key: cord-0942299-uoeg727c authors: Arutyunova, Elena; Bashir Khan, Muhammad; Fischer, Conrad; Lu, Jimmy; Lamer, Tess; Vuong, Wayne; van Belkum, Marco J.; McKay, Ryan T.; Lorne Tyrrell, D.; Vederas, John C.; Young, Howard S.; Joanne Lemieux, M. title: N-Terminal finger stabilizes the S1 pocket for the reversible feline drug GC376 in the SARS-CoV-2 Mpro dimer date: 2021-04-22 journal: J Mol Biol DOI: 10.1016/j.jmb.2021.167003 sha: 2a062002108a2461ba6da58b3b41c25477267d88 doc_id: 942299 cord_uid: uoeg727c The main protease (Mpro, also known as 3CL protease) of SARS-CoV-2 is a high priority drug target in the development of antivirals to combat COVID-19 infections. A feline coronavirus antiviral drug, GC376, has been shown to be effective in inhibiting the SARS-CoV-2 main protease and live virus growth. As this drug moves into clinical trials, further characterization of GC376 with the main protease of coronaviruses is required to gain insight into the drug’s properties, such as reversibility and broad specificity. Reversibility is an important factor for therapeutic proteolytic inhibitors to prevent toxicity due to off-target effects. Here we demonstrate that GC376 has nanomolar Ki values with the Mpro from both SARS-CoV-2 and SARS-CoV strains. Restoring enzymatic activity after inhibition by GC376 demonstrates reversible binding with both proteases. In addition, the stability and thermodynamic parameters of both proteases were studied to shed light on physical chemical properties of these viral enzymes, revealing higher stability for SARS-CoV-2 Mpro. The comparison of a new X-ray crystal structure of Mpro from SARS-CoV complexed with GC376 reveals similar molecular mechanism of inhibition compared to SARS-CoV-2 Mpro, and gives insight into the broad specificity properties of this drug. In both structures, we observe domain swapping of the N-termini in the dimer of the Mpro, which facilitates coordination of the drug’s P1 position. These results validate that GC376 is a drug with an off-rate suitable for clinical trials. In late 2019, a respiratory infection initially detected in China, was sparking fear of a viral outbreak 1 . This respiratory infection attributed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), led to an ongoing coronavirus disease 2019 (COVID- 19) pandemic with millions infected worldwide (https://coronavirus.jhu.edu/map.html). This respiratory illness was similar to a previous infection by SARS-CoV that led to a SARS outbreak in 2002/3 as well as the Middle East respiratory infection (MERS) outbreak in 2012 2; 3 . All of these outbreaks stem from related betacoronavirus infections, suggesting these strains will likely lead to future viral outbreaks 4 . Vaccines have been developed and will be important for prevention of new infections in the future. However, even with a 95% immunity rate, there will be a significant proportion of people worldwide who will require therapeutic treatment. Antiviral development remains a priority because of importance of immediate mitigation of acute infections, vaccine hesitancy, and the inability to vaccinate some individuals. The outbreak of SARS in 2003 and MERS in 2012 along with the current pandemic reminds us that paninhibitors may provide a means for initial control of outbreaks, thereby preventing or quickly controlling pandemics in the future 5 . SARS-CoV-2 is a 30-kb positive-sense single-stranded RNA virus that is translated by the host's cellular machinery to generate two alternatively spliced long polypeptides, PP1a and PP1ab. These long polypeptides release non-structural proteins (nsps), including the RNAdependent RNA polymerase, that are essential for viral replication after proteolytic cleavage by proteases from domain nsp3 and nsp5, respectively, a papain-like (PL pro ) protease and a chymotrypsin-like main protease (M pro or 3CL pro ) 6 . Similar to SARS-CoV, the SARS-CoV-2 M pro enzyme recognises the sequence of Leu-Gln↓Ser-Ala-Gly, where ↓ marks the cleavage site and this sequence is widely employed for generation of substrates for kinetic analysis and for development of peptidomimetic specific probes and inhibitors 7 8; 9 . The essential role of the M pro in viral replication has resulted in a great deal of crystallographic and in silico studies working towards the development of antiviral therapies to treat COVID-19 10; 11; 12; 13; 14 . Proteolytic inhibitors have been used successfully as antiviral therapeutics 15 It has been recently shown by our group, as well as by other teams, that M pro of SARS-CoV-2 is a promising drug target for the development of SARS-CoV-2 antivirals 11; 12; 14; 16 . We demonstrated that the proteolytic inhibitor GC376 (a bisulphite prodrug) used to treat feline coronavirus infection and its related aldehyde inhibitor, GC373, are effective at decreasing viral load of SARS-CoV-2 in cell culture 14 . These drugs have previously been shown to be effective inhibiting the M pro of picornavirus, norovirus and coronavirus, and furthermore have been validated in animal models for both SARS and MERS 17; 18; 19 . Even though we have a considerable understanding of the efficacy of GC376 and GC373 with both SARS-CoV and SARS-CoV-2 M pro 17; 18; 20; 21; 22; 23; 24 , detailed mechanistic and functional insight into the inhibitor binding process is still essential for directing broad-spectrum inhibitors in clinical trials. For example, one of desirable features for peptidomimetic proteolytic inhibitors is the reversible nature of binding since it reduces the risk of strong off-target effects and potential toxicity 25; 26 . In addition, in light of the new variants, we need a clear understanding of the efficacy of GC373 and GC376 with other coronavirus M pro , and importantly a crystal structure of these inhibitors with the SARS-CoV M pro has not been determined. In this study, we compare inhibition of the M pro of SARS-CoV and SARS-CoV-2 by GC376 using kinetic and structural approaches. We determine K i values are in the low nanomolar range for both SARS-CoV and SARS-CoV-2 M pro . After inhibition with GC376, NMR and activity assays demonstrate the reversible nature of inhibition for both proteases. In addition, the restoration of activity of M pro after inhibition reveal a high kinetic and thermodynamic stability for these viral proteases. We determine the crystal structures of SARS-CoV M pro inhibited with the dipeptidyl inhibitor, GC376, and aldehyde form, GC373, both of which reveal a covalent mode of inhibition similar to SARS-CoV-2 M pro . We highlight in both structures the role of the N-terminus in stabilizing the S1 subsite from domain swapping, and how this facilitates drug binding. This comparative analysis of M pro from SARS-CoV and SARS-CoV-2 provides additional insight into the mechanism of inhibition by this anti-coronaviral drug. Determining K i values that are reflective of drug binding affinity is a prerequisite for the prediction and evaluation of drug interactions. In our previous report, we determined the halfmaximal inhibitor concentrations (IC 50 ), values, which describe the functional strength of the inhibitor, for the feline drug GC376 with both M pro of SARS-CoV and SARS-CoV-2 14 . Here we determine K i values for the prodrug GC376 with both M pro of SARS-CoV and SARS-CoV-2. For K i determination, the inhibitory effects of increasing concentrations of GC376 on M pro from both SARS-CoV and SARS-CoV-2 were tested using the synthetic peptide FRET-substrate Abz-SVTLQSG-Y(NO 2 )-R followed by Michaelis-Menten kinetics. Data was presented as double reciprocal plot of reaction rate versus substrate concentration (primary Lineweaver-Burk plot) and the slopes (Km/Vmax) were determined by linear regression analysis. The slopes were plotted versus the concentration of GC376 to determine the inhibitory constant (K i as yintercept). The K i for GC376 was 0.02 µM for SARS-CoV M pro and 0.04 µM for SARS-CoV-2 M pro , (Figure 1 and Table 1 ). An important factor to consider when developing a therapeutic protease inhibitor is the reversibility of compound binding 25 . Irreversible protease drugs can yield long-lasting effects by permanently blocking proteases in cells that are not the intended target and thus causing detrimental consequences resulting in side effects and antigenicity of covalently modified proteins 27 . We previously demonstrated that the bisulfite prodrug GC376 converts to the peptide aldehyde GC373, which interacts covalently with the catalytic cysteine of SARS-CoV-2 M pro14 , but did not assess experimentally whether the inhibition was reversible. Reversibility of GC376 with SARS-CoV-2 M pro was evaluated first by NMR studies Figure 2D ) provided spectra to which the reversibility experiment could be compared. Evidence of binding reversibility was acquired by HSQC experiments conducted on a co-incubated sample containing both enzyme and inhibitor that was subsequently washed with buffer. The subsequent HSQC experiment using this sample showed a disappearance of the NMR signal corresponding to the bound inhibitor ( Figure 2E ). The disappearance of this signal would only be observed in the case of inhibitor dissociation. We then conducted a detailed study to provide the rate and percentage of reversibility, as well as the comparison of drug behaviour with SARS-CoV M pro and SARS-CoV-2 M pro . Reversibility was tested by measuring catalytic activity post dialysis. Incubation of SARS-CoV M pro and SARS-CoV-2 M pro with the GC376 followed by dialysis resulted in increase of enzymatic activity over time, indicative of a reversible dissociation of inhibitor ( Figure 3 ). We observed a recovery of 10% of activity after 22 hours of dialysis, which reached 30 -40% of initial activity for SARS-CoV and 40-60% for SARS-CoV-2 after 4 days of dialysis, suggesting over time the substrate competed for the enzyme binding site. To ensure the proteins remained stable over this time period, we also monitored the stability of uninhibited enzymes, which was compared with the activity of recovered enzymes. After 4 days the residual protease activity for the uninhibited M pro of SARS-CoV and SARS-CoV-2 was 30-40%, which allowed us to conclude that the drug was fully reversible. After observing the high kinetic stability of both viral proteases at room temperature, we characterized their thermal stability and assessed their thermodynamic parameters including activation energies of inactivation. Thermal stability is a characteristic used to describe the kinetic stability of enzymes, and many individual proteins or protein complexes are known to have high kinetic stability 28; 29; 30; 31; 32 . For viral proteins, particularly the structural ones, this feature is crucial because virus particles must be able to resist harsh environmental conditions until they find a new host to infect and also remain stable during infection 11; 14; 33 . For example, determination of thermodynamic parameters of the HIV protease in the presence of various inhibitors was used to reveal the differences in protein stability upon forming inhibitor-protein complexes, which informed on inhibitor design 34 . Thermal inactivation of SARS-CoV M pro (Figure 4A and 4B) and SARS-CoV-2 M pro ( Figure 4D and 4F ) was studied at the temperature range of 24-70 º C in a time-dependant manner. The semilogarithmic plots of residual activity versus incubation time were linear at all temperatures for both proteins, which was indicative of a simple first-order monophasic kinetic process. From the slopes of semilogarithmic plots inactivation rate constants were calculated and are given in Table 2 . For both proteases, the rate constant progressively increased with increasing temperatures, whereas half-life (t 1/2 ) and the decimal reduction time (Dt), two important parameters used in characterization of enzyme stability, decreased. The dependence of inactivation rate constants on temperature was plotted using the Arrhenius equation (Figure 4C and 4F), from which apparent activation energies of inactivation (Ea) were calculated. Interestingly, Arrhenius plots for both proteases were not linear and showed upward curvature suggesting two denaturation processes, each with its own temperature dependence and activation energy. At temperatures above 37 º C inactivation is a result of protein unfolding with high activation energy, with the rate of this process strongly dependant on temperature. At temperatures of 37 º C and below this rate becomes insignificant and other processes with low activation energy prevail. The activation energies for the high temperature range were found to be high and similar for SARS-CoV M pro (Ea=243.6 kJ/mol) and SARS-CoV-2 M pro (Ea=234.2 kJ/mol). However, for the low temperature range the activation energies were 10-20% of those determined at high temperature, confirming that M pro inactivation involves both high-and low-activation energy processes. Interestingly, the parameters of the inactivation process at low temperature range (24-37 º Determination of all thermodynamic parameters of inactivation can provide further information on enzyme stability. ΔG value, the Gibbs free energy, which is the energy barrier for enzyme inactivation, is directly related to protein stability. We see a significant decrease in ΔG for the temperatures above 55 º C indicating that the destabilization process occurs rapidly in this temperature range ( Table 2) . To gain a deeper insight into the driving forces of SARS-CoV M pro and SARS-CoV-2 M pro stability, the Gibbs free energy was decomposed into its enthalpic and entropic contributions. Enthalpy, ΔH, measures the number of non-covalent bonds broken during transition state formation for enzyme inactivation, allowing us to compare the energy landscapes of both SARS-CoV M pro and SARS-CoV-2 M pro . For temperature ranging from 37 º C to 70 º C we observed consistent high ΔH values, which is in agreement with a temperature-dependent inactivation process. Interestingly, at the 24 º C and 37 º C temperature interval a significant jump in ΔH occurred for both proteases, however, with different initial enthalpy values for SARS-CoV M pro and SARS-CoV-2 M pro at 24 º C (13.9 and 38.9 kJ/mol respectively), again highlighting higher stability of latter at physiological temperatures ( Table 2 ). The compactness in the protein molecular structure as well as enzyme and solvent disorder can be inferred through the quantitative analysis of entropy ΔS values 35; 36 . Small negative entropy values at 24 º C for both SARS-CoV M pro and SARS-CoV-2 M pro confirmed no disorder in protein structure upon inactivation; however, at higher temperatures all values of ΔS were positive and similar, suggesting that unfolding is a rate-limiting step at this range ( Table 2) . We previously reported increased catalytic activity of SARS-CoV-2 M pro in comparison to SARS-CoV M pro with the catalytic turnover rate being almost 5 times higher for the former using a FRET-peptide as substrate 14 . We were interested in structural comparison of the M pro from SARS-CoV and SARS-CoV-2, for both apo and drug-bound forms to reveal differences that account for the enhancement in activity. Crystal structures of apo-M pro from SARS-CoV and SARS-CoV-2, and bisulphite prodrug (GC376) and the aldehyde drug (GC373) bound forms were determined. The two proteins share 96% sequence identity with only 12 out of 306 residues being different ( Figure S1 ). Therefore, as expected, there is little change in the overall structures of apo-SARS-CoV and SARS-CoV-2 M pro (Figure 5) , with an RMSD of 0.6 Å. We observed a new helical feature at ƞ2 (residues 47-50) in SARS-CoV-2, which is unfolded in SARS-CoV, (Figure S1 and S2). It is located at the entrance to the active site, near a non-conserved residue between SARS-CoV, and SARS-CoV-2 ( Figure S2 ). In the GC373-bound form of proteins, however we observed the opposite; this helix is found in the M pro of SARS-CoV but not in SARS-CoV-2 ( Figure S3) , suggesting a dynamic nature of this structural element. Both SARS-CoV and SARS-CoV-2 M pro form dimers, and while monomers have very low activity, dimerization is necessary for full enzymatic activity and virulence 37; 38 . Comparative analysis of the biological dimer of the two proteases revealed that the main differences are located at the dimer interface. In the M pro of SARS-CoV-2, we observed a slight shift of the chymotrypsin-like domains away from each other, compared to the M pro of SARS-CoV ( Figure 5B) , which are not attributed to crystal packing. However, the biggest change is the difference in association between the dimerization domains ( Figure 5C and 5D) . The dimer interface of SARS-CoV and SARS-CoV-2 M pro is facilitated by several interactions between the two protomers, one of which is between the helical domain III of each protomer comprising of residues 284-286, specifically Ser-Thr-Ile (STI) in SARS-CoV M pro and Ser-Ala-Leu (SAL) in SARS-CoV-2 M pro . This unstructured loop self-associates between protomers in the dimer. Importantly, this region harbors a non-conservative residue in sequence at the dimer interface, where the Thr285 in SARS-CoV M pro is altered to Ala285 in SARS-CoV-2 M pro (Figure 5E and 5F ). The SAL-motif forms a tight van der Waals interaction and the residues from each protomer interdigitate to form a complementary interface that readily explains the observed enhanced stability. We recently presented the structure of GC373 with the SARS-CoV-2 M pro 14 . The structure of SARS-CoV-2 M pro with drug GC373, as well as prodrug GC376 that converts to GC373, reflects the specificity of the enzyme for a glutamine surrogate in the P1 position and a leucine, which is preferred in the P2 position. A benzyl group is in the P3 position. Here we determined the crystal structure of the SARS-CoV M pro with the prodrug GC376 and drug GC373 to examine features that determine its efficacy and compare this with the previously determined SARS-CoV-2 structure (Figure 6 ). SARS-CoV M pro was incubated with GC373 and GC376, prior to crystallization. The best crystals diffracted to 2.0 Å, and the data was refined with good statistics ( Table 3) . Overall comparison of SARS-CoV M pro and SARS-CoV-2 M pro structures with GC373 showed similar agreements with the apo-M pro structures, with an RMSD of 0.6 Å (Figure 6 ). The drug binding is supported by H-bonding with the main chains of oxyanion hole residues Asn142, Gly143 and Ser144, which are identical for both proteases (Figure 6B, S4 and S5) . A good fit was observed for both the P1 and P2 positions, supported structurally by hydrogen bonding and van der Waals interactions respectively with H-bonds for the P1 position being identical for M pro from SARS-CoV and SARS-CoV-2 ( Figure 6C, S4 and S5) . The N-terminal finger of the M pro stabilizes dimer formation and coordination of the S1 pocket that supports drug binding A distinctive feature of M pro dimer is the interaction of N-terminal residues ("N-finger") of protomer A with residues of domain II of protomer B. In the dimer for both protomers of SARS-CoV-2 M pro and SARS-CoV M pro , we observe the N-termini interact with residues near S1 substrate-binding subsite in a hairpin adjacent to the oxyanion hole of the active site ( Figure 7) . The NH-group of Ser1 from protomer A forms strong H-bonds with the carboxylate group of (Figure 8) , likely adding to its increased catalytic activity. The proper conformation of S1 pocket is also important for the drug binding, and importantly, P1 position of GC373 is stabilized by hydrogen bonding between the side chain of Glu166 (3.3 Å) and backbone carbonyl of Phe140 (3.3 Å) residues (Figure 8) . Thus, a hydrogen bond network between the dimer in M pro stabilizes the S1 substrate for substrate binding and hence inhibitor binding. Residues adjacent to the N-terminus also play a key role in dimerization, specifically Pro9 and Phe305 from protomer A, which interact with residues Pro122 and Ser123 in a strand on protomer B. We also observe these interactions in all of our SARS-CoV M pro and SARS-CoV-2 M pro structures bound to the inhibitor (Figure S7) . Mutation of Pro9 to Thr results in a monomeric species of SARS-CoV-2 M pro 40 . Together this data suggests a strong role for the Nterminus of the protease not only in function and stability, but also with inhibitor coordination. Here we show that the feline antiviral prodrug GC376 is reversible and inhibits M pro of both SARS-CoV and SARS-CoV-2 with low nanomolar K i values. While IC 50 42 . These drugs are reversible serine protease inhibitors whose development was facilitated by SAR studies 42; 43 . Our K i data further supports GC376 being a broad-spectrum inhibitor 17; 18; 21; 23 , and demonstrates it is in the inhibitory range to be considered as a viable antiviral for clinical trials. M pro from SARS-CoV and SARS-CoV-2 have 96% sequence identity and variant residues, with the exception of Ala285 discussed above, are conservative ( Figure S1 ). Therefore, it was not surprising that both proteins revealed similar physical chemical properties such as high thermal stability at temperatures above 37 o C with high activation energies and enthalpy independent of temperature ( Table 2) (Figure 5) . This mutation leads to residues in the domain III interface forming a hydrophobic zipper clearly aligning the two domains, and thus likely enhancing the t 1/2 at low temperatures as we have observed above. The high degree of stability of the enzymes for both SARS-CoV and SARS-CoV-2 is an interesting feature that likely contributes to viral potency. Another structural feature that might explain the increased activity and stability is a closer association between the N-finger Ser1 and Phe140 in the oxyanion loop in the M pro of SARS-CoV-2 compared to SARS-CoV (Figure 8) . This interaction plays a critical role for activity since it sustains the correct conformation of the oxyanion loop, therefore precise coordination of the N-finger in both M pro of SARS-CoV and SARS-CoV-2 is a prerequisite for function. Previous work demonstrated that enzymatic activity of SARS-CoV M pro was diminished with non-native affinity tags proving the need for native N-and C-termini 7; 39 . The effect was most pronounced with additional residues at the N-terminus, with the activity of the wild-type being 20-fold greater than a variant with an additional glycine at the N-terminus 39 . While GC376 has been crystallized with the main protease of the similar betacoronavirus MERS 19 , as well as other viral proteases, including norovirus and porcine diarrhea virus (PEDV) 46 , no N-finger association was observed in those crystal structures. This structural motif, however, was observed in a SARS-CoV M pro crystal structure with a Michael acceptor inhibitor, however the N-finger interaction was diminished with the addition of residues at the native Nterminus 39 . We demonstrated that the NH group of Ser 1 donates H-bonds to Phe140 and Glu166, the residues that coordinate the N-termini of each protomer in the dimer. Importantly, these residues also interact with the P1 position of GC373 in both SARS-CoV and SARS-CoV-2, demonstrating a strong hydrogen bond network near the active site, and stabilization of the S1 subsite pocket. This likely contributes to the high K i values for these inhibitors. The precise structural and mechanistic elucidation of the inhibitor-protease interaction and implications for M pro dimerization is paramount for the fine-tuned design of universally active inhibitor drugs. In this regard, the current study provides a rationale for the precise nature of a gamma-lactam group in the P1 position of the GC373/GC376 inhibitor. properties, yet demonstrate comparable efficacies of GC376 with both proteases. Furthermore, reversible inhibition with the drug further supports the clinical potential of the GC376 compound. The results presented here support the use of GC376 as an antiviral with broad specificity against coronaviruses. Purifications of proteases were performed as described earlier 14 Inhibitors GC373 and GC376, and the FRET substrate Abz-SVTLQSG-Y(NO 2 )-R were synthesized according to methods previously described 14 . The activity determination of both proteases was performed as previously described 14 using FRET-based cleavage assay with a synthesized fluorescent substrate containing the cleavage site The 13 C-labelled GC376 inhibitor was synthesized according to previously documented procedures, and initial HSQC NMR experiments involving only enzyme, only inhibitor, and both co-incubated were prepared as previously described 14 . The sample used for the reversibility experiment was prepared by subjecting a previously co-incubated sample containing both enzyme and inhibitor to washing steps with buffer (D 2 O, 50 mM phosphate, pD 7.5 with 20 mM DTT). This involved depositing the sample in an Amicon micro-spinfilter with a 10 kDa cutoff and spinning down the sample at 6600 g for 15 min. The sample was then diluted to 300 µL and the spin down and dilution steps were repeated once more, to a final volume of 300 µL. This sample was then analyzed by NMR in an HSQC experiment, following protocols identical to those previously described 14 . Reversibility of 3CL protease inhibition with GC376 was determined by dialysis method. The proteases (2 µM) were incubated with a single concentration (20 µM) of the GC376 compound for 15 min at RT to allow for full inhibition. Then the enzyme-inhibitor mixture was placed in a 6-8 kDa MWCO dialysis membrane (Fisher Scientific, Canada) and dialyzed against 2 L of 50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 5% glycerol, 1mM DTT at RT. The dialysis buffer was changed every 24 hours. Control experiments, which included dialyzing apo-proteases at the same concentration in the same dialysis buffer but different beakers, were performed simultaneously. The aliquots of dialyzing samples were taken out at certain time points and used for activity measurements. The data was represented as a percent of initial protease activity at a zero time point. The thermal stability was determined by heating 2 µM solution of M pro SARS CoV or M pro SARS-CoV-2 in 50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 5% glycerol, 1mM DTT buffer in a thermostatted water-bath at various temperatures. 30 µl protein samples were taken out at specific time points and immediately incubated on ice until activity measurements were performed as described above. Residual activities were expressed as relative to the maximal activity, which was the activity of proteases at zero time point. The enzyme inactivation over time is described by a first-order equation: where A represents enzyme activity at time t, A 0 is the initial activity at time zero, k is the rate constant (min −1 ), and t is time (min). Inactivation rate constants (kd) were obtained from slopes of semi-logarithmical plots of residual activity versus incubation time at each temperature. Calculated rate constants were replotted in Arrhenius plots as natural logarithms of k versus the reciprocal of absolute temperature. Arrhenius law describes the temperature dependence of rate constant as ln(k)=-Ea/RT+c (2) where Ea is the activation energy, R is the universal gas constant (8.31 J mol -1 K -1 ), and T is the absolute temperature. Ea was calculated from the slope of Arrhenius plot. The half-life of proteases (t 1/2 ), defined as time after which activity is reduced to 50% of initial value 47 , was determined as Another common way to present inactivation rate is as D value -decimal reduction time, which is the time required to reduce activity to 10% of the original value and calculated as: The activation free energy (ΔG, kJ mol -1 ) , enthalpy (ΔH o , kJ mol -1 ) and entropy (ΔSº, kJ mol -1 K - The coordinates and structural factors reported in this study have been deposited in the PDB database under accession code 7LCP (SARS-CoV-1 M pro with GC373) and 7LCQ (SARS-CoV-2 M pro with GC376). CRediT authorship contribution statement: Table 3 . Data collection and refinement statistics (molecular replacement) for SARS-CoV M pro with drug GC373 and prodrug GC376. Writing -review & editing. Conrad Fischer: Investigation; Methodology Investigation; Methodology; Writing -review & editing. Tess Lamer: Investigation; Methodology; Writing -review & editing. Wayne Vuong: Investigation; Methodology; Writing -review & editing Tyrrell: Conceptualization; Funding acquisition; Writingreview & editing. John C. Vederas: Conceptualization; Funding acquisition; Writing -review & editing. Howard S. Young: Conceptualization; Funding acquisition. M. Joanne Lemieux: Conceptualization; Funding acquisition Roles/Writing -original draft contributed to inhibitor synthesis. 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Muhammad Bashir Khan: Investigation Writing -review & editing. Conrad Fischer: Investigation; Methodology Investigation; Methodology; Writing -review & editing. Tess Lamer: Investigation; Methodology; Writing -review & editing. Wayne Vuong: Investigation; Methodology; Writing -review & editing Tyrrell: Conceptualization; Funding acquisition; Writingreview & editing. John C. Vederas: Conceptualization; Funding acquisition; Writing -review & editing. Howard S. Young: Conceptualization; Funding acquisition. M. Joanne Lemieux: Conceptualization; Funding acquisition Roles/Writing -original draft; declare the following financial interests/personal relationships which may be considered as potential competing interests The authors declare no competing interests.