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Chemical screen uncovers novel structural classes of inhibitors of the papain-like protease of coronaviruses

Summary The papain-like protease (PLpro) of coronaviruses is an attractive antiviral target to inhibit both viral replication and interference of the host immune response. We have identified and characterized three novel classes of small molecules, thiophene, cyanofuran, and triazoloquinazoline, as PLpro inhibitors. Thiophene inhibited the PLpro of two major coronaviruses, Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV) including SARS-CoV-2, while cyanofuran and triazoloquinazoline more selectively inhibited MERS-CoV PLpro. Unlike GRL0617, a known PLpro inhibitor, all three compounds contain no naphthyl group but like GRL0617 were predicted to fit on the cleft near the BL2 loop. Docking studies further revealed that the location and direction of the binding determined their specificity to different coronaviruses. Together, our work demonstrates that the BL2 loop and nearby regions are outstanding druggable targets, and our three inhibitors can be applicable to the development of therapeutics for coronavirus infection.


INTRODUCTION
Coronaviruses have caused diseases in humans and have led to several epidemic and pandemic crises. To date, seven human coronaviruses (HCoVs) have been reported: HCoV-OC43, Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2, which belong to the betacoronavirus genus, as well as HCoV-229E and HCoV-NL63, which belong to the alphacoronavirus genus (Hartenian et al., 2020;van der Hoek et al., 2004). The well-known HCoV-229E and HCoV-OC43 were first reported in 1960 and for decades were considered to be major disease-causing coronaviruses. Identification of SARS-CoV in 2002 (Rota et al., 2003) and MERS-CoV in 2012  proved that these air-borne pathogens can easily transfer between species, spread within the community, and result in an epidemic; and the recently identified SARS-CoV-2 has caused an unprecedented global pandemic and has posed a serious threat to public health and the world economy. By the fall of 2021, over 200 million infection cases and four million deaths by SARS-CoV-2 infection have been reported globally, and casualties are still mounting despite the recent vaccine drive (Grubaugh et al., 2021;Zhou et al., 2020). All these indicate that MERS-CoV and SARS-CoV are highly pathogenic and detrimental to humans. In addition, there is a high likelihood of further retransmission from various zoonotic reservoirs which can result in another serious pandemic. Although several antiviral drugs including neutralizing antibodies (Corti et al., 2021) and molnupiravir (Sheahan et al., 2020;Toots et al., 2019), a nucleoside analog that inhibits viral genome replication, have been developed, additional drugs with novel antiviral mechanisms of action still need to be developed for ready clinical use. This is because the virus can develop resistance mechanisms against nucleoside analogs, and also because a combination of drugs with different mechanisms of action is commonly used in the clinic to improve treatment outcomes.
MERS-CoV and SARS-CoV are single-stranded positive-sense RNA viruses with a genome of $30 kb enveloped with spike proteins (de Groot et al., 2013). For entry into host cells, the spike proteins of MERS-CoV and SARS-CoV recognize their respective host cell receptors, dipeptidylpeptidase 4 (DPP4 or CD26) and angiotensin-converting enzyme 2 (ACE2). Upon entry, the viral RNA genome is released from the capsid and undergoes translation to produce viral polyproteins, pp1a and pp1b. The polyproteins are further processed by two cysteine proteases, papain-like protease (PLpro) and a 3-C-like protease (3CLpro), which constitute an essential step for the maturation of 16 non-structural viral proteins (Hartenian et al., 2020;Perlman and Netland, 2009). Therefore, the protease activity of the coronavirus has been implicated as one of the most favorable antiviral drug targets. Especially, PLpro, in addition to being essential for viral proliferation, is involved in other important cellular processes including de-ISGylation and deubiquitination (Lindner et al., 2005;Ratia et al., 2014), thereby repressing host's innate immunity triggered by interferon regulatory factor 3 (IRF-3) (Bailey-Elkin et al., 2014;Barretto et al., 2005;Clementz et al., 2010;Frieman et al., 2009). Therefore, PLpro is an outstanding antiviral target to inhibit both viral replication and viral interference of the host innate immune response to the viral invasion. To date, several classes of PLpro inhibitors have been identified: the thiopurines 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) (Chen et al., 2009;Chou et al., 2008); the natural products tanshinones (Park et al., 2012) and geranylated flavonoids (Cho et al., 2013); zinc ion and zinc-conjugated inhibitors (Han et al., 2005); and naphthalene drugs (Ratia et al., 2008). Importantly, a potent naphthalene inhibitor GRL0617 and its naphthalene analogs were reported to specifically bind to the region near the BL2 loop, a region situated near the catalytic cleft, thereby playing an important role in gating the cleft toward the catalytic site of PLpro (Fu et al., 2021;Ghosh et al., 2009;Ratia et al., 2008). Therefore, PLpro, specifically its flexible BL2 loop, is an attractive target for the development of antiviral drugs against HCoVs; and owing to the aforementioned risk of development of viral resistance mechanisms to existing drugs and the effectiveness of combination therapy, uncovering novel classes of compounds as PLpro inhibitors can be an option for therapeutic measures for coronavirus infection.
Here, in our effort to uncover effective antiviral compounds against coronaviruses, we adapted a robust high-throughput compound assay to screen over 29,938 compounds, including FDA-approved drugs. We identified three effective compounds of novel structural classes: thiophene (1), cyanofuran (2), and triazoloquinazoline (3). We found that these three compounds showed differential activities against MERS-CoV PLpro (MERS-PLpro), SARS-CoV PLpro (SARS-PLpro), and SASR-CoV-2 PLpro (SARS-2-PLpro), which was validated with in vitro enzyme assays and immunofluorescence-based cell culture experiments. To further identify the structural properties important for their activity, we explored the series of analogs available, and through our molecular docking study and molecular dynamics simulation (MDS), found that their binding affinity to the cleft near the BL2 loop provides target selectivity against MERS-PLpro and SARS-PLpro. Together, our work identifies novel structural classes of PLpro inhibitors and elucidates their binding properties to the target, which provides essential information for the development of broad-spectrum anticoronavirus treatments.

Compounds of novel structural classes were identified as MERS-PLpro inhibitors
We screened for PLpro inhibitors using an in vitro enzymatic assay for which the results were validated by cell-based infection assays ( Figure 1A). For this, we codon-optimized and expressed the MERS-PLpro protein in a bacterial strain and adapted a fluorescence-based enzymatic assay where a short peptidelinked AMC (7-amido-4-methylcoumarin) was used as the substrate, as previously described (Kilianski et al., 2013;Ratia et al., 2008). As noted, although MERS-PLpro recognizes K/R-L/I-I/K/V-G-GYA, we chose a short peptide with the sequence of R-L-R-G-G-AMC instead, because this sequence generated a more robust fluorescence signal ( Figure 1B). We tested a series of peptide substrate concentrations (0.8-100 mM) and MERS-PLpro enzyme concentrations (0.3-10 mM) to set up optimal concentrations for the screen at room temperature. Concentrations of 2.5 and 40 mM were selected for MERS-PLpro and the substrate, respectively ( Figure S1A). The substrate concentration chosen is lower than the Michaelis constant (K M ) which makes it suitable for a screen for competitive inhibitors. The chosen concentrations of MERS-PLpro and the peptide substrate were confirmed using a known PLpro inhibitor, 6-TG (Figure S1C). Using the optimized conditions, we screened a collection of 29,938 compounds including FDA-approved drugs, sourced from ChemDiv, ChemBridge, and Selleckchem, at a single concentration of 10 mM. We performed a high-throughput screen using a 384-well format, and compounds that inhibited the protease activity were selected as positive hits. Our screen was found to be very robust with an average Z 0 -factor of 0.91 G 0.01 ( Figure S2A). We defined compounds with over 40% inhibition as positive hits and a subset was cherry picked and retested ( Figure 1C). Of the 29,938 compounds screened, 257 compounds were identified as primary hits for inhibitors of MERS-PLpro, which is a hit rate of $0.86% ( Figure 1A). Of the primary hit compounds, we excluded 81 compounds that showed over 20% cytotoxicity ( Figure S2B). Then, we further validated the hit compounds by performing the in vitro MERS-PLpro assay at serially diluted compound concentrations, and of the validated hit iScience Article compounds, three compounds, thiophene (1), cyanofuran (2), and triazoloquinazoline (3) were selected for further assessment ( Figures 1D-1F). To further validate the efficacy of these compounds in the host cell, we measured the degree of cellular ubiquitination in the presence of the compounds as MERS-PLpro is known to inhibit cellular ubiquitination. As shown in Figure 2, we have found that all three compounds reversed the inhibition of cellular ubiquitination by MERS-CoV. All these results indicate that the selected compounds prevent the activity of MERS-PLpro in the host cells.

Thiophene (1) shows a broad-spectrum potency against coronaviruses
As other known competitive inhibitors such as 6-MP and 6-TG show efficacy against both SARS-PLpro and MERS-PLpro, we tested the potency of thiophene (1) against SARS-PLpro in addition to MERS-PLpro using the in vitro enzymatic assay. In the same manner as for MERS-PLpro, a series of peptide substrate concentrations (0.8-100 mM) and SARS-PLpro enzyme concentrations (0.02-0.63 mM) were tested, and 0.02 and 40 mM were selected as optimal concentrations for SARS-PLpro and the substrate, respectively (Figures 1D and S1B) and the chosen concentrations were validated using 6-TG ( Figure S1D). As shown in Figure 3B, we found that thiophene (1) has potency against MERS-PLpro as well as SARS-PLpro. The 50% inhibitory concentration (IC 50 ) value of each analog was calculated from the dose-response curve generated from the fluorescence-based PLpro protease assay, and the results indicate that thiophene (1) shows a comparably outstanding potency against MERS-PLpro with an IC 50 value of 14.2 mM and a moderate potency against SARS-PLpro with an IC 50 value of 156.3 mM ( Figure 3B). All 29,938 small molecules from the test library, which include FDA-approved drugs, were tested on MERS-PLpro using a fluorescence-based protease assay, and cytotoxicity of the selected 257 primary hit compounds was determined using a commercially available cytotoxicity kit, followed by the final assessment of in vivo efficacy using an immunofluorescence-based MERS-CoV infection assay or viral RNA quantification in virus-infected Vero cells. In addition, the structure-activity relationship (SAR) was examined using compound analogs and molecular docking analysis. To identify the structural regions of thiophene important for its affinity to PLpro, we evaluated the available analogs of 1 ( Figure 3A). We tested the analogs with the modification of the para-bromo on the phenyl group of 1 with fluorination at different positions of the phenyl ring (-para or -ortho) on the benzamide region. Analog 1a with fluorine at the para position showed a 3-fold decrease in efficacy against MERS-PLpro and a complete loss of efficacy against SARS-PLpro, while 1b with the ortho-fluoro substituent still retained potency against both MERS-PLpro and SARS-PLpro although the potency decreased ( Figure 3B). Next, we tested the role of the carboxylic acid at the 3-position on the thiophene scaffold. As shown in Figure 3B, esterification of the carboxylic acid with methyl (1c) or ethyl (1d) moieties resulted in a complete loss of activity against both proteases. This suggests that the carboxylic acid moiety is crucial for the affinity of thiophene (1) to the target protein. Also, we tested analogs with modification on the 4-position of the thiophene scaffold: 1e, 1f, 1g, and 1h. Analog 1e with a tolyl group replacing the thiophene group showed an improved potency on both proteases; 1f with an isopropyl moiety still showed potency, although moderately decreased, on both enzymes; 1g with an introduction of a methyl moiety showed potency only on SARS-PLpro with no activity against MERS-PLpro; 1h with complete removal of the thiophene scaffold showed a 2-fold decrease in potency against MERS-PLpro with no change in potency against SARS-PLpro ( Figure 3B).
Although an in vitro protease assay can provide useful target-specific information, it does not validate the antiviral potency of the compounds in the host cells. Recently, we adapted an immunofluorescence-based assay to monitor MERS-CoV infection in host cells. In this assay, we infected Vero cells with MERS-CoV at a MOI of 0.0625 in the presence of the compounds, then two days after infection, we quantified the viral infection by immunofluorescence using a MERS-CoV spike-specific primary antibody and a fluorescenceconjugated secondary antibody ( Figure 1A). As shown in Figure 3C, treatment with both compound 1 and chloroquine, a lysosomotropic compound that prevents endosomal acidification and appears to have antiviral activity , resulted in the depletion of the MERS-CoV fluorescence signal in the host cells demonstrating antiviral activity. We performed this image-based analysis for 1 and its analogs on MERS-CoV-infected cells and calculated 50% efficient concentration (EC 50 ) values (Figures 3B and  S3). Overall, their potencies in the cell-based experiment were similar to the efficacy measured by the in vitro enzymatic assay: 1 was found to have a marked efficacy against MERS-CoV infection (EC 50 , 2.33 mM), and analogs 1e (EC 50 , 1.25 mM) and 1f (EC 50 , 1.06 mM) showed an improved potency. As expected, the analogs 1c and 1d with modification at the carboxylic acid showed no efficacy in cell-based assays. Also, 1h had no activity against MERS-CoV infection. Interestingly, 1b showed an improved IC 50 against MERS-PLpro compared to 1a, but 1b had no potency against MERS-CoV infection, which suggests that the modification in 1b may have resulted in poor delivery into the host cells. To further confirm their antiviral activity in the host cells, we also examined the replication of MERS-CoV upon the treatment of 1 or 1e by measuring the copy number of the RNA of the upE gene encoding the E protein of MERS-CoV by real-time quantitative RT-PCR. As shown in Figures 3D and 3E, in vivo potency of 1 and 1e against MERS-CoV was confirmed by an $80% decrease in mRNA levels of the upE gene upon treatment with 1 mM of 1 or 1e.
As MERS-PLpro is crucial in generating mature proteins including the nsp3 protein which is essential for iScience Article viral replication (Barretto et al., 2005;Lindner et al., 2005), we reasoned that 1 could be highly effective at an early stage of infection rather than the later stages. To test this hypothesis, we performed a time-of-addition experiment. We treated MERS-CoV infected cells with a compound: prior to infection (À1 h), at the time of infection (0), and post-infection (1-6 h) ( Figure 3F). Both 1 and 1e resulted in over 60% protection from the MERS-CoV infection only at an early stage of infection, while drug treatments 2-6 h post-infection had only marginal or negligible effects; and these results were similar to those obtained from two known antiviral compounds, lopinavir (LPV) (Choy et al., 2020) and chloropyridine ester (CE-5) (Ghosh et al., 2008), which are known to prevent the maturation of viral polyproteins. iScience Article Next, as we found through the in vitro PLpro assay that 1 and several of its analogs have potency against SARS-PLpro as well, we wanted to validate this activity by analyzing their efficacy in preventing SARS-CoV infection in a cell-based experiment. To this end, we adapted the same immunofluorescence-based infection assay used for the MERS-CoV infection model. We first treated the Vero cells with a compound for 2 h and then infected the cells with SARS-CoV at an MOI of 0.0625. Following a 2-day infection, the cells were subjected to immunofluorescence analysis using a SARS-CoV spike S1 antibody. As shown in Figure S4, 1e was found to have potency against SARS-CoV infection while 1 had only negligible activity. As these results suggested that 1e may have a broad-spectrum inhibitory effect on the PLpro of coronaviruses, we reasoned that 1e could be potent against other coronaviruses including HCoV-OC43 and HCoV-229E. To test this possibility, we treated 1e on HCoV-229E or HCoV-OC43-infected Vero cells and measured viral replication by quantifying the viral RNA. We found that 1e has a marginal potency at 10 mM on both HCoV-OC43 and HCoV-229E while 1 had no antiviral effect ( Figure S5). These results suggest that thiophene is capable of inhibiting the PLpro activity of a rather broad spectrum of coronaviruses.
Thiophene may interact with the catalytic cleft near the BL2 loop blocking the access of substrate to the catalytic site As thiophene does not have a naphthyl group, we wanted to gain insight into the binding properties of 1 on the target protein PLpro. To this end, we explored possible interactions by a computational molecular docking study. The PLpro protein has a right-handed palm-thumb-finger shape and contains an additional ubiquitin-like (UBL) domain at the N-terminus ( Figure 3G) Lei et al., 2014). Although MERS-PLpro and SARS-PLpro share high similarity in their structural architecture, MERS-PLpro recognizes Leu/Ile-X-Gly-Gly-Y-Arg/Glu-X while SARS-PLpro preferentially recognizes Lys over Glu, making its recognition motif Leu-X-Gly-Gly-Y-Arg(or Lys)-X. As reported (Ratia et al., 2008),  Figure 3H). We also performed an MDS to further validate the molecular docking, and we found that compound 1 was predicted to not bind to the catalytic site of MERS-PLpro but to bind to the region near the BL2 loop as the docking predicted ( Figures S6C and  S6D). This suggests that 1 may be a non-competitive inhibitor similar to the known inhibitor of SARS-PLpro, GRL0617. As reported, the BL2 loop plays crucial role in gating access to the catalytic triad, and in PLpro, the BL2 moves toward an open position upon substrate binding (Henderson et al., 2020;Ratia et al., 2008). The cleft region near the BL2 loop of SARS-PLpro binds to a non-competitive inhibitor, GRL0617, through T-shaped stacking ( Figure 3G), while interestingly, as shown in Figure 3H, our docking study predicted that 1 binds to a cavity branched from the cleft at the opposite side of the BL2 loop in a ''vertically pining shape (VPS).'' This cavity is not involved in the interaction of GRL0617 to SARS-PLpro, and notably, MERS-PLpro has different residues constituting the cleft region when compared to SARS-PLpro ( Figure 3G). These differences may provide structural restrictions, which might potentially contribute to the unique binding properties of MERS-PLpro and SARS-PLpro. In particular, Y269 of SARS-PLpro is missing in MERS-PLpro; instead, it has T274 which might not be able to form interactions with the naphthyl group of GRL0617 as the phenyl group of the tyrosine does. Also, in place of Q270 of SARS-PLpro which provides a hydrogen bond to GRL0167, MERS-PLpro has A275, which might further contribute to the difference in the binding shape of 1 to MERS-PLpro when compared to the binding shape of GRL0617 to SARS-PLpro. Collectively, our molecular docking study suggested that 1 could be a non-competitive inhibitor, and unlike GRL017, 1 was predicted to bind to the cleft near the BL2 of MERS-PLpro in a different mode of interaction generating a VPS.

Cyanofuran (2) and triazoloquinazoline (3) inhibit MERS-CoV PLpro selectively over SARS-CoV PLpro
Unlike thiophene (1), we found cyanofuran (2) and triazoloquinazoline (3) to be highly potent only against MERS-PLpro with no activity against SARS-PLpro (Figures 4 and 5). To identify the structural regions important for the affinity of 2 and 3 to PLpro, we tested the available analogs with important structural modifications (Figures 4A and 5A). For the 3-cyano-furan scaffold (2), we tested analogs with the modification of the chloride on the benzamide group with either fluorine (2b) or iodine (2c) and found that their potency remained unchanged. However, the introduction of trimethoxy (2d)  iScience Article is essential for its affinity to the target protein. Also, we tested an analog with para-chloride on the phenyl ring at the 5-position of the furan scaffold (2a), and its potency decreased by $40% which suggests that the phenyl group without any substituent is crucial for binding ( Figure 4B). To validate antiviral efficacy against the authentic virus, we examined the efficacy of 2 in cells infected by MERS-CoV, and it was found to be highly potent (IC 50 , 0.93 mM) as shown in Figures 4B, 4C, and S7. The in vitro efficacy of 2 was further validated by real-time RT-PCR analysis ( Figure 4D). Consistent with the PLpro assay, analogs of 2 showed comparable potency to 2 against MERS-CoV infection while their activity was lower. Interestingly, although 2d had no efficacy in the in vitro PLpro assay, it appeared to retain antiviral efficacy in a cell-based assay ( Figure 4B). Although we used a range of substrate concentrations to calculate the IC 50 of compound 2d in the in vitro PLpro assay, 2d might have efficacy at a concentration not included in the range tested. Next, we examined whether cyanofuran (2) is as effective at an early stage of infection as thiophene. As shown in Figure 4E, 2 shows a high potency only when added at the beginning of the MERS-CoV infection (À1 h, 0 h) comparable to the effect of chloroquine. Lastly, we performed molecular docking as shown in Figure 4F, and molecular docking of 2 into MERS-PLpro revealed that it was predicted to interact with the key residues of the cleft of MERS-PLpro including Y269 but in a different direction compared to the SARS-PLpro inhibitor, GRL0167. Like compound 1, compound 2 was calculated to bind to the cavity branching from the cleft of MERS-PLpro by interacting more significantly with the  S8B). Also, in MDS performed with compound 2 and MERS-PLpro, we found that compound 2, similar to compound 1, was predicted to not bind to the catalytic site of MERS-PLpro although it was predicted to bind to a region upper than the region predicted by molecular docking (Figures S8C  and S8D).
In the case of triazoloquinazoline (3), we tested a total of 15 available analogs as shown in Figure 5A. First, we examined several analogs with modifications in the region containing the benzamide moiety. Replacing this region significantly affected potency: 3a-9 with the modification of a simple carboxylic acid at this site decreased the potency by 3-fold; 3a-4 with an N-isopropyl group resulted in a 4-fold decrease; 3a-2 with a thiophene moiety had further decreased efficacy; and 3a-3 with a substituted cyclohexyl group resulted in a complete loss of potency ( Figure 5B). This data suggests that the benzamide group may bind to the limited space of the cleft of MERS-PLpro. Next, we tested several analogs with modifications on the meta-fluorophenyl ring at the 3-position of the triazole scaffold, and none, except for 3c-1, showed efficacy. Finally, we also tested an analog of 3b with a para-methoxy phenyl ring at the 3-position of the triazole scaffold, and no efficacy was found. To examine whether triazoloquinazoline analogs have antiviral activity, we tested all analogs against MERS-CoV infection ( Figures 5B, 5C, and S9), and most compounds that showed no efficacy in the PLpro assay showed no efficacy in the cellbased assays, as expected. Interestingly, although 3a-5, 3a-6, and 3a-7 have decreased in vitro potency on MERS-PLpro, their efficacy in the cell-based assay appeared to remain comparable to 3. These results suggest that the region may be important for their delivery into the effective locale in the host cell. In the case of the analog 3a-8 with a meta-methoxy phenyl ring, its potency in the MERS-PLpro assay increased more than 4-fold, however, antiviral efficacy was not detected by the in vivo cell-based assay. Together iScience Article with the 3a-5, 3a-6, and 3a-7 results, this suggests that the phenyl ring might play crucial roles in the potency of triazoloquinazoline in the host cell. Like 1 and 2, the effectiveness of 3 was confirmed using real-time quantitative RT-PCR ( Figure 5D). To obtain molecular insight into possible molecular interactions of 3, molecular docking was performed. As shown in Figure 5E, 3 was predicted to bind through a VPS to an upper region of the cavity further away from the BL2 loop, when compared to 2 and 3, and similar to compounds 1 and 2, compound 3 did not bind to the catalytic site (Figures S10A and S10B). Also, in the MDS analysis of compound 3 and MERS-PLpro, compound 3 was predicted to not bind to the catalytic pocket of MERS-PLpro but to bind to the region near BL2, which is similar to the result generated by the docking analysis (Figures S10C and S10D). Our docking study and MDS suggest that the cavity and its upper regions may contribute to the selectivity of compounds to MERS-PLpro and SARS-PLpro. Importantly, we found that the introduction of mutations in two residues T251 and T310 of MERS-PLpro, specifically substitution with alanine, resulted in a complete loss of expression in the case of the T310A mutant and T310A-T251A double mutant or a significant loss (>50%) of the enzyme activity in the case of the T251A mutant ( Figure S11A). When we tested three compounds with T251A mutant, compound 1 showed no potency at a tested concentration (0-50 mM), and the potency of compounds 2 and 3 was decreased significantly (Figures S11B-S11D). These results suggest that T251 plays a crucial role in the interaction of MERS-PLpro with our compounds, especially compound 1 that shows potential as a broad-spectrum PLpro inhibitor. Importantly, these results are in line with those of our molecular docking studies where compound 1 was expected to bind to the cleft most near to the BL2 loop whereas compounds 2 and 3 which were expected to bind to the region further away from the BL2 loop did not show a significant reduction in interaction with MERS-PLpro T251A. In total, our results demonstrate that the residues near the gating of the BL2 loop are crucial for both activity and structural stability of the protease. Therefore, residues near the loop including T251 and T310 can be effective druggable targets.

Inhibitors show potency against SARS-2-PLpro and SARS-CoV-2 infection
Owing to the significance of the recent pandemic caused by SARS-CoV-2, we wanted to test if the selected compounds are efficient in SARS-CoV-2 infection. As shown in Figures 6A-6C, the compounds iScience Article show a marginal potency against SARS-2-PLpro, and the IC 50 of these compounds was barely calculated in the range of tested concentrations. In addition, similar to MERS-CoV and SARS-CoV, we adapted an immunofluorescence-based viral infection assay in cells . The assay conditions were validated with the known antiviral drug remdesivir. Treatment with remdesivir resulted in the depletion of the SARS-CoV-2 fluorescence signal in the host cells ( Figure S12). Interestingly, when we examined these compounds in a cell-based infection model for SARS-CoV-2, we found that they showed potency at a concentration below their cytotoxicity although their IC 50 was over 10-fold lower than those of MERS-CoV infection (Figures 6D-6F). To further validate the potency, we performed a cleavage assay using a 67 kDa protein substrate containing the 10 amino acids at the junction of nsp 2-3 with a cleavage site. Upon PLpro-mediated cleavage at this site, products of two sizes, 25 and 42 kDa, are generated. As shown in Figure 7A, we first confirmed that our recombinant SARS-2-PLpro cleaved the substrate and produced two fragments. When we treated the reaction with the three test compounds, marginal inhibition of SARS-2-PLpro cleavage was observed at the high concentration of 200 mM ( Figure 7B).

Conclusions
We identified novel PLpro inhibitors with no naphthalene group, which is important for the binding of GRL0617 to SARS-PLpro; and we found by molecular docking that each compound has unique binding properties toward the target protein contributing to target selectivity. Like GRL0617, all three inhibitors were predicted to be non-competitive inhibitors, as in our molecular docking study and MDS they do not fit on the catalytic triad but instead fit on the cleft near the BL2 loop. This result suggests that the BL2 loop is an important target for the treatment of coronaviruses. Furthermore, each class of coronaviruses shows variation in key residues constituting the cleft and the region near the BL2 loop; and this variation can provide structural restriction for accessing the BL2 loop thereby resulting in target specificity. Our compound 1 was found to inhibit both MERS-PLpro and SARS-PLpro/SARS-2-PLpro, while 2 and 3 were selective toward MERS-PLpro. We note that the expected binding of 1 to the target can provide information on structurally important scaffolds for the development of broad-spectrum inhibitors for the PLpro of coronaviruses. Unlike 1, our docking study revealed that 2 and 3 were expected to bind to a cavity branching from the cleft away from the BL2 loop of MERS-PLpro. This binding property can therefore provide insight to develop MERS-PLpro-specific inhibitors. Vaccines are prophylactic and are readily available in limited countries. Currently, in addition to neutralizing antibodies, several non-specific medications including the combinatory treatment of lopinavir-ritonavir and interferon-b1b have been used in the clinic. However, their efficacies can be compromised. Therefore, in view of an urgent need to develop therapeutics targeting viral proteins, our compounds may be promising candidates for drug development.

Limitations of the study
In our present study, we have identified novel structural classes of MERS-PLpro inhibitors, and in our docking analysis and MDS, we have shown that these compounds interact with the region near the BL2 loop of PLpro. Importantly, we found that the mode of binding to the region near the BL2 loop is essential for broad-spectrum potency, which was validated with a cell-based infection model including SARS-CoV-2. iScience Article However, our study could expand further; although we expect that our compounds could be further engineered as a therapeutic option against SARS-CoV-2, we have no animal data required for pharmaceutical deferment. In addition, performing co-crystallography using our compounds and the PLpro could further validate the mode of action of our inhibitors.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Materials availability
This study did not generate new unique reagents.
Data and code availability d All data reported in this paper will be shared by the lead contact upon request.
d This paper does not report original code.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Virus strains
Patient-derived MERS-CoV (passage 4, MERS-CoV/KOR/KNIH/002_05_2015; GenBank: KT029139.1) was provided by the Korea Centers for Disease Control and Prevention. The SARS-CoV (strain HK39849) was kindly provided by Prof. JSM Peiris from the University of Hong Kong. SARS-CoV-2 for this study was provided by the National Culture Collection for Pathogens (NCCP43326). Human coronavirus 229E and OC43 (HCoV-229E and HCoV-OC43) were purchased from ATCC. The viral strains are also listed in KRT. All experiments with MERS-CoV, SARS-CoV, and SARS-CoV-2 were performed in BSL-3 facility at Institut Pasteur Korea following the guidelines of the Korea National Institute of Health (KNIH) using the BSL-3 protocol approved by the KCDC.

Chemical compounds and analogs
A 29,938-compound library originated from an in-house library was used for the high-throughput screen. The library consists of 16,960 compounds selected from the Chembridge library on the basis of quantitative estimates of drug-likeness (QED) scores, 3,200 compounds from the antiviral library (ChemDiv), 4,480 from the protease inhibitor library (ChemDiv or Life Chemicals), 3,040 from the natural product standard library (Analyticon or Chemstrong), 1,280 from the LOPAC library (Sigma-Aldrich), and 978 from the FDA-approved library (Selleckchem iScience Article of 10 mM in DMSO and the natural products were at that of 2 mg/mL in DMSO. RLRGG-AMC peptides were purchased from Bachem. Selected compounds for the analog study were purchased as described in Figure S13.
The introduced mutations were verified by sequencing. Then, the plasmids were transformed into the T7 express lysY/Iq competent E. coli strain (NEB). Ampicillin-resistant colonies were grown at 37 C and induced by the addition of 0.5 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) at OD $0.7. After additional culture for 14 h at 20 C, cells were harvested by centrifugation (5,000 g, 10 min) and stored at À70 C. Cell pellets were resuspended in binding buffer (50 mM Tris-Cl, pH 8.0, 500 mM NaCl, 5% glycerol, 20 mM imidazole, and 5 mM b-mercaptoethanol) and lysed by sonication using a Sonic Dismembrator (Fisher Scientific). The cleared lysate was subjected to affinity chromatography using Ni Sepharoseâ 6 Fast Flow beads (Cytiva). Stepwise gradient elution was carried out using the elution buffer with increasing concentrations of imidazole (50-500 mM). Fractions containing pure C-terminal hexahistidine-tagged PLpro proteins (>95%) were pooled and dialyzed against the storage buffer containing 20 mM Tris-Cl, pH 8.0, 10% glycerol, 100 mM NaCl and 5 mM b-mercaptoethanol, followed by concentration using Amiconâ Ultra-15 centrifugal filter units (Merck). The purity and concentration of proteins were determined by SDS-PAGE and a NanoDrop spectrophotometer, respectively.

Fluorescence-based PLpro protease assay
The general procedures were performed as described (Lee et al., 2015;Ratia et al., 2008). To optimize the MERS-PLpro protease assay conditions, varying concentrations of Z-RLRGG-AMC substrate (0.8-100 mM) diluted in assay buffer containing 50 mM HEPES, pH 7.5, 0.01% Triton X-100, 0.1 mg/mL BSA and 5 mM dithiothreitol (DTT) were mixed with varying concentrations of purified MERS-PLpro (0.3-10 mM) diluted in the reaction buffer. Reaction mixtures with a total volume of 40 mL were added to each well of a 384-well non-binding black plate (Greiner Bio-one). Following 1 h incubation at room temperature, fluorescence intensity was monitored at 360 nm (excitation) and 450 nm (emission) using a FlexStationâ 3 microplate reader (Molecular Device). The data were plotted using a Prismâ 6 analysis tool (GraphPad Software). For optimization of the SARS-PLpro protease assay, all the procedures were the same except for the enzyme concentration range (0.02-0.63 mM) and buffer composition (2 mM reduced L-glutathione instead of 5 mM DTT). The 50% inhibitory concentration (IC 50 ) of a reference compound (6-TG) was determined in 40 mL reaction mixtures containing 40 mM Z-RLRGG-AMC substrate, 2.5 mM MERS-PLpro or 0.02 mM SARS-PLpro/SARS-2-PLpro and varying concentrations of 6-TG compound (1.953-1,000 mM), which were subjected to the same measurement and analysis procedures mentioned above. For determination of efficacy of test compounds on MERS-PLpro T251A, 0-50 mM of compounds and final concentrations of 2.5 mM and 3.125 mM were used for the enzyme and AMC, respectively. For determination of efficacy of test compounds on SARS-2-PLpro, 0-200 mM of compounds and final concentrations of 0.625 mM and 3.125 mM were used for the enzyme and AMC, respectively. The assay was performed in the same assay buffer as for MERS-PLpro.

Primary high-throughput screening
The 29,938-compound library was screened against MERS-PLpro. The screen was carried out in monoplicate. Twenty microliters of MERS-PLpro enzyme (2 mM final concentration) diluted in the assay buffer was added to the wells to which 30 mL of the compounds were dispensed using a JANUS Automated Workstation (Perkin-Elmer). Then, 10 mL of Z-RLRGG-AMC (40 mM final concentration) diluted in the assay buffer was added to initiate the enzyme reaction, after which the assay plates were incubated at room temperature for 1 h. Fluorescence intensity was measured in the same manner as mentioned above. The screening data were processed by the Gyeonggi Bio-center Assay Management System (GAMS) derived from the Pipeline Pilot protocols (Accelrys iScience Article Technology) at 1:1,000 dilution and rabbit anti-B actin antibody (Cell Signaling Technology) at a dilution of 1:5,000 for loading control. Secondary antibodies were HRP-linked horse anti-mouse IgG (Cell Signaling Technology) and goat anti-rabbit IgG (Cell Signaling Technology) antibodies. Immunoreactivity was detected using SuperSignal West Pico PLUS chemiluminescent substrate (Fisher Scientific). Protein bands were visualized by Chemidoc XRS (Bio-Rad) and intensities of protein bands were quantified using ImageJ.

Gel-based polyprotein cleavage assay
The gel-based polyprotein cleavage assay was performed as previously described (Lim et al., 2021). TLKGG//APTKV (nsp2/3 junction) used as a substrate was modified by attaching the GST-tag and MBPtag to the N-and C-terminus, respectively (Addgene ID 169195) after which the constructs were expressed in BL21(DE3). The protein was purified as described above in PLpro protease expression and purification section. The enzyme was pre-incubated at a concentration of 1 mM with the selected inhibitor compounds for 10 min. Gel-based assay substrates were added at a concentration of 5 mM and incubated at RT for 24 h in an assay buffer (50 mM HEPES, pH 7.6, 2 mM DTT, 10% glycerol, 0.02% Tween-20) before being run through a gel.

Molecular docking study
Protein sequences and their structure information (SARS-PLpro PDB: 3E9S (Ratia et al., 2008), MERS-PLpro PDB: 4RF1 (Bailey-Elkin et al., 2014)) were downloaded from the Protein Data Bank followed by the removal of water molecules and metal ions using the Schrodinger (v. 2019-2) protein preparation wizard. Test compounds were drawn using the Schrodinger 3D builder and further converted to ligands using the ligand preparation wizard. For docking, the grid box was created using the Schrodinger receptor grid generation wizard with the inner box size set to 16 3 16 3 40 and outer box size set to 52 3 52 3 76. Docking was performed using the Schrodinger XP Glide ligand docking tool, and the generated images were further modified to indicate the important residues and structures.

Molecular dynamics simulation
To evaluate the stability of the molecular docking of three compounds with MERS-PLpro, molecular dynamics simulation (MDS) was performed using Cresset's Flare 6.0.1. The MDS was initiated using the molecular docking poses of each compound. Under the Amber force field (version GAFF2), charges were calculated by the AM1-BCC method (Jakalian et al., 2002). A solvent system of MDS was built by using a truncated octahedron shape box. Explicit SPC/E water and two chloride molecules were used for solvent and neutralization, respectively. For the equilibration, the default values of each parameter were used. Each simulation was performed for 80 ns at the 300K and 1 bar using a graphics processing unit (GPU) platform. To measure the stability of the protein-ligand complex, the root mean square deviation (RMSD) was calculated every 10 ps.

QUANTIFICATION AND STATISTICAL ANALYSIS
Student's t-test was performed for statistical analyses between experimental groups using Prismâ 6 software. The results were expressed as the mean GSEM or GSD for duplicate, triplicate, or quadruple assays. Differences between groups were inferred as significant for p < 0.05. Statistical details of individual experiments can be found in the figure legends.