure the M^pro is in the reducing condition. All compounds were tested at 20 μM, except compound 26, which was tested at 2 μM due to its fluorescent background. Encouragingly, four inhibitors (24, 28, 29 and 43) showed more than 60% inhibition against M^pro at 20 μM. Among the hits, simeprevir (24), boceprevir (28), and narlaprevir (29) are HCV NS3-4A serine protease inhibitors, and compound MG-132 (43) inhibits both proteasome and calpain.

Secondary screening of a focused library of calpain/cathepsin inhibitors and known viral 3CL^pro inhibitors

Given the encouraging results from the primary screening, we then further characterized the four hits (24, 28, 29, and 43) in a consortium of assays including dose-response titration, thermal shift binding assay (TSA), and counter screening assays with two other viral cysteine proteases, the enterovirus A71 (EV-A71) 2A and 3C proteases, both of which are cysteine proteases (Table 2). The HCV NS3-4A protease inhibitors boceprevir (28) and narlaprevir (29) inhibited M^pro with IC₅₀ values of 4.13 and 4.73 μM, respectively (Table 2), more potent than simeprevir (24) (IC₅₀ = 13.74 μM). Both compounds 28 and 29 also showed strong binding towards M^pro and shifted the melting temperature of the protein (ΔT_m) by 6.67 and 5.18 °C, respectively, at 40 μM. Despite their potent inhibition against the HCV NS3-4A serine protease and the SARS-CoV-2 cysteine M^pro, boceprevir (28) and narlaprevir (29) did not inhibit the EV-A71 2A and 3C proteases (IC₅₀ > 20 μM), suggesting they are not non-specific cysteine protease inhibitors. The calpain inhibitor MG-132 (43) had an IC₅₀ value of 3.90 μM against the M^pro, and was not active against the EV-A71 2A and 3C proteases (IC₅₀ > 20 μM). The binding of MG-132 (43) to M^pro was also confirmed in the TSA assay with a ΔT_m of 4.02 °C.

Table 2:

Characterization of HCV and calpain proteases inhibitors against SARS-CoV-2 M^pro using a consortium of secondary assays^a

^a:Value = mean ± S.E. from 3 independent experiments;

^b:N.T. = not tested;

^c:The IC₅₀ of PSI (64) on SARS CoV-2 M^pro was calculated by end point reading of 1 hour digestion, instead of the initial velocity.

In light of the promising results of the calpain inhibitor MG-132 (43), we then pursued to testing other calpain and cathepsin inhibitors that are commercially available (56-63) (Table 2). These compounds were not included in the initial library because they have not been advanced to clinical studies. Among this series of analogs, calpain inhibitor II (61) and XII (62) are the most potent M^pro inhibitors with IC₅₀ values of 0.97 and 0.45 μM, respectively. Binding of compounds 61 and 62 to M^pro shifted the melting curve of the protein by 6.65 and 7.86 °C, respectively. Encouragingly, both compounds 61 and 62 did not inhibit the EV-A71 2A and 3C proteases (IC₅₀ > 20 μM). Calpain inhibitor I (59) and MG-115 (60) also showed potent inhibition against M^pro with IC₅₀ values of 8.60 and 3.14 μM, respectively. Calpeptin (56) and PSI (63) had moderate activity against M^pro with IC₅₀ values of 10.69 and 10.38 μM, respectively. In contrast, calpain inhibitors III (57) and VI (58) were not active (IC₅₀ > 20 μM).

We also included two well-known viral 3CL protease inhibitors GC-376 (64) and rupintrivir (65) in the secondary screening. GC-376 (64) is an investigational veterinary drug that is being developed for feline infectious peritonitis (FIP).^{13, 14} GC-376 (64) was designed to target the viral 3CL protease and had potent antiviral activity against multiple viruses including MERS, FIPV, and norovirus.^{13, 15} Rupintrivir (65) was developed as a rhinovirus antiviral by targeting the viral 3CL protease, but it was discontinued in clinical trials due to side effects.¹⁶ In our study, we found that GC-376 (64) was the most potent M^pro inhibitor with an IC₅₀ value of 0.03 μM. It shifted the melting curve of M^pro by 18.30 °C upon binding. In contrast, rupintrivir (65) was not active against M^pro (IC₅₀ > 20 μM). Previous report also showed that rupintrivir was not active against the SARS-CoV 3CL^pro (M^pro) (IC₅₀ > 100 μM).¹⁷ Both compounds 64 and 65 were not active against the EV-A71 2A protease, but showed potent inhibition against the EV-A71 3C protease, which is consistent with previously reported results.^{15, 18, 19}

When plotting the IC₅₀ values (log scale) of the inhibitors against M^pro from the FRET enzymatic assay with the melting temperature shifts (ΔT_m) from thermal shift binding assay (Fig. 3A), a linear correlation was observed, and the r² of the linear regression fitting is 0.94. This suggests that there is a direct correlation between the enzymatic inhibition and protein binding: a more potent enzyme inhibitor also binds to the protein with higher affinity. The stabilization of the M^pro against thermal denaturation was also compound concentration dependent (Fig. 3B).

Figure 3: Binding of inhibitors to SARS-CoV-2 M^pro using thermal shift binding assay and native mass spectrometry.

(A) Correlation of inhibition efficacy (IC₅₀) with ΔT_m from thermal shift binding assay. Data in Table 2 were used for the plot. The r² of fitting is 0.94. (B) Dose-dependent melting temperature (T_m) shift. Native MS reveals binding of SARS-CoV-2 M^pro to (C) GC-376 (64), (D) Calpain inhibitor II (61), (E) calpain inhibitor XII (62), and (F) Boceprevir (28). All ligand concentrations are 12.5 μM except E, which is 25 μM. Peak are annotated for dimer (blue circle), dimer with one bound ligand, (yellow down triangle), and dimer with two bound ligands (red up triangle). Other minor signals are truncated dimers, which bind ligands at the same ratios. Charge states are annotated in C, and insets show the deconvolved zero-charge mass distribution.

The binding of four most potent inhibitors boceprevir (28), calpain inhibitors II (61), XII (62), and GC-376 (64) to SARS-CoV-2 M^pro was further characterized by native mass spectrometry (MS) (Figs. 3C–F). Native MS analysis showed that the M^pro formed a dimer complex with a mass of 69,722 Da, indicating a cleaved N-terminal methionine (Fig. S1). A small amount of monomer and dimer with a single C-terminal truncation of the His-tag was also observed, but the intact dimer was the predominant signal (Fig. S1). Addition of all four ligands tested, boceprevir (28), calpain inhibitors II (61), XII (62), and GC-376 (64), showed binding of up to two ligands per dimer (Fig. 3C–F), suggesting a binding stoichiometry of one drug per monomer.

Mechanism of action of hits

To elucidate the mechanism of action of hits against SARS-CoV-2 M^pro, we focus on five most potent compounds prioritized from the primary and secondary screenings including boceprevir (28), MG-132 (43), calpain inhibitor II (61), calpain inhibitor XII (62), and GC-376 (64). For this, we performed enzyme kinetic studies with different concentrations of inhibitors (Fig. 4). A biphasic enzymatic progression curve in the presence but not in the absence of inhibitor is typically a hallmark for a slow covalent binding inhibitor. In the Fig. 4, left column shows the progression curves up to 4 hours. Biphasic progression curves were observed for all 5 inhibitors at high drug concentrations. Significant substrate depletion was observed when the proteolytic reaction proceeded beyond 90 minutes, we therefore chose the first 90 minutes of the progression curves for curve fitting (Fig. 4 middle column). We fit the progression curves in the presence different concentrations of GC-376 (64) with the two-step Morrison equation (equation 3 in methods section). GC-376 (64) binds to SARS-CoV-2 M^pro with an equilibrium dissociation constant for the inhibitor (K_I) of 59.9 ± 21.7 nM in the first step. After initial binding, a slower covalent bond is formed between GC-376 (64) and M^pro with the second reaction rate constant (k₂) being 0.00245 ± 0.00047 s⁻¹, resulting an overall k₂/K_I value of 4.08 x 10⁴ M⁻¹ s⁻¹ (Fig. 4A). However, when we tried to fit the proteolytic progression curves for boceprevir (28), MG-132 (43), calpain inhibitors II (61) and XII (62) using the same two-step reaction mechanism, we could not obtain accurate values for the second rate constant k₂. This is presumably due to significant substrate depletion before the equilibrium between EI and EI*, leading to very small values of k₂. Accordingly, for these four inhibitors 28, 43, 61, and 62, only the dissociation constant K_I values from the first step were determined (Figs. 4B–4E). The inhibition constants (K_I) for boceprevir (28), MG-132 (43), calpain inhibitors II (61) and XII (62) are 1.18 ± 0.10 μM, 1.57 ± 0.13 μM, 0.40 ± 0.02 μM, and 0.13 ± 0.02 μM, respectively.

Figure 4: Proteolytic reaction progression curves of M^pro in the presence or the absence of compounds.

In the kinetic studies, 5 nM M^pro was added to a solution containing various concentrations of protease inhibitors and 20 μM FRET substrate to initiate the reaction, the reaction was then monitored for 4 hrs. Left column shows the reaction progression up to 4 hrs; middle column shows the progression curves for the first 90 minutes, which were used for curve fitting to generate the plot shown in the right column. Detailed methods were described in the Method section. (A) GC-376 (64); (B) Boceprevir (28); (C) MG-132 (43); (D) Calpian inhibitor II (61); (E) Calpain inhibitor XII (62).

Cellular antiviral activity and cytotoxicity of hits

To test the hypothesis that inhibiting the enzymatic activity of M^pro will lead to the inhibition of SARS-CoV-2 viral replication, we performed cellular antiviral assays for the five promising hits 64, 28, 43, 61, and 62 against SARS-CoV-2. For this, we first tested the cellular cytotoxicity of these compounds in multiple cell lines (Table S1). GC-376 (64), boceprevir (28), and calpain inhibitor II (61) were well tolerated and had CC₅₀ values of over 100 μM for all the cell lines tested. MG-132 (43) was cytotoxic to all the cells with CC₅₀ values less than 1 μM except A549 cells. Calpain inhibitor XII (62) had acceptable cellular cytotoxicity with CC₅₀ values above 50 μM for all the cell lines tested (Table S1).

Next, we chose four compounds boceprevir (28), calpain inhibitors II (61), XII (62), and GC-376 (64) for the antiviral assay with infectious SARS-CoV-2. MG-132 (43) was not included due to its cytotoxicity. Gratifyingly, all four compounds showed potent antiviral activity against SARS-CoV-2 in the primary viral cytopathic effect (CPE) assay with EC₅₀ values ranging from 0.49 to 3.37 μM (Table 3). Their antiviral activity was further confirmed in the secondary viral yield reduction (VYR) assay. The most potent compound was calpain inhibitor XII (62), which showed EC₅₀ of 0.49 μM in the primary CPE assay and EC₉₀ of 0.45 μM in the secondary VYR assay. In comparison, remdesivir was reported to inhibit SARS-CoV-2 in the VYR assay with an EC₅₀ of 0.77 μM.²⁶ None of the compounds inhibited the unrelated influenza virus A/California/07/2009 (H1N1) virus (EC₅₀ > 20 μM) (Table S1), suggesting the antiviral activity of the four compounds (boceprevir, calpain inhibitors II, XII, and GC-376) against SARS-CoV-2 is specific. In comparison with recently reported SARS-CoV-2 M^pro inhibitors (Table 3), the hits identified herein represent one of the most potent and selective drug candidates with broad chemical diversity.

Table 3:

Antiviral activity of hits against SARS-CoV-2 and the comparison with recently reported M^pro inhibitors.

^aCPE EC₅₀, VYR EC₉₀, and cytotoxicity CC₅₀ values are mean ± S.D. of 3 independent experiments.

^bResults were retrieved from recent publications.^{9, 11, 12}

N.T. = not tested. N. A. = not available

Complex crystal structure of SARS-CoV-2 M^pro with GC-376 (64)

The crystal structure of the SARS-CoV-2 M^pro in complex with GC-376 (64) was solved in the p3₂21 space group at 2.15 Å resolution (Table S2). There are three monomers per asymmetric unit (ASU), with two constituting a biological dimer and the third forming a dimer with a crystallographic symmetry related neighboring protomer (Fig. S2). The presence of three monomers in our crystal structure allowed us to capture different binding configurations of GC-376 (64) (Fig. 5), a unique feature that was not observed in previous X-ray crystal structures ^{9, 11, 12}. The pairwise r.m.s.d among the monomer backbone Cα atoms ranges from 0.435 Å to 0.564 Å. Previously, SARS-CoV M^pro and SARS-CoV-2 M^pro crystal structures have been solved most frequently as a monomer per ASU, and occasionally a dimer ^{9, 11, 12, 28, 29}. In its native state, M^pro requires dimerization to become catalytically active ^{30, 31}, which is supported by our native MS data (Fig. S1). In our crystal structures, all three protomers appear catalytically competent, with the third protomer activated by the N-finger from an adjacent asymmetric unit (Fig. S2B).

Figure 5: Molecular recognition of GC-376 (64) by SARS-CoV-2 M^pro.

Complex of SARS-CoV-2 M^pro and GC-376 (64) with (A, B) protomer A and (C, D) protomer C. Unbiased F_o-F_c map, shown in grey, is contoured at 2 σ. Hydrogen bonds are shown as red dashed lines. (E) Surface representation of SARS-CoV-2 M^pro + GC-376 (64) (white) superimposed with the SARS-CoV M^pro natural, N-terminal substrate (PDB ID 2Q6G, with residues P1’-P4 in different colors). The SARS-CoV-2 M^pro cleaves between the P1’ and P1 residues. (F) Superimposition of the three protomers in the asymmetric subunit of SARS-CoV-2 M^pro with GC-376 (64). Significant conformational flexibility is observed, particularly in the TSEDMLN loop.

GC-376 (64) forms an extensive network of hydrogen bonds with the active site while also exhibiting excellent geometric complementarity (Fig. 5). These interactions are coupled with the thermodynamic payoff of covalent adduct formation between the aldehyde bisulfite warhead and Cys145, making GC-376 (64) one of the most potent SARS-CoV-2 M^pro inhibitors in vitro with an IC₅₀ of 0.030 ± 0.008 μM (Table 3). Along with other known M^pro inhibitors N3, 13b, 11a and 11b (Table 3), GC-376 (64) mimics the peptide substrate that is cleaved by this enzyme (Fig. 5E) ^{9, 27, 29, 32}. The glutamine surrogate γ-lactam ring is a cyclized derivative of the P1 glutamine side chain that normally occupies the S1 site; here it forms hydrogen bonds with the His163 and Glu166 side chains and the main chain of Phe140 (Figs. 5A & C). An amide bond connects the γ-lactam side chain to an isobutyl moiety that embeds itself in the hydrophobic S2 site formed by His41, Met49, and Met169. Normally, this S2 site in SARS-CoV-2 M^pro can accommodate a variety of hydrophobic substitutions such as isobutyl in GC-376 (64) and N3, cyclopropyl in 13b, cyclohexyl in 11a, and 3-fluorophenyl in 11b (Table 3) ^{33, 34}. A carbamate bond in GC-376 (64), which forms hydrogen bonds with the main chain of Glu166 and the side chain of Gln189, connects the P2 isobutyl group to a phenylmethyl ester that interacts with the aliphatic S4 site. Compared with previous inhibitors, the phenylmethyl ester of GC-376 exhibits high complementarity with the S4 site, and the extensive non-polar interactions may contribute significantly to the potency of this compound (Fig. 5E).

Three copies of GC-376 (64) were found in the crystal structure, one in each protomer active site (Figs. 5A, 5C, S3). The configurations of G-C376 (64) were consistent in protomers A and B, where the thioacetal hydroxide is positioned in the “oxyanion hole” formed by the backbone amides of Gly143, Ser144, and Cys145 (Figs. 5A & S3), resulting in the (S)-configuration. It is noted that aldehydes 11a and 11b also bind in the active site of SARS-CoV-2 M^pro in the (S)-configuration (PDB: 6M0K and 6LZE) (Figs. S4A).¹² In protomer C, however, the same hydroxide group orients outwards from the oxyanion hole, forming hydrogen bonds with His41 (Fig. 5C), which gives the (R)-configuration. This (R)-configuration is consistent with the binding mode of α-ketoamide 13b in the active site of SARS-CoV-2 M^pro (PDB: 6Y2F) (Fig. S4B).⁹ These two unique configurations R and S might be a result of the Cys145 thiol nucleophilic attacking the aldehyde from two different faces (Figs. 5B & D). The fact that GC-376 (64) can adapt two different configurations R and S upon binding to the active site might explain its high binding affinity towards the target.

An additional difference between the configurations of GC-376 (64) in A, B and C is observed in the orientation of the phenylmethyl ester. In protomer C, the CH₂ of the phenylmethyl points towards the main chain of Leu167 in a ‘cis’ conformation (Fig. 5C), whereas in protomers A and B this same CH₂ points downwards in a ‘trans’ conformation (Figs. 5A & S3). Consequently, this influences the rotameric configuration of the Leu167 isobutyl moiety, where a rotational adjustment of 180° occurs at its “β” carbon. Furthermore, large rearrangements are observed in the flexible TSEDMLN loop consisting of residues 45-51 (TAEDMLN in SARS-CoV-2 M^pro) that form the S2 and S3’ subsites (Fig. 5F), explaining the broad substrate scope in the P2 site (Table 3). The loop conformations in protomers B and C may be influenced by crystal packing interactions with protomers from adjacent asymmetric units and resemble the conformations in previously determined structures ^{11, 12}. Meanwhile, the conformation of protomer A is less restrained and exhibits the most significant conformational divergence. The different loop conformations offer a glimpse of the protein plasticity that allows M^pro to accommodate peptides with differing amino acid composition, and underscores the importance of considering this flexibility when analyzing and modeling protein-ligand interactions for M^pro.

DISCUSSION

Coronaviruses have caused three epidemics/pandemics in the past twenty years including SARS, MERS, and COVID-19. With the ongoing pandemic of COVID-19, scientists and researchers around the globe are racing to find effective vaccines and antiviral drugs.⁷ The viral polymerase inhibitor remdesivir holds the greatest promise and it is currently being evaluated in several clinical trials.^{35, 36} The HIV drug combination lopinavir and ritonavir recently failed in a clinical trial for COVID-19 with no significant therapeutic efficacy was observed.³⁷ To address this unmet medical need, we initiated a drug repurposing screening to identify potent inhibitors against the SARS-CoV-2 M^pro from a collection of FDA-approved protease inhibitors. The M^pro has been shown to be a validated antiviral drug target for SARS and MERS.³⁸ As the SARS-CoV-2 M^pro shares a high sequence similarity with SARS and to a less extent with MERS, we reasoned that inhibiting the enzymatic activity of SARS-CoV-2 M^pro will similarly prevent viral replication.^{9, 11}

Noticeable findings from our study include: 1) Boceprevir (28), an FDA-approved HCV drug, inhibits the enzymatic activity of M^pro with IC₅₀ of 4.13 μM, and has an EC₅₀ of 1.90 μM against the SARS-CoV-2 virus in the cellular viral cytopathic effect assay. The therapeutic potential of boceprevir (28) should be further evaluated in relevant animal models and human clinic trials. Since boceprevir (28) is a FDA-approved drug, the dose, toxicity, formulation, and pharmacokinetic properties are already known, which will greatly speed up the design of follow up studies; 2) GC-376 (64), an investigational veterinary drug, showed promising antiviral activity against the SARS-CoV-2 virus (EC₅₀ = 3.37 μM ). It has the highest enzymatic inhibition against the M^pro with an IC₅₀ value of 0.03 μM. This compound has promising in vivo efficacy in treating cats infected with FIP, and has favorable in vivo pharmacokinetic properties. Therefore, GC-376 (64) is ready to be tested in relevant animal models of SARS-CoV-2 when available. Importantly, the X-ray crystal structure of SARS-CoV-2 M^pro in complex with GC-376 (64) provides a molecular explanation of the high binding affinity of aldehyde-containing compounds as they can adapt two configurations R and S. The conformational flexibility at the TSEDMLN loop explains the broad substrate scope at the P2 position of M^pro inhibitors; 3) Three calpain/cathepsin inhibitors, MG-132 (43), calpain inhibitors II (61) and XII (62), are potent inhibitors of M^pro and inhibit SARS-CoV-2 with single-digit to submicromolar efficacy in the enzymatic assay. Calpain inhibitors II (61) and XII (62) also inhibit SARS-CoV-2 in the CPE assay with EC₅₀ values of 2.07 and 0.49 μM, respectively. This result suggests that calpain/cathepsin inhibitors are rich sources of drug candidates for SARS-CoV-2. Indeed, previous studies have shown that calpain and cathepsin are required for the proteolytic processing of the coronavirus S protein, a step that is essential for the viral fusion and genome release during the early stage of viral replication.³⁹ Calpain and cathepsin inhibitors such as MDL28170 (calpain inhibitor III)³⁹, MG-132⁴⁰, calpain inhibitor VI⁴¹ have been shown to inhibit SARS-CoV replication in cell culture. Other than the increased potency of targeting both M^pro and calpain/cathepsin, an additional benefit of such dual inhibitors might be their high genetic barrier to drug resistance. A significant number of calpain/cathepsin inhibitors have been developed over the years for various diseases including cancer, neurodegeneration disease, kidney diseases, and ischemia/reperfusion injury.⁴² Given our promising results of calpain inhibitors II (61) and XII (62) in inhibiting the SARS-CoV-2 M^pro and their potent antiviral activity in cell culture, it might be worthwhile to repurposing them as antivirals for SARS-CoV-2.

All potent SARS-CoV-2 M^pro inhibitors contain reactive warheads such as α-ketoamide (boceprevir (28), calpain inhibitor XII (62)) or aldehyde (MG-132 (43), calpain inhibitor II (61)) or aldehyde prodrug, the bisulfite (GC-376 (64)). This result suggests that reactive warheads might be essential for SARS-CoV-2 M^pro inhibition. The compounds identified in this study represent one of the most potent and selective hits reported so far, and are superior than recently reported SARS-CoV-2 M^pro inhibitors ebselen, N3, and 13b (Table 3). Calpain inhibitor XII (62) had similar potency as the recently disclosed compounds 11a and 11b (Table 3).¹² Notably, calpain inhibitor II (61) and XII (62) have different chemical scaffolds as GC-376 (64), N3, 13b, 11a, and 11b, therefore providing new opportunities for designing more potent and selective SARS-CoV-2 M^pro inhibitors.

Aside from the above positive results, we also showed that ritonavir (9) and lopinavir (10) failed to inhibit the SARS-CoV-2 M^pro (IC₅₀ > 20 μM, Fig. 2), which might explain their lack efficacy in clinical trials for COVID-19.³⁷ Camostat (39) was recently proposed to inhibit SARS-CoV-2 entry through inhibiting the host TMPRSS2, a host serine protease that is important for viral S protein priming.⁴³ However, the antiviral activity of camostat has not been confirmed with infectious SARS-CoV-2 virus. In our study, we found camostat (39) has no inhibition against the SARS-CoV-2 M^pro (IC₅₀ > 20 μM).

Figure 2: Screening of known protease inhibitors against SARS-CoV-2 M^pro using the FRET assay.

20 μM of compounds (26 was tested at 2 μM) was pre-incubated with 100 nM of SARS-CoV-2 M^pro for 30 minutes at 30 °C, then 10 μM FRET substrate was added to reaction mixture to initiate the reaction. The reaction was monitored for 2 hours. The initial velocity was calculated by linear regression using the data points from the first 15 minutes of the reaction. The calculated initial velocity with each compound was normalized to DMSO control. The results are average ± standard deviation of two repeats.

In summary, this study identified several potent SARS-CoV-2 M^pro inhibitors with potent enzymatic inhibition as well as cellular antiviral activity. Further development based on these hits might lead to clinically useful COVID-19 antivirals. They can be used either alone or in combination with polymerase inhibitors such as remdesivir as a means to achieve potential synergic antiviral effect as well as to suppress drug resistance.

MATERIALS AND METHODS

Details of materials and methods can be found in the supporting information.

Table 1.

List of protease inhibitors tested against SARS-CoV-2 M^pro in the primary FRET assay.