Hepatitis C virus drugs that inhibit SARS-CoV-2 papain-like protease synergize with remdesivir to suppress viral replication in cell culture
- https://www.cell.com/cell-reports/fulltext/S2211-1247(21)00472-1Los fármacos contra el virus de la hepatitis C que inhiben la proteasa similar a la papaína del SARS-CoV-2 actúan en sinergia con el remdesivir para suprimir la replicación viral en cultivo celularEl control eficaz de COVID-19 requiere antivirales dirigidos contra el SARS-CoV-2. Hemos evaluado 10 fármacos inhibidores de la proteasa del virus de la hepatitis C (VHC) como posibles antivirales del SARS-CoV-2. Existe una sorprendente similitud estructural entre las hendiduras de unión al sustrato de la proteasa principal del SARS-CoV-2 (Mpro) y la proteasa NS3/4A del VHC. Los experimentos de acoplamiento virtual muestran que estos fármacos contra el VHC pueden unirse potencialmente a la hendidura de unión al sustrato de Mpro. Demostramos que siete fármacos contra el VHC inhiben tanto la actividad de la proteasa Mpro del SARS-CoV-2 como la replicación del virus del SARS-CoV-2 en células Vero y/o humanas. Sin embargo, sus actividades inhibidoras de la Mpro no se correlacionan con sus actividades antivirales. Este enigma se resuelve demostrando que cuatro fármacos inhibidores de la proteasa del VHC, simeprevir, vaniprevir, paritaprevir y grazoprevir, inhiben la proteasa similar a la papaína (PLpro) del VRS-CoV-2. Los fármacos contra el VHC que inhiben la PLpro actúan en sinergia con el inhibidor de la polimerasa viral remdesivir para inhibir la replicación del virus, aumentando la actividad antiviral de remdesivir hasta 10 veces, mientras que los que sólo inhiben la Mpro no actúan en sinergia con remdesivir.
Summary
Effective control of COVID-19 requires antivirals directed against SARS-CoV-2. We assessed 10 hepatitis C virus (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 main protease (Mpro) and HCV NS3/4A protease. Virtual docking experiments show that these HCV drugs can potentially bind into the Mpro substrate-binding cleft. We show that seven HCV drugs inhibit both SARS-CoV-2 Mpro protease activity and SARS-CoV-2 virus replication in Vero and/or human cells. However, their Mpro inhibiting activities did not correlate with their antiviral activities. This conundrum is resolved by demonstrating that four HCV protease inhibitor drugs, simeprevir, vaniprevir, paritaprevir, and grazoprevir inhibit the SARS CoV-2 papain-like protease (PLpro). HCV drugs that inhibit PLpro synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, increasing remdesivir’s antiviral activity as much as 10-fold, while those that only inhibit Mpro do not synergize with remdesivir.Graphical abstract
Keywords
Introduction
Effective control of the SARS-CoV-2 coronavirus that causes COVID-19 requires antivirals. Considering the urgency to identify effective antiviral drugs, and the usually lengthy process involved in approving candidate drugs for human use, our goal is to identify existing drugs already approved for use in humans that can be repurposed as safe and effective therapeutics for treating COVID-19 infections, and which may also be useful as lead molecules for novel drug development.SARS-CoV-2 is an enveloped RNA virus, which causes COVID-19 (). Its genome comprises a single, large positive-sense single-stranded RNA, which is directly translated by host cell ribosomes. The SARS-CoV-2 genome encodes 4 structural proteins, 16 non-structural proteins (NSPs), which carry out crucial intracellular functions, and 9 accessory proteins (; ). Many of these proteins, and their host binding partners, are potential targets for development of antivirals for SARS-CoV-2. For example, the repurposed drug remdesivir, which inhibits the viral RNA-dependent RNA polymerase, is the current FDA-approved antiviral standard of care for COVID-19 (; ).Translation of the viral genomic RNA results in the biosynthesis of two polyproteins that are processed into the 16 separate NSPs by two virus-encoded cysteine proteases, the papain-like protease (PLpro) and a 3C-like protease (3CLpro). The latter is also referred to as the main protease (Mpro). Mpro and PLpro are essential for the virus life cycle and hence are attractive targets for antiviral development. These two viral proteases are required for the production of functional viral RNA polymerases. Mpro cleavages are predicted to generate several NSPs, including the three subunits nsp7, nsp8, and nsp12 that constitute the viral RNA polymerase complex (), as well as integral membrane proteins nsp4 and nsp6. PLpro cleavages generate other NSPs, including nsp3 (). The nsp3-nsp4-nsp6 complex is a key component of the replication organelles, also known as double-membrane vesicles (DMVs), that are required for the function of the viral polymerase in infected cells (; ; ; ; , ). Considering that both Mpro and PLpro generate either the RNA polymerase itself or the replication organelles required for polymerase function, we reasoned that inhibitors of one or both of these proteases might be synergistic with inhibitors of the viral polymerase, such as remdesivir.We observed that the substrate binding cleft and active site of the SARS-CoV-2 Mpro have remarkable structural similarity with the active site of the hepatitis C virus (HCV) NS3/4A protease, suggesting that drugs that inhibit the HCV protease might also inhibit SARS-CoV-2 Mpro (). Consistent with this hypothesis, subsequent studies have reported that three of these HCV drugs, boceprevir, narlaprevir, and telaprevir, inhibit Mpro proteolytic activity and bind into its active site (; ; ; ). Boceprevir has also been reported to inhibit SARS-CoV-2 replication in Vero cells (; ; ). Other HCV protease inhibitors have also been reported to inhibit Mpro proteolytic activity (; ; ; ) and/or viral replication () to various extents, while other studies report that some of these same HCV protease inhibitors did not significantly inhibit Mpro ().In this study, we assess the ability of 10 available HCV protease inhibitors to suppress SARS-CoV-2 replication. Virtual docking experiments predict that all 10 of these HCV drugs can bind snuggly into the Mpro binding cleft with docking scores comparable to a known Mpro inhibitor, suggesting that any of these 10 HCV drugs are potential inhibitors of Mpro. Seven of these HCV drugs inhibit both SARS-CoV-2 Mpro protease activity, and SARS-CoV-2 virus replication in Vero and/or human 293T cells expressing the SARS-CoV-2 ACE2 receptor. Surprisingly, we found that four HCV drugs also inhibit PLpro protease activity (including one that did not inhibit Mpro). Consequently, HCV drugs that inhibit Mpro and/or PLpro can suppress SARS-CoV-2 virus replication, viz, boceprevir (BOC), narlaprevir (NAR), vaniprevir (VAN), telaprevir (TEL), paritaprevir (PAR), simeprevir (SIM), grazoprevir (GRZ), and asunaprevir (ASU).Further, we demonstrate that the four HCV drugs that inhibit the proteolytic activity of PLpro, SIM, GRZ, PAR, and VAN, also act synergistically with remdesivir to inhibit SARS-CoV-2 virus replication, thereby increasing remdesivir antiviral activity as much as 10-fold. In addition, the PLpro -specific inhibitor, GRL0617, also synergizes with remdesivir. In contrast, the HCV drugs BOC and NAR, which inhibit Mpro but not PLpro, as well as the Mpro-specific inhibitor GC-376, act additively rather than synergistically with remdesivir to inhibit virus replication. Our results suggest that the combination of a HCV protease inhibitor with a RNA polymerase inhibitor could potentially function as an antiviral against SARS-CoV-2. More generally, our results strongly motivate further studies of the potential use of PLpro protease inhibitors in combination with RNA polymerase inhibitors as antivirals against SARS-CoV-2.Results
Similarity of the substrate-binding clefts and active sites of SARS-CoV-2 Mpro and HCV protease NS3/4A
The SARS-CoV-2 main protease (Mpro) is a 67.6 kDa homodimeric cysteine protease with three domains (; ). Domains I and II adopt a double β-barrel fold, with the substrate binding site located in a shallow cleft between two antiparallel β-barrels of domains I and II. The fold architecture of domains I and II are similar to picornavirus cysteine proteases and chymotrypsin serine proteases (; ). A three-dimensional structural similarity search of the Protein Data Bank using the DALI program (, ), with domains I and II (excluding domain III) of the SARS-CoV-2 Mpro as the query, identified several proteases, including the HCV NS3/4A serine protease, as structurally similar. These HCV and SARS-CoV-2 enzymes have a structural similarity Z score (, ) of +8.4 and overall backbone root-mean-square deviation for structurally similar regions of ∼3.1 Å. Superimposition of the analogous backbone structures of these two proteases results in superimposition of their substrate binding clefts and their active-site catalytic residues, His41/Cys145 of the SARS-CoV-2 Mpro cysteine protease and His57/Ser139 of the HCV NS3/4A serine protease (Figure 1A). Because of these structural similarities, we proposed that some HCV protease inhibitors might bind well into the substrate-binding cleft of the SARS-CoV-2 Mpro and inhibit virus replication ().
Figure 1SARS-CoV-2 Mpro binds HCV NS3/4A protease inhibitorsHCV protease inhibitors are predicted to bind into the substrate binding cleft of Mpro
Based on these structural similarities, we carried out docking simulations of 10 HCV NS3/4A protease inhibitor drugs (), using AutoDock (; ). These 10 drugs have been approved for at least phase 1 clinical trials, and some are FDA-approved prescription drugs used to treat HCV-infected patients (Table 1). To test the validity of our docking protocol, we first carried out docking of a previously described inhibitor of the SARS-CoV-2 Mpro, compound 13b (Figure S1A), that also inhibits virus replication (, ). Details of these control docking studies are provided in the STAR Methods and Figures S1B–S1D. The resulting docking scores are summarized in Figure 1B and Table S1. These results demonstrate that all 10 of these HCV protease inhibitors have the potential to bind snuggly into the binding cleft of Mpro, with extensive hydrogen-bonded and hydrophobic contacts, and predicted AutoDock energies of −8.37 to −11.01 kcal/mol, comparable to those obtained for Mpro inhibitor 13b (∼ −9.0 kcal/mol).Table 1HCV protease inhibitorsInhibitor (trade name) Identifier of protease inhibitor Trade name; manufacturer Drug status Vaniprevir VAN MK-7009; Merck investigational drug Simeprevir SIM Olysio/Medivir; Janssen prescription drug Paritaprevir PAR Veruprevir/ABT-450; Abbott Laboratories prescription drug Danoprevir DAN Ganovo; Array/Pfizer, Roche/Ascletis investigational drug Narlaprevir NAR Arlansa; Merck/R-Pharm prescription drug Grazoprevir GRZ Zepatier; Merck prescription drug Glecaprevir GLE Mavyret Maviret; AbbVie/Enanta prescription drug Boceprevir BOC Victrelis; Merck prescription drug Telaprevir TEL Incivek/Incivo; Vertex/J&J prescription drug Asunaprevir ASU Sunvepra; Bristol-Myers Squibb investigational drug a Mavyret (or Maviret) is a multidrug formulation including glecaprevir and pibrentasvir.The 1.44-Å X-ray crystal structure of the complex of BOC bound to the SARS-CoV-2 Mpro was recently released in the Protein Data Bank [PDB: 6WNP (; )]. The BOC pose observed in this X-ray crystal structure is almost identical to the lowest energy pose (−9.13 kcal/mol) predicted by AutoDock (Figure 1C). In addition, both the docked and crystal structure binding poses for BOC near the active site of Mpro are very similar to its binding pose in the substrate binding cleft of HCV protease (Figure 1D), with an essentially identical hydrogen-bonding network between BOC and corresponding residues in each protease (shown in Figures S1E and S1F). Recently, also reported an X-ray crystal structure of the SARS-CoV2 Mpro-BOC complex, as well as the structures of Mpro complexed with HCV inhibitors NAR and TEL. The predicted binding modes of BOC and NAR are also an excellent match to these subsequently determined experimental structures (Figures S1G and S1H), while for TEL poses similar to the crystal structure are included among the best-scoring AutoDock poses (Figures S1I and S1J). BOC, NAR, and TEL are alpha-ketoamides which form covalent bonds with the active site residue Cys145 of Mpro. Although this docking protocol does not include energetics or restraints for covalent bond formation, in the low-energy poses of both BOC and NAR bound to Mpro, the alpha-keto amide carbon is positioned within 3.8 Å of the active site thiol sulfur atom. These blind tests support the predictive value of docking results for the other HCV protease inhibitors. From these virtual docking studies and comparison with subsequently determined X-ray crystal structures, we conclude that all 10 of these HCV protease inhibitors have the potential to bind into the substrate-binding cleft of Mpro, and to inhibit binding of its substrates.Seven HCV drugs inhibit Mpro protease activity
Inhibition activity of HCV drugs against Mpro was initially assessed using a protease assay based on Föster resonance energy transfer (FRET) using the peptide substrate Dabsyl-KTSAVLQ/SGFRKME-(Edans), containing a canonical Mpro protease recognition site. Under the conditions of these FRET assays, there is little or no inner filter effect for most of the drug-peptide substrate assays (Table S3), and the half-life for the proteolytic reaction is about 30 min (Figures S2A and S2B). HCV protease inhibitors NAR, BOC, and TEL have significant enzyme inhibition activity, with IC50 values of 2.2 ± 0.4 μM, 2.9 ± 0.6 μM, and 18.7 ± 6.4 μM, respectively (Figure 1E). In contrast, little or no inhibition activity was detected in this FRET assay with the other seven HCV protease inhibitors (Figures S3A and S3B).We also developed a 1D 1H-NMR assay, using the peptide substrate KTSAVLQ/SGFRKME that lacks the Dabsyl and Edans N-terminal and C-terminal tags (Figure S4). In this 1D 1H-NMR assay, NAR, BOC, and TEL have substantial enzyme inhibition activity, as was the case in the FRET assay. In addition, in the NMR assay substantial inhibitory activity was also observed for VAN, and moderate inhibitory activity was observed for GRZ, SIM, and ASU (Figures 1F and 1G). This significant inhibition activity of VAN, and moderate inhibition activities of GRZ, SIM, and ASU, was not detected in the FRET assay. DAN, PAR, and GLE had little or no detectable Mpro inhibitory activity. From these studies, we conclude that seven HCV drugs (viz BOC, NAR, TEL, VAN, GRZ, SIM, and ASU) inhibit SARS-CoV-2 Mpro strongly or moderately under the conditions tested.Eight HCV drugs inhibit SARS-CoV-2 replication in Vero and/or human 293T cells
The motivation for the docking and biophysical studies described above was to identify HCV drugs with the potential to inhibit SARS-CoV-2 virus replication. For antiviral assays, Vero E6 cells or human 293T cells expressing the ACE2 receptor, were grown in 96-well plates and were incubated with various levels of a HCV protease inhibitor for 2 h. Cells were then infected with SARS-CoV-2 virus at the indicated multiplicity of infection (MOI, plaque-forming units [PFU)/cell) and incubated for the indicated times at 37°C in the presence of the inhibitor. Virus-infected cells were identified by immunofluorescence using an antibody specific for the viral nucleoprotein. Inhibition of viral replication was quantified by determining the percentage of positive infected cells at the end of the incubation period in the presence of the compound, as compared with the number of infected cells in its absence. To determine whether a HCV drug was cytotoxic, uninfected Vero E6 or human 293T cells were incubated with the same levels of the compounds for the same length of time, and cytotoxicity was measured using an MTT assay (Roche). In all of these replication assays, remdesivir was used as a positive control.Viral replication inhibition data in Vero E6 cells are summarized in Figure 2; Table S2. Five of the HCV protease inhibitors tested, PAR, NAR, GRZ, ASU, and BOC, inhibited SARS-CoV-2 virus replication at concentrations significantly lower than the concentrations that cause cytotoxicity. Two other HCV protease inhibitors, SIM and VAN, inhibited virus replication with low IC50 values, but some cytotoxicity was also observed. These seven HCV drugs have IC50 values for inhibiting SARS-CoV-2 replication of 4.2 to 19.6 μM. The remaining three HCV drugs tested, TEL, GLE, and DAN, did not inhibit virus replication in Vero E6 cells, even at a drug concentration of 50 μM.
Figure 2Antiviral activity of HCV protease inhibitors in Vero E6 cellsNext, we also determined whether the HCV drugs exhibited similar antiviral activities in human cells, specifically human 293T cells expressing the ACE2 receptor (Figure 3; Table S2). Again, PAR, SIM, and VAN were the most effective inhibitors of virus replication, with IC50 values of 0.55 to 3.0 μM, and with considerably reduced cytotoxicity as compared to assays in Vero cells. BOC and NAR, which are relatively strong Mpro inhibitors (Figures 1E–1G), were less effective inhibitors of virus replication, with IC50s of 5.4 and 15 μM. The other covalent Mpro inhibitor, TEL, which did not inhibit in Vero cells had a IC50 of 20.5 μM in human cells, while GRZ had less potent antiviral activity in human cells than in Vero cells (cf. Figures 2F, 2I, 3G, and 3H). Thus, seven HCV drugs inhibited SARS-CoV-2 replication in human 293T cells, with IC50 values ranging from 0.55 to 20.5 μM. GLE and DAN did not inhibit virus replication in human cells, as was also the case in Vero cells. ASU, a modest inhibitor in Vero cells, did not inhibit viral replication in human cells at concentrations lower than those exhibiting cytotoxicity.
Figure 3Antiviral activity of HCV protease inhibitors in HEK293T cellsWe also determined whether the inhibition of SARS-CoV-2 replication by representative HCV protease inhibitors occurs at steps after virus entry, as would be expected for inhibitors of viral proteases that are produced after infection. Accordingly, we performed time-of-addition assays in human cells, using BOC (50 μM), GRZ (25 μM), NAR (50 μM), VAN (5 μM), and SIM (5 μM), at concentrations that do not show detectable cytotoxicity as measured by MTT assay (Table S2) or by DAPI staining in infected cells. In a single cycle (MOI of 2 PFU/cell over a total infection time of 8 h) of infection, drugs were added 2 h prior to infection, at the time of infection, or at 2 or 4 h post-infection. Virus replication was inhibited by the addition of these drugs as late as 4 h post-infection (Figure L), indicating that these drugs can inhibit viral infection after the initial phase of virus entry. These results indicate that these HCV drugs inhibit virus-encoded proteases synthesized in infected cells. The results do not, however, rule out that these drugs may also inhibit other proteases or enzymes, including any involved in virus entry. In contrast, addition of the RNA polymerase inhibitor remdesivir 4 h after the initiation of infection decreased its ability to suppress virus infection, demonstrating the crucial role of viral RNA synthesis at early times of infection.Simeprevir and grazoprevir synergize with remdesivir to increase inhibition of SARS-CoV-2 virus replication
Because Mpro and PLpro generate either the RNA polymerase itself or the proteins that constitute the replication organelles required for polymerase function, we predicted that HCV drugs that inhibit one or both of these viral proteases might be synergistic with inhibitors of the viral polymerase like remdesivir. To test this hypothesis, we carried out antiviral combination assays of SIM, GRZ, and BOC, respectively, with remdesivir in Vero cells. To assess synergy, two analyses are required. In one analysis the IC90 of the remdesivir was measured in the presence of increasing concentrations of each of these three HCV drugs (Figures 4A–4C). These results demonstrate that SIM and GRZ increase the antiviral activity of remdesivir. For example, in the presence of 1.25 μM SIM, approximately 10-fold less remdesivir is required for the same antiviral effect achieved in the absence of SIM (Figure 4A). A similar 10-fold enhancement in antiviral activity of remdesivir is observed in the presence of GRZ, though at higher (6.25 μM) GRZ concentrations (Figure 4B). Surprisingly, although BOC is a much better inhibitor of Mpro than either SIM or GRZ, BOC did not significantly affect the antiviral activity of remdesivir (Figure 4C). In the second analysis, the IC90 concentration of each HCV drug was determined in the presence of increasing concentrations of remdesivir (Figures 4D–4F). Remdesivir increased the antiviral activity of SIM and GRZ. For example, addition of 1.25 μM remdesivir substantially reduces the concentration of SIM or GRZ needed to achieve IC90 conditions (Figures 4D and 4E). In contrast, remdesivir did not significantly affect the antiviral activity of BOC (Figure 4F).
Figure 4Simeprevir and grazoprevir are synergistic with remdesivir in Vero E6 cellsThese antiviral assays indicate that SIM and GRZ, but not BOC, act synergistically with remdesivir to inhibit virus replication. As confirmation, we subjected these results to analysis by the zero interaction potency (ZIP) model for synergy (). In the landscapes generated by this model (Figures 4G–4I), red denotes a synergistic interaction, and green denotes an additive interaction. In this model a synergistic interaction between drugs has a score greater than +10, an additive interaction has a score between −10 to +10, and an antagonistic interaction has a score of less than −10. The landscapes for the interaction of remdesivir with both SIM and GRZ are red, with synergy scores of +30.2 and +25.0, respectively, denoting moderate synergism. In contrast, the landscape for the interaction of remdesivir with BOC does not indicate synergy (Figure 4I); the synergy score, −7.6, indicates an additive interaction. We also carried out combination antiviral assays in human 293T cells. The interaction between remdesivir and GRZ in inhibiting virus replication in the human cells was also synergistic, with a red landscape and a synergy score of +20.3 (Figure S5). Consequently, at least two HCV drugs, SIM and GRZ, act synergistically with remdesivir to inhibit SARS-CoV-2 virus replication in Vero and/or human 293T cells.Four HCV protease inhibitors that are synergistic with remdesivir inhibit PLpro
The preceding results demonstrate that several HCV inhibitors inhibit viral replication, and that for some of these drugs inhibition is synergistic with the viral replication inhibition activity of remdesivir. Most of these drugs also inhibit SARS-CoV-2 Mpro. However, Mpro and SARS-CoV-2 inhibition by these drugs were not consistently correlated. For example, PAR does not inhibit Mpro in either the FRET or NMR assays but is an effective inhibitor of SARS-CoV-2 in both Vero (IC50 = 6 μM) and 293T (IC50 = 0.55 μM) assays. In addition, the HCV protease inhibitors SIM and GRZ, which are only moderate inhibitors of Mpro, synergize with remdesivir, while BOC, which is an excellent inhibitor of Mpro, acts additively rather than synergistically with remdesivir to inhibit virus replication. Although the lack of strong correlation between protease inhibition activity and viral inhibition activity could result from various effects, including the efficiency of transport of drugs into the cell and/or metabolism of the drugs in the cell-based assays, these results suggested that there might be a second viral target for some of these HCV drugs, for which inhibition may provide the basis for the observed synergy.Aside from both being Cys proteases, the active site of PLpro does not share structural similarity with the HCV NS3/4A or Mpro proteases. However, it has recently been reported that SIM, GRZ, and ASU inhibit PLpro (). Accordingly, we carried out virtual docking studies of these same 10 HCV drugs into the substrate-binding cleft of PLpro, using protocols similar to those developed in virtual docking studies with Mpro. The known PLpro inhibitor GRL0617 () was used to assess the docking protocol, providing a reference AutoDock score of −7.54 kcal/mol. The scores of docking poses for HCV drugs, summarized in Figure 5A and Table S1, range from –5.56 kcal/mol for BOC and NAR, to much more favorable values of < –8 kcal/mol for others, including VAN, GRZ, SIM, and PAR. These proof-of-concept docking studies suggest that, surprisingly, some HCV protease inhibitors may bind in the substrate-binding clefts of both Mpro and PLpro.
Figure 5HCV protease inhibitors also inhibit SARS-CoV-2 PLproBased on these docking predictions, we anticipated that several HCV protease inhibitors, not including BOC, NAR, or TEL, might effectively inhibit PLpro protease activity. To test this hypothesis, fluorescence assays of PLpro inhibition were carried out, using the substrate zRLRGG/AMC (z - carboxybenzyl; AMC - 7-Amino-4-methylcoumarin) () containing a natural canonical PLpro protease recognition site (XLXGG). Of the HCV drugs tested, four drugs predicted to bind into the active site of PLpro, VAN, SIM, PAR, and GRZ, do in fact inhibit PLpro protease activity (Figure 5B; Figure S6). Hence, VAN, SIM, and GRZ inhibit both Mpro and PLpro, while PAR inhibits PLpro, but not Mpro. Under these assay conditions, VAN and SIM are more effective PLpro inhibitors than GRZ or PAR.These results strengthened our hypothesis that synergy between these HCV drugs and remdesivir arises primarily from their activities in inhibiting PLpro, rather than from inhibiting Mpro. To further test this, combination assays with remdesivir were carried out also for NAR, PAR, and VAN. As predicted, PAR which moderately inhibits PLpro but not Mpro, is synergistic with remdesivir (Figure 5C, synergy score +17.3). VAN, which inhibits both Mpro and PLpro, is also modestly synergistic, with synergy score + 10.9, while NAR, which inhibits Mpro but not PLpro, is additive with synergy score –3.6 (Figures 5D and 5E).Data for HCV protease drugs on SARS-CoV-2 protease inhibition and synergy are summarized in Figure 6A and in Table S2. Two drugs, BOC and NAR, which are relatively good inhibitors of Mpro but do not inhibit PLpro, have additive interactions with remdesivir. Three drugs, GRZ, SIM, and VAN, which inhibit both Mpro and PLpro, are synergistic, with synergy scores of +10.9 to +30.2. Interestingly, among these three, VAN, which is a relatively strong inhibitor of both Mpro and PLpro, has the weakest synergy. A fourth HCV drug, PAR, which inhibits PLpro but not Mpro, also has synergy with remdesivir, with synergy score +17.3. These data demonstrate a correlation between the PLpro inhibiting activities of these drugs and their ability to function synergistically with remdesivir to suppress viral replication.
Figure 6PLpro inhibition and synergy with remdesivir activities are correlatedIn order to further test this model of synergy, we also carried out biochemical and viral replicase assays with the molecule GC-376, an Mpro inhibitor (; ), and an analog of GRL0617, referred to as compound 6, an established PLpro inhibitor (; ; ). While GC-376 has been reported to be specific for Mpro relative to PLpro (), the relative specificity of GLR0617 (or compound 6) for PLpro relative to Mpro has not previously been reported. Using the same fluorescence assays outlined above, we validated the high specificity of GC-376 for Mpro inhibition compared to PLpro (Figure 6B, left) and documented high specificity of compound 6 for PLpro inhibition compared to Mpro (Figure 6B, right). As predicted, combination viral inhibition assay of GC-376 with remdesivir shows an additive interaction (Figure 6C; synergy score +5.9), while the GRL0617 analog (compound 6) has a synergistic interaction with remdesivir (Figure 6D; synergy score +18.6). We conclude that inhibitors of the SARS-CoV-2 PLpro protease function synergistically with remdesivir to inhibit viral replication, whereas specific inhibitors of the Mpro protease act additively with remdesivir.Discussion
To provide antiviral drugs that can be rapidly deployed to combat the COVID-19 pandemic, we carried out the present study to identify currently available drugs that could potentially be repurposed as inhibitors of the SARS-CoV-2 virus that causes the COVID-19 disease. A total of eight HCV drugs were identified that inhibit virus replication in Vero and/or human 293T cells expressing the ACE2 receptor.We initiated our search based on the striking similarity of the substrate binding clefts of the SARS-CoV-2 Mpro and HCV NS3/4A proteases (). The substrate-binding clefts and active sites of Mpro and HCV proteases superimpose remarkably well (Figure 1A), despite having very low sequence similarity (Figure S7) and significantly different structural topologies (). Our virtual docking experiments showed that 10 HCV protease inhibitors can be docked snuggly into the substrate binding cleft of Mpro and hence have the potential to inhibit binding of the Mpro substrate, thereby inhibiting proteolytic cleavage of the viral polyprotein to form NSPs. For BOC and NAR, these predicted docking poses () are consistent with the subsequently determined X-ray crystal structures (; ; ); for TEL, some predicted binding poses are also similar to the corresponding crystal structure (). Four of these HCV drugs, BOC, NAR, TEL, and VAN, are relatively strong inhibitors of SARS-CoV-2 Mpro protease activity (IC50 of 2 to ∼20 μM), and three other HCV drugs, GRZ, SIM, and ASU, moderately inhibit Mpro activity. BOC, NAR, and TEL are α-keto amides, which can form a covalent bond with the active site Cys thiol of Mpro . The other four HCV drugs are non-covalent inhibitors of the Mpro protease and cannot form a covalent bond with the active site Cys thiol.Other groups have also recently reported that some of these same HCV protease inhibitors can inhibit Mpro protease activities (; ; ; ; ). While all of these studies report BOC as a moderately potent inhibitor of Mpro, there are inconsistent reports of the effectiveness of some of the other HCV protease inhibitors reported here as inhibitors of Mpro and/or PLpro. These inconsistencies likely arise from details of the different assays, including the specific protein constructs and polypeptide substrates.The significant intrinsic fluorescence of these non-covalent inhibitor drugs complicates the Mpro FRET assay (see STAR Methods), particularly for VAN, SIM, and GRZ (see Figure S3A). For this reason, we also used 1D 1H-NMR assay for Mpro inhibition, which confirmed that BOC, NAR, and TEL inhibit Mpro. In the NMR assay VAN also has inhibitory activity comparable to TEL, while GRZ, SIM, and ASU are moderate inhibitors of Mpro. The ability of the NMR assay to detect inhibitory activity that was not detected by the FRET assay is attributable to several factors, including differences in substrate and enzyme concentrations used in these assays, and differences in the substrates themselves. The ability of HCV drugs to inhibit Mpro also depends on other details of the assay conditions, most notably the enzyme, substrate, and drug concentrations and details of the Mpro construct ().Although the active site of PLpro does not share structural similarity with the HCV NS3/4A protease, we observe that four HCV drugs, SIM, GRZ, VAN, and PAR, inhibit PLpro protease activity in vitro. None of these four inhibitors can form covalent bonds with the active-site Cys residue of PLpro. VAN is a good inhibitor of both Mpro and PLpro, presumably accounting for its strong inhibition of virus replication. All four of these HCV drugs function synergistically with remdesivir to inhibit SARS-CoV-2 virus replication in Vero and/or human cells.Particularly interesting in this set is PAR, which has the strongest potency in the human cell assay (IC50 = 0.55 μM), strong synergy with remdesivir (synergy score + 17.3), and low cytotoxicity (CC50 > 100 μM) in both the Vero and human cell assays (Table S2). However, PAR only moderately inhibits PLpro and does not inhibit Mpro . One possibility is that inhibition of virus replication by PAR could result, at least in part, from its inhibition of a third target. Inhibition of such a putative third target might also play some role in the inhibition of virus replication by the other HCV drugs.In addition to its function in cleavage of viral polyproteins to generate crucial viral non-structural proteins, PLpro also removes the ubiquitin-like ISG15 protein from viral proteins (; ). ISG15 conjugation in infected cells results in a dominant-negative effect on the functions of viral proteins (); i.e., ISGlyation disrupts a wide range of viral functions. In addition, the resulting free ISG15 is secreted from infected cells and binds to the LFA-1 receptor on immune cells, causing the release of interferon-γ and inflammatory cytokines (, ). The release of these cytokines could contribute to the strong inflammatory response, the so-called cytokine storm, that has been implicated in the severity of COVID-19 disease (). Inhibition of PLpro by a HCV drug should also inhibit the release of interferon-γ and inflammatory cytokines, potentially mitigating the cytokine storm.Viral replication assays using combinations of drugs allowed us to assess whether the interactions between HCV drugs and remdesivir are additive or synergistic. We found that these inhibitory effects are additive or synergistic depending on which HCV drug is used to inhibit virus replication. In particular, HCV drugs that inhibit PLpro synergize with remdesivir to inhibit SARS-CoV-2 replication in Vero and 293T cells. These HCV drugs include SIM, VAN, GRZ, and PAR. The conclusion that inhibition of PLpro alone is sufficient for synergy with remdesivir was confirmed by a combination assay with compound 6 (a GRL0617 analog) that specifically inhibits PLpro but not Mpro. On the other hand, we show that the HCV drugs BOC and NAR that inhibit only Mpro have an additive rather than a synergistic interaction with remdesivir in inhibiting SARS-CoV-2 replication. The conclusion that selective inhibition of Mpro has an additive interaction with remdesivir was confirmed by a synergy assay with compound GC-376, that specifically inhibits Mpro but not PLpro. Another Mpro inhibitor (PF-00835231) has been reported to act in combination with remdesivir, but it was not clear whether this interaction was additive or synergistic (). It was also recently reported that SIM acts synergistically with remdesivir but that this synergy results from inhibition of Mpro and/or other targets ().The mechanism through which PLpro inhibition, but not Mpro inhibition, results in synergy with remdesivir is not yet known. One rational mechanism involves the critical role of PLpro in the formation of replication organelles (DMVs). Studies of DMV formation by SARS-CoV nsp3, nsp4, and nsp6 proteins demonstrate a requirement for all three proteins, and for a catalytically active PLpro nsp3 construct (). HCV drugs that inhibit PLpro in infected cells should therefore inhibit formation of DMVs that are required for polymerase function, reducing the amount of functional viral RNA polymerases, and hence reducing the amount of remdesivir needed for inhibition of virus replication. This hypothetical mechanism could explain why drugs that inhibit PLpro (e.g., SIM, VAN, PAR, and GRZ) act synergistically with remdesivir. In contrast, the drugs that inhibit Mpro, but not PLpro (e.g., BOC and NAR), are not synergistic with remdesivir. Mpro inhibitors are expected to reduce the amount of the three subunits, nsp7, nsp8, and nsp12, of the viral polymerase in infected cells. However, the reduction in the amounts of these polymerase subunits might not reduce the level of the viral polymerase sufficiently to exhibit synergy with remdesivir if there is relatively large pool of these subunits in infected cells. While Mpro also generates the nsp4 and nsp6 proteins that contribute to DMV formation (), it is not known whether this function of Mpro is required for DMV formation.Synergy between PLpro and viral polymerase inhibitors could also involve other viral or host targets of these protease inhibitors. Removal of ISG15 from viral or host proteins by PLpro could potentially restore their functions, and inhibition of the de-ISGlyaton function of PLpro could provide another mechanism for synergy between inhibitors of PLpro and inhibitors of other viral or host protein functions, including remdesivir.HCV drugs that are strongly synergistic with remdesivir are most pertinent for the goal of the present study. Repurposed drugs may not have sufficient inhibitory activity on their own to achieve clinical efficacy. Synergy with remdesivir increases the potency of both the proposed repurposed HCV drugs and remdesivir. We identified four HCV drugs, SIM, VAN, PAR, and GRZ, that act synergistically with remdesivir to inhibit SARS-CoV-2 virus replication. Of these four, SIM, PAR, and VAN are particularly interesting as repurposed drugs because they effectively inhibit SARS-CoV-2 virus replication in human cells at lower concentrations than GRZ. Consequently, the combination of an FDA-approved PLpro inhibitor, such as SIM or PAR, and remdesivir, could potentially function as an antiviral against SARS-CoV-2, while more specific and potent SARS-CoV-2 antivirals are being developed. SIM, VAN, PAR, and GRZ are orally administered drugs that might also be combined with an oral polymerase inhibitor rather than with remdesivir, which has to be administered intravenously. One such oral polymerase inhibitor, molnupiravir (MK-4482) (), which is currently in late-stage clinical trials, could potentially be combined with one of these four HCV protease inhibitors for clinical applications. For example, a combination of SIM and molnupiravir could be assessed for outpatient use. Beyond the proposed repurposing of these FDA-approved HCV inhibitors as antivirals for COVID-19, our results indicate that the SARS-CoV-2 PLpro is an important target for future antiviral drug development that when used in conjunction with polymerase inhibitors could provide potent efficacy and protection from SARS-CoV-2, especially for virus variants that are resistant to vaccine-generated antibodies.STAR★Methods
Key resources table
REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-N protein Dr. Thomas Moran (MSSM) mAb 1C7 Bacterial and virus strains USA-WA1/2020 BEI Resources NR-52281 Biological samples Not applicable Chemicals, peptides, and recombinant proteins Recombinant SARS-CoV-2 PLpro protein; residues 1564 to 1881 of the SARS-CoV-2 replicase polyprotein 1a (Uniprot id P0DTD1 (R1A_SARS2)), with a C-terminal purification tag LEHHHHHH Laboratory of Prof. J. Hunt, Columbia University References
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Article Info
Publication History
Published: May 18, 2021Accepted: April 23, 2021Received in revised form: March 18, 2021Received: November 16, 2020Identification
DOI: https://doi.org/10.1016/j.celrep.2021.109133
https://notistecnicas.blogspot.com/2021/09/el-remdesivir-reduce-un-87-el-riesgo-de.html
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