key: cord-0756981-cy9pl8qi
authors: Bafna, Khushboo; White, Kris; Harish, Balasubramanian; Rosales, Romel; Ramelot, Theresa A.; Acton, Thomas B.; Moreno, Elena; Kehrer, Thomas; Miorin, Lisa; Royer, Catherine A.; García-Sastre, Adolfo; Krug, Robert M.; Montelione, Gaetano T.
title: Hepatitis C Virus Drugs Simeprevir and Grazoprevir Synergize with Remdesivir to Suppress SARS-CoV-2 Replication in Cell Culture
date: 2020-12-14
journal: bioRxiv
DOI: 10.1101/2020.12.13.422511
sha: 3870db775d082a5dede24017749de8a954aeee83
doc_id: 756981
cord_uid: cy9pl8qi

Effective control of COVID-19 requires antivirals directed against SARS-CoV-2 virus. Here we assess ten available HCV protease inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS- CoV-2 Mpro and HCV NS3/4A proteases, and virtual docking experiments show that all ten HCV drugs can potentially bind into the Mpro binding cleft. Seven of these HCV drugs inhibit SARS-CoV-2 Mpro protease activity, while four dock well into the PLpro substrate binding cleft and inhibit PLpro protease activity. These same seven HCV drugs inhibit SARS-CoV-2 virus replication in Vero and/or human cells, demonstrating that HCV drugs that inhibit Mpro, or both Mpro and PLpro, suppress virus replication. Two HCV drugs, simeprevir and grazoprevir synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, thereby increasing remdesivir inhibitory activity as much as 10-fold. Highlights Several HCV protease inhibitors are predicted to inhibit SARS-CoV-2 Mpro and PLpro. Seven HCV drugs inhibit Mpro enzyme activity, four HCV drugs inhibit PLpro. Seven HCV drugs inhibit SARS-CoV-2 replication in Vero and/or human cells. HCV drugs simeprevir and grazoprevir synergize with remdesivir to inhibit SARS- CoV-2. eTOC blurb Bafna, White and colleagues report that several available hepatitis C virus drugs inhibit the SARS-CoV-2 Mpro and/or PLpro proteases and SARS-CoV-2 replication in cell culture. Two drugs, simeprevir and grazoprevir, synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, increasing remdesivir antiviral activity as much as 10-fold.

The COVID-19 pandemic has caused more than one million deaths worldwide 9 and crippled the global economy. Effective control of the SARS-CoV-2 coronavirus that 10 causes COVID-19 requires antivirals, especially until safe and effective vaccines are 11 available. Considering the urgency to identify effective antiviral drugs, and the usually 12 lengthy process involved in approving candidate drugs for human use, our goal is to 13 identify existing drugs already approved for use in humans that can be repurposed as 14 safe and effective therapeutics for treating COVID-19 infections, and which may also be 15 useful as lead molecules for novel drug development. we reasoned that inhibitors of one or both of these proteases might be synergistic with 18 inhibitors of the viral polymerase, such as remdesivir. 19

We observed that the substrate binding cleft and active site of the SARS-CoV-2 20 M pro has remarkable structural similarity with the active site of the hepatitis C virus 21 (HCV) NS3/4A protease, suggesting that drugs that inhibit the HCV protease might also 22 inhibit SARS-CoV-2 M pro (Bafna et al., 2020). Consistent with this hypothesis, recent 23

replication. Simeprevir provides synergy with remdesivir at significantly lower 1 concentrations than grazoprevir. Our results suggest that the combination of a HCV 2 protease inhibitor with a RNA polymerase inhibitor could potentially function as an 3 antiviral against SARS-CoV-2. In particular, the combination of two FDA-approved 4 drugs, namely, simeprevir and remdesivir, could potentially function as a therapeutic for 5 COVID-19 while more specific SARS-CoV-2 antivirals are being developed. More 6 generally, our results strongly motivate further studies of the potential use of M pro and/or 7 PL pro protease inhibitors in combination with RNA polymerase inhibitors as antivirals 8 against SARS-CoV-2. Data Bank using the DALI program (Holm and Sander, 1993, 1999) , with domains I and 21 II (excluding domain III) of the SARS-CoV-2 M pro as the three-dimensional structural 22 query, identified several proteases, including the HCV NS3/4A serine protease, as 1 structurally similar. These two enzymes have a structural similarity Z score (Holm and 2 Sander, 1993, 1999) of +8.4, and overall backbone root-mean-squared deviation for 3 structurally similar regions of ~ 3.1 Å. The HCV serine protease also has a double β-4 barrel fold, with relative orientations of several secondary structure elements similar to 5 those of the SARS-CoV-2 M pro cysteine protease, and a substrate binding site located in 6 a shallow cleft between its two six-to eight-stranded antiparallel β-barrels ( Figure 1B) . 7

Superimposition of the analogous backbone structures of these two proteases results in 8 superimposition of their substrate binding clefts and their active-site catalytic residues, 9

His41 / Cys145 of the SARS-CoV-2 M pro cysteine protease and His57 / Ser139 of the 10 HCV NS3/4A serine protease ( Figure 1C ). Because of these structural similarities, we 11

proposed that some HCV protease inhibitors might bind well into the substrate binding In the 1.75-Å X-ray crystal structure of the 13b-M pro complex (Zhang et al., 2020b) , 1 approximately 50% of the 13b compound forms a reversible thiohemiketal covalent 2 bond with the S g atom of catalytic residue Cys145 of the protease (Zhang et al., 2020b) . 3

The contribution of this partial covalent bond formation is difficult to assess, and our 4 docking calculations determine only how well these drugs fit into the active site without 5 the additional stabilization energy that may result from any covalent bond formation. 6

The best-scoring docked conformation of 13b (Supplementary Figure S1C) , has an 7 AutoDock binding energy of -9.17 kcal/mol, and is similar but not identical to the pose 8 observed in the X-ray crystal structure. Because the experimentally-determined pose is 9

often not the one with the lowest docking energy, but rather is found among other 10 highly-ranked poses ), we also examined other 11 low energy poses. One such pose, which has a slightly higher binding energy of -9.03 12 kcal/mol, is almost identical to the pose observed in the crystal structure 13 (Supplementary Figure S1D) . Hence, binding poses very similar to that observed in the 14 crystal structure are indeed included among the low energy poses generated by our 15 AutoDock protocol. Having validated the use of AutoDock for docking of inhibitor 13b to M pro , and 20 determining a representative AutoDock score for functionally-inhibitory binding (~ -9.0 21 kcal / mol) we carried out docking simulations with 10 HCV NS3/4A protease inhibitor 22 drugs (Ozen et al., 2019). These 10 drugs have been approved for at least Phase 1 1 clinical trials, and some are FDA-approved prescription drugs used in treating HCV-2 infected patients (Table 1 ). The resulting docking scores are summarized in Figure 1D Table S1 ; and details of these docking poses are summarized in 4 Supplementary Table S2 . These results demonstrate that all of these HCV protease 5

inhibitors have the potential to bind snuggly into the binding cleft of M pro , with extensive 6 hydrogen-bonded and hydrophobic contacts, and predicted AutoDock energies of -8.37 7 to -11.01 kcal/mol, comparable to those obtained for the functional 13b inhibitor ( ~ -9.0 8 kcal/mol). 9

As we were preparing our work for publication, a 1.44 Å X-ray crystal structure of 10 the complex of boceprevir bound to the SARS-CoV-2 M pro was released in the Protein 11

Data Bank [PDB id 6WNP (Anson et al., 2020; Anson and Mesecar, 2020)]. The 12 boceprevir pose observed in this X-ray crystal structure is almost identical to the lowest 13 energy pose (-9.03 kcal/mol) predicted by AutoDock ( Figure 1E ). In addition, both the 14 docked and crystal structure binding poses for boceprevir near the active site of M pro are 15 very similar to its binding pose in the substrate binding cleft of HCV protease ( Figure  16 1F), with an essentially identical hydrogen-bonding network between boceprevir and 17 corresponding residues in each protease. Boceprevir is a alpha-ketoamide which 18 potentially forms a covalent bond with the active site residue Cys145. Although this 19 docking protocol does not include energetics or restraints for covalent bond formation, 20 in the low energy pose of boceprevir bound to M pro , the alpha-keto amide carbon is 21 positioned within 3.8 Å of the active site thiol sulfur atom. These blind tests of our 22

docking results for boceprevir support the predictive value of docking results for the 1 other HCV protease inhibitors. 2

Detailed images of the lowest-energy pose for seven of the ten complexes are 3 shown in Supplementary Figure S2 , and three representative complexes, for vaniprevir, 4 simeprevir, and narlaprevir are illustrated in Figure 1G -I. In addition, a summary of 5

AutoDock scores (Supplementary Table S1) , key intermolecular contacts in each of the 6 ten complexes (Supplementary Table S2 ), and descriptions of these binding poses, are 7 presented in Supplementary Information. In this analysis, we paid particular attention to 8 key details of the lowest energy pose for each inhibitor, including interactions with the 9 sidechains of catalytic dyad residues His41 and Cys145, and hydrogen-bonded 10 interactions with the backbone amides of Gly143, Ser144 and Cys145, which form the 11 oxyanion hole of this cysteine protease (Zhang et al., 2020b) . All these HCV protease 12 inhibitors form extensive hydrogen-bonded networks and hydrophobic interactions 13 within the substrate binding cleft, and would occlude access of polypeptide substrates to 14 the active-site residues His41 and Cys145 (as shown for example in Figure 1G -I). 15

While the three HCV protease inhibitors with alpha-keto amide functional groups, 16 namely, boceprevir, narlaprevir, and telaprevir, have lowest-energy or low-energy poses 17 that could form covalent bonds with the active-site Cys145 thiol, the seven other HCV 18 protease inhibitors are non-covalent inhibitors that would not form such thioester bonds. 19

From these virtual docking studies, we conclude that all ten of these HCV protease 20

inhibitors have the potential to bind snuggly into the substrate binding cleft of M pro , and 21

to inhibit binding of its substrates. 22

Inhibition activity of HCV drugs against M pro was initially assessed using a 2 protease assay based on Föster resonance energy transfer (FRET) using the peptide 3 substrate Dabsyl-KTSAVLQ/SGFRKME-(Edans), containing a canonical M pro protease 4 recognition site. Rates of hydrolysis were determined for 90 minutes, and initial 5 velocities were used to assess the relative inhibitory activities of the ten HCV protease 6

inhibitors. The percent M pro activity denotes the ratio of initial hydrolysis velocities in the 7 presence of 20 µM drug to the velocities in the presence of only the DMSO solvent. 8

These assays used 10 nM M pro and 20 µM peptide substrate, at a final DMSO 9 concentration of 1.0%. Under these conditions, HCV protease inhibitors narlaprevir, 10 boceprevir, and telaprevir have significant enzyme inhibition activity, with IC50 values of 11 2.2 ± 0.4 µM, 2.9 ± 0.6 µM, and 18.7 ± 6.4 µM, respectively (Figures 2A, B) . In contrast, 12 little or no inhibition activity was detected with the other seven HCV protease inhibitors 13 ( Figure 2A and Supplementary Figure S3A) . 14 While very sensitive, this FRET assay is complicated by the fact that, aside from 15 narlaprevir, boceprevir, and telaprevir, the other HCV drugs have significant intrinsic 16 fluorescence that interferes with FRET measurements using this peptide substrate 17 (compare fluorescence intensity at time t = 0 sec in Supplementary Figure S3A) . 18

Consequently, M pro inhibition by some of these drugs might be masked or interfered with 19 by fluorophore interactions. To overcome this, we also developed a 1D 1 H-NMR assay, 20 using the peptide substrate KTSAVLQ/SGFRKME that lacks the Dabsyl and Edans N-21

terminal and C-terminal tags, as outlined in Figure 2C and Supplementary Figure S4 . In 22 this 1D 1 H-NMR assay, M pro activity was defined as the ratio of hydrolyzed peptide 23 observed after 30 min in the presence of drug, to the amount of hydrolyzed peptide 1 observed when the enzyme is incubated with DMSO alone. These assays used 100 nM 2 M pro , 50 µM peptide substrate, and 50 µM drug (or DMSO solvent alone), at a final 3 DMSO concentration of 1.0%. Under these conditions, narlaprevir, boceprevir, and 4 telaprevir have substantial enzyme inhibition activity, as was the case in the FRET 5 assay. In addition, in the NMR assay substantial inhibitory activity was also observed for 6 vaniprevir, and moderate inhibitory activity was observed for grazoprevir, simeprevir, 7 and asunaprevir ( Figure 2D ). This moderate inhibition was not detected in the FRET 8 assay. Danoprevir, paritaprevir, and glecaprevir had little or no detectable M pro inhibitory 9

activity. From these studies we conclude that seven HCV drugs (viz boceprevir, 10 narlaprevir, telaprevir, vaniprevir, grazoprevir, simeprevir, and asunaprevir) inhibit 11 SARS-CoV-2 M pro strongly or moderately under the conditions tested. 12 13 Three HCV protease inhibitors inhibit not only M pro but also PL pro 14

Although the active site of PL pro does not share structural similarity with the HCV 15 protease, it is possible that some HCV protease inhibitors can also bind into the active 16 site of PL pro . Accordingly, we carried out virtual docking studies of these same ten HCV 17 drugs into the substrate binding cleft of PL pro , using protocols similar to those developed 18 in virtual docking studies with M pro . The PL pro inhibitor GRL-0617, for which a crystal 19 structure bound to PL pro is available in the PDB (Fu and Huang, 2020), was used to 20 assess the docking protocol and to determine a reference AutoDock score of -7.54 kcal 21 / mol. The scores of docking poses for HCV drugs, summarized in Figure 3A and 22 Supplementary Table S1, range from -5.56 kcal / mole for boceprevir and narlaprevir,  1 to much more favorable values of < -8 kcal / mol for others including vaniprevir, 2 grazoprevir, simeprevir, and paritaprevir. The most favorable docking poses for 3 simeprevir and vaniprevir are shown in Figures 3B -C , and for the remaining complexes 4 in Supplementary Figure S5 . These results indicate that, surprisingly, some HCV 5 protease inhibitors may bind in the active sites of both M pro and PL pro . 6

Based on these docking results, we anticipated that several HCV protease 7 inhibitors, not including bocepriver or narlaprevir, might inhibit PL pro protease activity. To 8 test this hypothesis, fluorescence assays of PL pro inhibition were carried out, using the 9 substrate ZRLRGG/AMC (Z -carboxybenzyl; AMC -7-Amino-4-methylcoumarin) 10 

The motivation for the docking and biophysical studies described above was to 21 identify HCV drugs with the potential to inhibit SARS-CoV-2 virus replication. For 22 antiviral assays, Vero E6 cells, or human 293T cells expressing the SARS-CoV-2 ACE2 1 receptor, were grown in 96-well plates, and were incubated with various levels of a HCV 2 protease inhibitor for 2 hours. Cells were then infected with SARS-CoV-2 virus at the 3 indicated multiplicity of infection [moi, plaque-forming units (pfu)/cell] and incubated for 4 the indicated times at 37 0 C in the presence of the inhibitor. Virus-infected cells were 5 then identified by immunofluorescence using an antibody specific for the viral 6 nucleoprotein. Inhibition of viral replication was quantified by determining the 7 percentage of positive infected cells at the end of the incubation period in the presence 8 of the compound, as compared with the number of infected cells in its absence. To 9 determine whether a HCV drug was cytotoxic, uninfected Vero E6 or human 293T cells 10 were incubated with the same levels of the compounds for the same length of time, and 11 cytotoxicity was measured using an MTT assay (Roche). In all of these replication 12 assays, remdesivir was used as a positive control. Further details of these replication 13 assays are provided in STAR Methods. 14 Viral replication inhibition data in Vero E6 cells are presented in Figure 4 . Three 15

of the HCV protease inhibitors tested, grazoprevir, asunaprevir, and boceprevir, 16 inhibited SARS-CoV-2 virus replication at concentrations lower than the concentrations 17 that cause significant cytotoxicity. Two other HCV protease inhibitors, simeprevir and 18 vaniprevir, inhibited virus replication with even lower IC50 values, but some cytotoxicity 19 was also observed. These five HCV drugs have IC50 values for inhibiting SARS-CoV-2 20 replication of 4.2 to 19.6 µM. The remaining three HCV drugs tested, telaprevir, 21 glecaprevir, and danoprevir, did not inhibit virus replication in Vero E6 cells, even at a 22 maximum drug concentrations of 50 µM. 23

We next determined whether the inhibition of SARS-CoV-2 replication by a 1 representative HCV drug occurs at steps after virus entry, as would be expected for 2 inhibitors of a viral protease that is produced only after infection. Accordingly, we 3 performed a time of addition assay using grazoprevir as the inhibitor. In a single cycle 4 We also determined whether the HCV drugs exhibited similar antiviral activities in 12 human cells, specifically human 293T cells expressing the ACE2 receptor ( Figure 5 ). 13

We included in these antiviral assays another HCV protease inhibitor, narlaprevir, which 14 we observed to be a strong M pro inhibitor ( Figure 2B ). Again, simeprevir and vaniprevir 15

were the most effective inhibitors of virus replication, with IC50 values of 2.3 and 3.0 µM, 16 respectively, and with considerably reduced cytotoxicity as compared to that in Vero 17 cells. Boceprevir and narleprevir, which are strong M pro inhibitors (Figure 2A Grazoprevir had considerably lower antiviral activity in human 293T cells than in Vero 21 cells (compare Figures 4D and 5F ). Thus, six HCV drugs inhibited SARS-CoV-2 22 replication in human 293T cells, with IC50 values ranging from 2.3 to 20.5 µM. 23

Glecaprevir and danoprevir did not inhibit virus replication in 293T cells, as was also the 1 case in Vero cells. 2 3 Simeprevir, and grazoprevir synergize with remdesivir to increase inhibition of 4

Because M pro and PL pro generate either the RNA polymerase itself, or the 6 proteins that constitute the replication organelles required for polymerase function, we 7

predicted that HCV drugs that inhibit one or both of these viral proteases might be 8 synergistic with inhibitors of the viral polymerase like remdesivir. To test this hypothesis, 9

we carried out antiviral combination assays in Vero cells of simeprevir, grazoprevir, or 10 boceprevir with remdesivir ( Figure 6 ). To assess synergy, two analyses are required. In 11 one analysis the IC90 of the remdesivir was measured in the presence of increasing 12 concentrations of each of these HCV drugs ( Figure 6A -C). These results demonstrate 13 that simeprevir or grazoprevir increase the antiviral activity of remdesivir. For example, 14

in the presence of 1.25 µM simeprevir, approximately 10-fold less remdesivir is required 15 for the same antiviral effect achieved in the absence of simeprevir ( Figure 6A ). 16

Surprisingly, although boceprevir is a much better inhibitor of M pro than either simeprevir 17 or grazoprevir, boceprevir did not significantly affect the antiviral activity of remdesivir 18 ( Figure 6C ). In the second analysis, the IC90 concentration of each HCV drug was 19 determined in the presence of increasing concentrations of remdesivir ( Figures 6D-F) . 20

Remdesivir increased the antiviral activity of simeprevir and grazoprevir. For example 21 addition of 1.25 µM remdesivir substantially reduces the concentration of simeprevir 22 needed to achieve IC90 conditions ( Figure 6D ). In contrast, remdesivir did not 1 significantly affect the antiviral activity of boceprevir ( Figure 6F ). 2

These combination antiviral assays indicate that simeprevir and grazoprevir, but 3 not boceprevir, act synergistically with remdesivir to inhibit virus replication. As 4 confirmation, we subjected these results to analysis by the Zero Interaction Potency 5 (ZIP) model for synergy (Ianevski et al., 2020) . In the landscapes generated by this 6 model that are shown in Figure 6G -I, red denotes a synergistic interaction, and green 7 denotes an antagonistic interaction. In this model a synergistic interaction between 8 drugs has a score greater than +10; an additive interaction has a score between -10 to 9 +10; and an antagonistic interaction has a score of less than -10. The landscapes for 10 the interaction of remdesivir with both simeprevir and grazoprevir are red, with synergy 11 scores of +30.2 and +25.0, respectively, denoting moderate synergism. In contrast, 12 although the landscape for the interaction of remdesivir with boceprevir suggests weak 13 antagonism ( Figure 6I ), the score of the interaction is -7.6, which we interpret as an 14 additive interaction. 15

We also carried out combination antiviral assays in human 293T cells. The 16

interaction between remdesivir and grazoprevir in inhibiting virus replication in the 17 human cells was also synergistic, with a red landscape and a synergy score of +20.3 18 Figure S6A) . The interaction between remdesivir and vaniprevir in 19

inhibiting virus replication was also analyzed in 293T cells; it was weakly synergistic, 20 with a score of +10.9. Vaniprevir has an additive effect or, at best, very weak synergy 21 with remdesivir in human 293T cells. Consequently, two HCV drugs, simeprevir and 22 grazoprevir, act synergistically with remdesivir to inhibit SARS-CoV-2 virus replication in 1

Vero and/or human 293T cells. To provide antiviral drugs that can be rapidly deployed to combat the COVID-19 5 pandemic, we carried out the present study to identify currently available drugs that 6 could potentially be repurposed as inhibitors of the pandemic SARS-CoV-2 virus that 7 causes COVID-19 disease. Instead of screening libraries of current drugs, we took a 8 different approach to identify candidate drugs. Specifically, we initiated our search 9 based on the striking similarity of the substrate binding clefts of the SARS-CoV-2 M pro 10 and HCV proteases. The HCV protease is a serine-protease, with catalytic triad His57, 11 As predicted by the striking similarity of the substrate binding clefts of the SARS-4

CoV-2 M pro and HCV proteases, our virtual docking experiments showed that ten HCV 5 protease inhibitors can be docked snuggly into the substrate binding cleft of M pro , and 6 hence would have the potential to inhibit binding of the M pro substrate, thereby inhibiting 7 proteolytic cleavage of the substrate. In fact, we showed that four HCV drugs, 8 boceprevir, narlaprevir, telaprevir, and vaniprevir strongly inhibit SARS-CoV-2 M pro 9

protease activity, and that three other HCV drugs, grazoprevir, simeprevir and 10 asunaprevir moderately inhibit M pro activity. Boceprevir, narleprevir and telaprevir are a- reported here as inhibitors of M pro and/or PL pro . These inconsistencies likely arise from 6 details of the different assays that have been used. For example, our 1 H-NMR assay 7 could identify moderate inhibition of M pro by simeprevir, grazoprevir, and asunaprevir 8 HCV drugs that was not detected by our FRET assay. This is not surprising, since both 9 the FRET and NMR assays are competition assays, which will give different results for 10 substrates that have different binding affinities. The ability of HCV drugs to inhibit M pro 11 may also depend on other details of the assay conditions, most notably the enzyme, likely be reduced. Viral replication assays using combinations of drugs allowed us to 10 assess if the interactions between HCV drugs and remdesivir are additive or synergistic, 11

i.e. resulting in an inhibition of virus replication that is greater than the sum of the 12 inhibitions caused by the HCV drug and remdesivir. Surprisingly, we found that these 13 inhibitory effects are additive or synergistic depending on which HCV drug is used to 14 inhibit virus replication. Boceprevir, which strongly inhibits only M pro , and vaniprevir, 15 which strongly inhibits both M pro and PL pro , act additively with remdesivir to inhibit virus 16 replication. In contrast, simeprevir and grazoprevir, which moderately inhibit M pro and 17 either strongly or moderately inhibit PL pro , act synergistically with remdesivir to inhibit 18 virus replication. The basis for the different interactions between remdesivir and 19 particular HCV drugs, and the mechanism(s) of synergy, needs to be further explored. 20

The HCV drugs that are strongly synergistic with remdesivir are most pertinent 21

for the goal of the present study to identify available drugs that can be repurposed as 22 SARS-CoV-2 antivirals and/or as lead molecules for new drug development. 23

Repurposed drugs like the HCV drugs in the present study may not have sufficient 1 inhibitory activity on their own to achieve clinical efficacy. Synergy with remdesivir 2 increases the potency of both the repurposed HCV drug and remdesivir. We identified 3 two HCV drugs, simeprevir and grazoprevir, that act synergistically with remdesivir to 4 inhibit SARS-CoV-2 virus replication. Of these two, simeprevir may be the better choice 5 as a repurposed drug because it effectively inhibits SARS-CoV-2 virus replication in 6 human cells at much lower concentrations than grazoprevir. Consequently, the 7 combination of simeprevir and remdesivir could potentially function as an antiviral 8 against SARS-CoV-2 while more specific SARS-CoV-2 antivirals are being developed. 9

Simeprevir, which is an oral drug, might also be combined with an oral polymerase 10 inhibitor rather than with remdesivir, which has to be administered intravenously. One surfaces an antagonistic interaction. In this model a synergistic interaction between drugs has a 9 score greater than +10; an additive interaction has a score between -10 to +10; and an 10 antagonistic interaction has a score of less than -10 (Ianevski et al., 2020). 

BioMagResDB accession number: 50568 Title: " 1 H and 15 N assignments for 14-residues 2 peptide that is cleaved by SARS-CoV-2 M pro ." 3 4 BioMagResDB accession number: 50569. Title: " 1 H and 15 N assignments for 14-residue 5 peptide after cleavage by SARS-CoV-2 M pro " 6 7 Resource Availability 8

Further information and requests for resources and reagents should be directed to and 10 will be fulfilled by the designated contacts Gaetano T. Montelione (monteg3@rpi.edu). 11 12

All materials generated in this study are available upon request. The computational docking program AutoDock v4.2.6. is based on an empirical free-1 energy force field and uses a search method based on Lamarckian genetic algorithm 2 (Morris et al., 1998) . Target protein coordinates were obtained from SARS-CoV-2 M pro 3 X-ray crystal structure (PDB id 6Y2G) (Zhang et al., 2020) , and structural water was 4 removed. Three-dimensional coordinates for ligand molecules were obtained from PDB 5 (http://www.rcsb.org/) or from chemical structure databases, ChemSpider 6 (http://www.chemspider.com/) and DrugBank (https://www.drugbank.ca/). Protein and 7 ligand coordinates were then prepared using AutoDockTools; polar hydrogens were 8 added to protein structures, and Gasteiger-Marsili empirical atomic partial charges were 9 added to ligands. Torsional degrees of freedom (dihedral angles) were identified for 10 each ligand. These data and parameters for each protein and ligand were saved as 11 individual PDBQT files. In these studies, ligand dihedral angles were allowed to vary 12 The docking protocol was same as above, except a larger grid of size 56, 56, and 58 16 points in the x, y and z direction respectively was used to compute electrostatic maps 17 for PL pro target. For a comparative analysis, docking simulations of PL pro inhibitor 18 GRL0617 (PDB id 7CJM) were also performed using the same protocol. 19 20 M pro expression and purification 21

The full length SARS-CoV-2 M pro gene was ordered from GenScript USA Inc. in pGEX-22

6P-1 vector, as previously described (Zhang et al., 2020) . This expression vector is 23 designated GTM_COV2_NSP5_001. This plasmid, expressing SARS-CoV-2 M pro as a 1 self-cleaving (using its native cleavage site) GST-fusion were transformed into 2 competent E. coli BL21(DE3) cells. A single colony was picked and inoculated in 2 mL 3 LB supplemented with 0.1 mg/ml Ampicillin at 37 ºC and 225 rpm. The 2 mL inoculum 4 was added to 1L LB broth with 0.1 mg/mL Ampicilin. The cells were allowed to grow to 5 an optical density of 0.6 at 600 nm at 37 ºC and 225 rpm, and induced with 1 mM IPTG. The proteolysis of substrate KTSAVLQ/SGFRKME was studied using Fluorescence 21

Resonance Energy Transfer (FRET) and Nuclear magnetic Resonance (NMR) assays. 22

For the FRET assay the substrate was labeled with Dabcyl and Glu-Edans FRET pair 23 on the N and C-termini of the peptide, respectively, as described elsewhere (Ma et al., 1 2020 ). Both, labeled and unlabeled substrates were ordered from GenScript USA Inc. 2 For all assays, the specific activity of the enzyme was checked at the beginning and end 3 of the data collection session, or back-to-back with each measurement, in order to avoid 4 spurious results due to enzyme inactivation during a measurement session. For the 1 H NMR proteolysis assay the reaction was performed at 100 nM M pro in 20 the same assay buffer described above, along with 5% D2O and 50 µM HCV inhibitors 21 dissolved in d6-DMSO (for the control experiments where no inhibitor is added, the 22 same quantity of d6-DMSO was added). 50 µM of unlabeled substrate was added and 23 immediately transferred to a 5-mm NMR tube. The final volume of each reaction mixture 1 was 600 µL. The NMR tube was quickly placed in a 600 MHz Bruker Avance II 2 spectrometer equipped with a 5-mm TCI cryoprobe, equilibrated at 298K. The 3 homogeneity of the magnetic field was adjusted by gradient shimming on the z-axis and 4 in each case an array of 24 1 H experiments was acquired with 1D 1 H NMR using 5 excitation sculpting for water suppression. The probe had previously been tuned and 6 matched with a sample of similar composition. The delay between initiation of the 7 reaction and starting acquisition was ~ 5 mins for most of the reaction conditions. The 8 duration of each NMR experiment was also taken into account to obtain accurate time 9

values. All 1 H spectra were acquired, processed, and analyzed in Bruker TopSpin 3.6.2 10 software. The regions of interest were integrated, and the values obtained were 11 transferred for further analysis and plotting. 12 13 These 1 H NMR spectra were used to monitor the evolution of substrate and 14 product as a function of time. Resonance assignments (discussed below) of the cleaved 15 and uncleaved KTSAVLQ/SGFRKME peptide identified the amide H N resonances that 16 were monitored during the reaction. The H N resonances for amino-acid residues Phe-10 17 (uncleaved) and Gln-7 (cleaved) were used to quantify substrate utilization and product 18 formation, respectively, during the reaction. The H N preak intensity of residue Glu-14, 19 which did not shift upon cleavage, was monitored as an internal control. 20

The percent substrate cleavage in the presence of inhibitors at 30 min was calculated 21 as a ratio of the H N resonance integrals of Gln-7 in presence of inhibitor to the 22 corresponding resonance integral with no inhibitor. Background] *100 and the DMSO control was then set to 100% infection for analysis. 22

The combination antiviral assay was performed in biologically independent triplicates. The apparent IC90 for each combination in the matrix was determined using the Prism 2 (GraphPad Software) software. The IC90 for HCV drugs and remdesivir were calculated 3 for each drug treatment alone and in combination. This combination data was analyzed 4

using SynergyFinder by the ZIP method (Ianevski et al., 2020) , and combination indices 5 were calculated as previously described (Amanat et al., 2020) . Catalytic residues of HCV NS3/4A (His57, Asp81 and Ser139) and SARS-CoV-2 M pro (His41

Validation of AutoDock protocols with inhibitor 13b. 3

In the 1.75-Å X-ray crystal structure of the 13b-M pro complex (Zhang et al., 2020), 4

13b makes hydrogen-bonded interactions with backbone amides of key residues, 5

Gly143, Ser144 and Cys145, in the canonical oxyanion hole of the active site. To 6 estimate the docking score for the pose observed in the X-ray crystal structure (Zhang 7 et al., 2020), we first carried out docking using a modified protocol in which the dihedral 8

angles of 13b were fixed to the values observed in the crystal structure. The lowest 9 energy pose obtained with this protocol matches the crystal structure almost exactly 10 (Supplementary Figure S1B) , with AutoDock binding energy of -7.19 kcal/mol. Next, we 11 assessed the docking protocol using flexible ligand dihedral angles, as was used for all 12 the other HCV protease inhibitors. The best-scoring docked conformation 13

(Supplementary Figure S1C) , has an AutoDock binding energy of -9.17 kcal/mol. 14 Because the experimentally-determined pose is often not the one with the lowest 15 docking energy, but rather is found among other highly-ranked poses 16 Kolb and Irwin, 2009), we also examined other low energy poses. One such pose, 17 which has a slightly higher binding energy of -9.03 kcal/mol, is almost identical to the 18 pose observed in the crystal structure (Supplementary Figure S1D) . Hence, binding 19

poses very similar to that observed in the crystal structure are indeed included among 20 the low energy poses generated by our AutoDock protocol. 21

Detailed descriptions of AutoDock lowest-energy binding poses observed for ten 23 HCV protease inhibitors bound to M pro . 24 25 26

Asunaprevir (ligand id ASU) 27

Molecular docking studies show that asunaprevir binds to the substrate binding cleft of 28 M pro with a predicted binding energy of -8.37 Kcal/mol ( Figure 1D and Table S1 ). The 29

key M pro atoms involved in hydrogen bonding with ASU are the side chain N ε atom of 30 catalytic His41, backbone N atom of residues Asn142, Glu166 and Arg188 ( Figure S2 ). 31

In addition, residues Thr25, Met165, Glu166 and Gln189 contribute towards binding 32 energy through hydrophobic interactions. In this binding conformation ASU does not 33

interact with the residues forming the oxyanion hole and catalytic residues Cys145 that 34 resides deep in the binding pocket. ASU rather binds in a conformation that can occlude 35 the access of catalytic Cys145 by the polyprotein substrate of the protease.

Boceprevir (ligand id BOC) 38

Boceprevir is an alpha-ketoamide inhibitor like 13b and is therefore capable of 39 covalently inhibiting the protease. In our docking studies BOC binds in the substrate 40 binding cleft of M pro with a predicted binding energy of -9.13 kcal/mol ( Figure 1D and 41 Table S1 ). The lowest scoring docking pose of BOC forms hydrogen bonds with side 42 chain N ε of catalytic His41 and backbone atoms of Gly143, Ser144 and Glu166 ( Figure  43 S2). Hydrophobic interactions with residues His41, Phe140, Met165, Glu166, Leu167, 44

Pro186 and Asp187 further stabilized the ligand in the binding cavity. The α-ketoamide 45 group is oriented towards residue Cys145 of the protease, with a distance between the 46 S atom of Cys145 and C of BOC of 3.8 Å. The lowest scoring pose for danoprevir in the substrate binding cleft of M pro has a 2 predicted binding energy of -9.99 kcal/mol ( Figure 1D and Table S1 ). The catalytic 3 residues His41 and Cys145 do not directly interact with DAN with either hydrogen 4 bonds or hydrophobic interactions. Apparently because of its large size and macrocyclic 5 nature, DAN cannot access these deeper residing catalytic residues. DAN occupies the 6 oxyanion hole of the protease, forming hydrogen bonds with Asn142 sidechain O δ and 7

Gly143 backbone amide N atom ( Figure S2 ). In addition, backbone amide N atoms of 8

Thr26 and Glu 166, and sidechain N ε of His163 also form hydrogen bonds with DAN. 9

The pose is also stabilized by hydrophobic interactions involving residues Thr25, Leu27, 10

Asn142 and Glu166. 11 12

Glecaprevir (ligand id GLE) 13

The predict binding energy of glecaprevir docked to the substrate binding cleft of M pro is 14 -9.51 kcal/mol ( Figure 1D and Table S1 ). The catalytic residue Cys145, which is deeper 15

in the binding pocket of M pro , is not accessible to GLE as observed for other macrocyclic 16

HCV inhibitors ( Figure S2 ). GLE forms hydrogen bonds with the sidechain N ε atom of 17 catalytic His41 along with backbone N atom of residues Asn142, Gly143 and Glu166.

Residues that form hydrophobically contribute towards GLE binding are Thr25, Leu141, 19

Met165, Glu166. 20 21

Grazoprevir (ligand id GRZ) 22

The lowest scoring pose of grazoprevir in the substrate binding cleft of M pro has a 23 predicted binding energy of -9.71 kcal/mol ( Figure 1D and Table S1 ). GRZ is a 24 macrocyclic compound and therefore occupies more extensive surface of the binding 25

pocket when compared to a-ketoamide inhibitor 13b and boceprevir. The catalytic 26

residue His41 does not directly interact with GRZ with either hydrogen bonds or 27 hydrophobic interactions. GRZ forms hydrogen bonds with backbone amide N atoms of 28

Gly143, Ser144, catalytic Cys145 occupying the oxyanion hole of the protease. It is also 29 observed to forms hydrogen bond with the residues Glu166 ( Figure S2 ). This binding 30 mode of GRZ also includes extensive hydrophobic interactions with the protease, 31

involving residues Thr25, Leu27, Met165, Pro168 and Gln189 that contribute to the 32 stabilization of the GRZ-M pro complex. 33 34

Narlaprevir (ligand id NAR) 35

The best scoring pose of narlaprevir has a predicted binding energy of 36 -9.80 Kcal/mol ( Figure 1D and Table S1 ). The NAR-protease interaction ( Figure S2) is 37 stabilized by a number of hydrogen bonds formed with N ε atom of His41, side chain O 38

and backbone N atoms of Asn142, backbone N atoms of Gly143, Ser144, Glu166, and 39

catalytic Cys145. Backbone carbonyl atom of Glu166 also forms two hydrogen bonds 40

with N atoms of NAR. Residues contributing to hydrophobic interactions are Thr25, 41

Leu27, Met165, Leu167, Gln192. Although the α-ketoamide group of NAR is oriented 42 towards residue Cys145 of the protease, the distance between the S atom of Cys145 43

and C of NAR is 4.4 Å, which is larger than the distance in the 13b-M pro crystal 44 structure.

Paritaprevir (ligand id PAR) 47

Paritaprevir is a macrocyclic compound, and the predicted binding energy of the lowest 48 scoring pose for PAR is -10.71 kcal/mol ( Figure 1D and Table S1 ). In this pose PAR 49 occupies the oxyanion hole of the protease and forms hydrogen bonds with backbone N 1 atoms of GLy143, Ser 144, Cys145 ( Figure S2 ). PAR can for hydrogen bonds with 2 sidechain O δ atom of Asn142, N δ of His 163 and carbonyl O atom of Glu166 as well. 3

This pose also appears to be stabilized by hydrophobic interactions involving residues 4

Leu27, Gln189 and Gln192. 5 6

Simeprevir (ligand id SIM) 7

The predicted binding energy of the best scoring complex of simeprevir is -10.75 8 kcal/mol ( Figure 1D and Table S1 ). In this pose, SIM sits in the binding cavity differently 9 than inhibitor 13b ( Figure S2 ). The key atoms involved in hydrogen bonding with SIM 10 are Thr25 sidechain hydroxyl OH, His45 backbone carbonyl O, Cys44 backbone N, and 11

Glu166 backbone N. In addition, sidechains of residues Thr24, Met165, Glu166, and 12

Gln189 form hydrophobic interactions with SIM. SIM does not occupy the binding site 13

near the catalytic residue Cys145; rather, SIM would occlude the access of Cys145 to 14 the polyprotein substrate of the protease. 15 16

Telaprevir (ligand id TEL) 17

The α-ketoamide group in the best ranking pose of telaprevir is not oriented towards 18

protease residue Cys145, whereas the second-ranking pose (binding energy -8.61) is 19

oriented towards Cys145 ( Figure S2 ). In the latter pose the distance between the S 20 atom of Cys145 and C of TEL is 2.9 Å, which is similar to the distance in the 13b-21 protease complex. This pose is stabilized hydrogen bonds of TEL with side chain of N ε 22

His41 and backbone N atoms of Gly143, Ser144, Cys145 and Glu166. Thr25 and 23

Gln189 residues of the protease further stabilize the binding of TEL through 24 hydrophobic interactions.

Vaniprevir (ligand id VAN) 27

Docking with vaniprevir provided a lowest scoring pose with predicted binding energy of 28 -10.95 kcal/mol ( Figure 1D and Table S1 ). In this pose ( Figure S2 ), VAN occupies a 29 much a larger portion of the binding cavity than inhibitor 13b. This could be attributed to 30 the large size and macrocyclic nature of VAN 

Microneutralization Assay for SARS-CoV-2 Serology and Drug Screening

The PyMOL Molecular Graphics System, Version 1.2r3pre

SynergyFinder 2.0: visual analytics of 17 multi-drug combination synergies

Boceprevir, GC-376, and calpain inhibitors II, 22 XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease

Automated docking using a Lamarckian genetic algorithm and 27 an empirical binding free energy function

AutoDock4 and AutoDockTools4: Automated docking with 33 selective receptor flexibility

PLIP: 37 fully automated protein-ligand interaction profiler

Supplementary References

Docking and 2 chemoinformatic screens for new ligands and targets

Docking screens: right for the right reasons

SynergyFinder 2.0: visual analytics of 9 multi-drug combination synergies

Crystal structure of SARS-CoV-2 main protease 14 provides a basis for design of improved alpha-ketoamide inhibitors