The complex structure of GRL0617 and SARS-CoV-2 PLpro reveals a hot spot for antiviral drug discovery

SARS-CoV-2 is the pathogen responsible for the COVID-19 pandemic. The SARS-CoV-2 papain-like cysteine protease (PLpro) has been implicated in playing important roles in virus maturation, dysregulation of host inflammation, and antiviral immune responses. The multiple functions of PLpro render it a promising drug target. Therefore, we screened a library of approved drugs and also examined available inhibitors against PLpro. Inhibitor GRL0617 showed a promising in vitro IC50 of 2.1 μM and an effective antiviral inhibition in cell-based assays. The co-crystal structure of SARS-CoV-2 PLproC111S in complex with GRL0617 indicates that GRL0617 is a non-covalent inhibitor and it resides in the ubiquitin-specific proteases (USP) domain of PLpro. NMR data indicate that GRL0617 blocks the binding of ISG15 C-terminus to PLpro. Using truncated ISG15 mutants, we show that the C-terminus of ISG15 plays a dominant role in binding PLpro. Structural analysis reveals that the ISG15 C-terminus binding pocket in PLpro contributes a disproportionately large portion of binding energy, thus this pocket is a hot spot for antiviral drug discovery targeting PLpro.

T he COVID-19 pandemic has caused devastating damage to the world and it has resulted in over 12 million confirmed cases and over half a million deaths as of July 14, 2020 1 . The novel SARS-CoV-2 coronavirus is the etiological agent responsible for the pandemic, and it belongs to the beta coronavirus family [2][3][4] . Similar to the two beta coronaviruses, SARS and MERS, which have caused pandemic or epidemic in human history, the novel SARS-CoV-2 also causes severe acute respiratory syndromes 5,6 . Unexpectedly, SARS-CoV-2 has been reported to have more mild symptoms but much higher transmission rate 7,8 , therefore it has caused the biggest catastrophe to the world healthcare since the Spanish flu in 1918-1920 9 . Four previously deemed promising antiviral drugs, i.e., Remdesivir, Hydroxychloroquine, Lopinavir, and Interferon, showed no or little effect on hospitalized COVID-19 patients, in WHO global solidarity clinical trials 10 . Encouragingly, the UK started rollout of an mRNA vaccine developed by Pfizer/BioNtech in early December 2020, with its long-term safety and efficacy to be assessed. Meanwhile, progress has been made in the discovery of antibodies for COVID-19. Therefore, anti-SARS-CoV-2 drugs are urgently needed.
As a positive strand RNA virus, SARS-CoV-2 encodes two functional proteases, i.e., the papain-like protease (PLpro) and the 3-chymotrypsin-like cysteine protease (Mpro or 3CLpro). The major function of Mpro is to cleave the viral polyproteins, which is critical for virus maturation, replication, and invasion. Several potent Mpro covalent inhibitors have been reported and their cocrystal structures provided potential opportunities for structurebased drug optimization [11][12][13][14] . SARS-CoV PLpro is a cysteine protease with multiple major functions, including processing of the viral polyprotein chain for viral protein maturation, dysregulating host inflammation responses through deubiquitylation, and impairing the host type I interferon antiviral immune responses by removing interferon stimulated gene 15 (ISG15) modifications [15][16][17] . ISG15 modification (ISGylation) is the covalent conjugation of ISG15 protein (MW = 17.1 kDa) to protein substrates and this process is known to inhibit virus replication [18][19][20] . SARS-CoV-2 PLpro (MW = 35.6 kDa), sharing 83% sequence identity with SARS-CoV PLpro, contains an Nterminal ubiquitin-like (UBL) domain and a C-terminal ubiquitin-specific protease (USP) domain with implicated catalytic functions of cleaving ubiquitin (Ub) or ISG15 modifications from host proteins 21 .
Besides Mpro, PLpro has been considered another potentially promising target for drug discovery to treat COVID-19 22 . Due to the urgent need of therapies for the pandemic, the strategy of repurposing approved drugs or optimizing new compounds has been employed to fight COVID-19 14,[23][24][25] . Accordingly, we describe our efforts in screening of a compound library of drugs approved by FDA or CFDA (China FDA) against SARS-CoV-2 PLpro, and structural characterization of the interactions between a promising drug lead and PLpro. The co-crystal structure of PLpro in complex with the compound GRL0617 and its antiviral effect provided direct proof of druggability of PLpro and the mechanism of action of the compound. Further structural and biophysical analysis reveals that the C-terminus of ISG15 plays a dominant role in its binding with PLpro through extensive hydrogen bonds and electrostatic interactions. Therefore, the ISG15-C-terminus binding cleft in PLpro is a hot spot for antiviral drug discovery.

Results
High-throughput screening of a library of approved drugs against PLpro. To repurpose existing drugs to inhibit the SARS-CoV-2 PLpro, we initiated screening of a 2040-compound library of drugs approved by FDA or CFDA against SARS-CoV-2 PLpro (Supplementary Table 1). First, we set up a FRET assay to characterize the enzymatic activity of SARS-CoV-2 PLpro based on an established assay for SARS-CoV PLpro 26 . The recombinant full-length SARS-CoV-2 PLpro protein was expressed in Escherichia coli, and subsequently purified using his-tag chromatography and size exclusion chromatography ( Supplementary  Fig. 1). A commercially available fluorogenic peptide substrate Arg-Leu-Arg-Gly-Gly-AMC (RLRGG-AMC), representing the Cterminal residues of ubiquitin, was used to report the enzymatic activity of PLpro. The first round of screening provided~30 compounds with over 50% inhibition at 100 μM. Because the FDA approved drug Tioguanine (6-TG) has been previously tested on SARS-CoV and MERS PLpro proteins with effective inhibitions 27,28 , we determined its potency against SARS-CoV-2 PLpro and the IC 50 value was 72 ± 12 μM, so we used it as a positive control throughout the screening. Hits from the first round of screening went into the second round of validation using the same enzymatic assay. After removing compounds with poor solubility, strong reactivity, or high intrinsic fluorescence, seven relatively potent compounds including 6-TG were measured for IC 50 . These seven drugs showed modest IC 50 values ranging from 29 to 91 μM ( Supplementary Fig. 3). Although these compounds can potentially provide a starting point for further optimization, their low potency implies a need of large amounts of resources and time input.
Identification of GRL0617 as an inhibitor for SARS-CoV-2 PLpro. Parallelly, we cherry-picked GRL0617 and its analog compound 6 from promising SARS-CoV PLpro inhibitors 26,29 based on high sequence identity between the SARS-CoV and SARS-CoV-2 PLpro proteins ( Supplementary Fig. 2). The in vitro IC 50 values of GRL0617 and compound 6 against SARS-CoV-2 PLpro were 2.1 ± 0.2 μM and 11 ± 3 μM, respectively (Fig. 1a). The compound 6, as an acetamide derivative of GRL0617, did not show improved potency in the in vitro FRET assay. Our data suggested that GRL0617 is a promising lead compound and therefore it was subjected to further antiviral, structural, and mechanistic studies. Our identification of GRL0617 and its analogs along with their potencies are in line with recent studies by the Pegan group 30 , the Dikic group 31 , and the Komander group 32 .
Inhibition of the in-cell deubiquitinating and deISGylating activity of PLpro by GRL0617. To assess whether GRL0617 can inhibit the in cyto deubiquitinating and deISGylating activity of SARS-CoV-2 PLpro, we transfected HEK293T cells with plasmids of PLpro and the ISGylation machinery (Ube1L, UbcH8, HECR5, and ISG15) and then treated with GRL0617 at different concentrations for 24 h. Our data (Fig. 1b) showed that the deubiquitinating activity of SARS-CoV-2 PLpro is weak but its deISGylating activity is relatively strong, which is consistent with recent publications [30][31][32][33] . SARS-CoV-2 PLpro is capable of reversing the ISGylation and polyubiquitination (to a much lesser extent) of cellular substrates (Fig. 1b). Furthermore, addition of GRL0617 caused inhibition of SARS-CoV-2 PLpro and resulted in partially recovered poly-ubiquitin-conjugates and ISG15conjugates (Fig. 1b). Moreover, we further used interferon β (IFN-β) to induce the ISGylation in HEK293T cells which generated a remarkable amount of ISG15-conjugated cellular substrates (Fig. 1c, lanes 1 and 2). The addition of 160 μM of GRL0617 alone to cell lysate had no observable effect to ISGylation (Fig. 1c, lanes 2 and 3), which demonstrated the selectivity of the compound over other deISGylating enzymes in cells, such as USP18. Indeed, GRL0617 showed no inhibition effect on the recombinant mouse USP18 protein ( Supplementary Fig. 5g), which is consistent with other studies 26,31 . Further addition of purified SARS-CoV-2 PLpro (100 nM) to cell lysate efficiently reduced ISG15-conjugated proteins (Fig. 1c, lanes 2 and 4). In contrast, addition of GRL0617 to cell lysate together with PLpro recovered the ISGylation in a dose-dependent manner (Fig. 1c, lanes 5-9) with significant band recovery seen at 40 μM of GRL0617. Clearly, GRL0617 inhibited the deISGylation activity of PLpro through an on-target effect.
Antiviral activity of the inhibitor GRL0617. To further validate the potential of PLpro as an antiviral drug target, we tested GRL0617 for its inhibitory activity in Vero E6 cells infected with SARS-CoV-2 at a multiplicity-of-infection (MOI) of 0.01. The mRNA copy numbers of the viral spike protein were monitored to evaluate antiviral activity of the compound. Based on the dosedependent response, GRL0617 showed a clear inhibition of viral replication and 100 μM of GRL0617 resulted in over 50% inhibition. No apparent cytotoxicity on Vero E6 cells was observed in our assay with concentrations up to 100 μM (Fig. 1d). The cytopathic effect (CPE) analysis revealed an EC 50 of 21 ± 2 μM (Fig. 1e). This in cyto antiviral potency of GRL0617 is also in line with other recent studies 30,31 .
The co-crystal structure of SARS-CoV-2 PLpro in complex with GRL0617. A co-crystal structure would be crucial to understand the mechanism of inhibition of SARS-CoV-2 by GRL0617, therefore we set out to solve the co-crystal structure. Fig. 1 Inhibitory activity of GRL0617 against SARS-CoV-2 PLpro. a The inhibitory activity of GRL0617 and compound 6 against PLpro was measured using the peptide RLRGG-AMC as a substrate. IC 50 was presented as mean ± SEM, n = 3 independent experiments. b In-cell deISGylating (left) and deubiquitinating (right) activities of PLpro, HEK293T cells were transfected for 24 h with plasmids encoding GFP-PLpro, ISG15, and E1(Ube1L)/E2(UbcH8)/ E3(HECR5) enzymes, alone or in combination. Cells were treated for an additional 24 h with indicated concentrations of GRL0617. Cell lysates were subjected to immunoblotting with anti-ubiquitin, anti-ISG15, and anti-GFP antibodies. GAPDH served as a loading control. A representative from three independent experiments is shown. c HEK293T cells were treated with or without 500 U/mL interferon β (IFN-β) for 48 h. The cell extracts were incubated with purified recombinant PLpro (100 nM) and indicated concentrations of GRL0617 for 60 min at 37°C, followed by immunoblotting analysis with anti-ISG15. A representative from three independent experiments is shown. d Antiviral activity of GRL0617 on SARS-CoV-2 and the cytotoxicity of GRL0617 on Vero E6 cells. Vero E6 cells were infected with SARS-CoV-2 using a multiplicity-of-infection (MOI) of 0.01. The quantification of absolute viral RNA copies (per mL) in the supernatant at 48 h post-infection was determined by qRT-PCR analysis. The cytotoxicity of GRL0617 on Vero E6 cells was measured using CCK8. All data are shown as mean ± SEM, n = 3 independent experiments. e EC 50 was presented as mean ± SEM, n = 3 independent experiments, the cytopathic effect (CPE) analysis revealed an EC 50 of 21 ± 2 μM. However, the crystals of wild-type SARS-CoV-2 PLpro were difficult to grow. Therefore, we turned to grow co-crystals of SARS-CoV-2 PLpro C111S in complex with GRL0617 by incubating the compound with the protein before setting up crystal trays. The obtained co-crystal diffracted at 3.2 Å (Fig. 2a-e and Supplementary Table 2). When our manuscript was in the review process, a few groups also reported the co-crystal structures of SARS-CoV-2 PLpro and GRL0617 or its analogs 34,35 . The crystal of PLpro/GRL0617 belongs to the space group I4 1 22 with one protein molecule in each asymmetric unit. SARS-CoV-2 PLpro has two domains, i.e., the N-terminal UBL domain and the Cterminal USP domain (Fig. 2a). Based on the B factor analysis, the palm and thumb regions in the USP domain have lower B factor values than the UBL domain and the fingers region of the USP domain, indicating that the palm and thumb regions are relatively more rigid than rest of the structure. As shown in the 2Fo-Fc electron density map (Fig. 2a, b) as well as in the difference Fo-Fc electron density map ( Supplementary Fig. 4b), GRL0617 resides in a pocket in the palm region of PLpro. GRL0617 is apart from the catalytical triad (including S111 in place of C111, H272, and D286) of PLpro with a minimum distance of 7.5 Å to S111 (from the methyl of the 4-Methylbenzenamine moiety of GRL0617 to the sidechain oxygen of S111). Therefore, GRL0617 inhibits SARS-CoV-2 PLpro in a non-covalent manner. The GRL0617bound PLpro structure is overall similar to the available apostructure of PLpro C111S (PDB 6WRH) with a backbone RMSD of 0.76 Å, except for two residues on the BL2 loop, i.e., Y268 and Q269 (Fig. 2d). Upon binding to GRL0617, the sidechains of Y268 and Q269 shifted toward GRL0617 to form polar and hydrophobic interactions with the compound and stabilized its binding (Fig. 2d). Specifically, the sidechain oxygen of Y268 forms a hydrogen bond with the amino group on the benzene ring of GRL0617, and another hydrogen bond of the backbone amino group of Q269 with the carbonyl oxygen of GRL0617 (Fig. 2b,   include the hydrogen bonds between D164 and the amide NH of GRL0617, as well as between Y264 and the carbonyl oxygen of GRL0617. In addition, hydrophobic integration also contributed to the binding of GRL0617 to PLpro, e.g., the naphthalene group of GRL0617 is involved in the interactions with aromatic residues Y264 and Y268, and the hydrophobic sidechains of P247 and P248 (Fig. 2b, c). Since GRL0617 is capable of inhibiting both SARS-CoV and SARS-CoV-2, it is of interest to understand its selectivity on coronaviral PLpro proteins from a structural biology perspective. It has been reported that naphthalene-based compounds have low to zero potency toward MERS PLpro 36,37 . The superposition of GRL0617 on a surface model of the MERS PLpro structure 37 indicated that the original pocket in MERS PLpro might be too shallow to allow GRL0617 to bind with extensive contacts, and the naphthalene moiety of GRL0617 would also be in a steric clash with T249 of MERS PLpro (Supplementary Fig. 5c). In contrast to SARS and SARS-CoV-2, the BL-2 loop of MERS is one residue longer, but it lacks the critical Y268 of SARS-CoV-2 which played a critical role in encircling GRL0617 in the cocrystal structure ( Supplementary Fig. 2). The extra residue of MERS PLpro may rearrange the hydrogen-bond interaction network of the BL2 loop and the lack of the aromatic tyrosine clearly resulted in the removal of the T-shaped π-π stacking and van der Waals interactions with the naphthalene group of GRL0617 ( Fig. 2e and Supplementary Fig. 5a-e). Our enzymatic assay confirmed the lack of inhibition of MERS PLpro by GRL0617 or compound 6, which is consistent with our structural analysis ( Supplementary Fig. 5f) and a recent study 31 .

SARS-CoV-2-PLpro C111S apo SRAS-CoV-2-PLpro
GRL0617 is a protein-protein interaction (PPI) inhibitor revealed by NMR. Because GRL0617 is a non-covalent inhibitor binding in the USP domain, i.e., the catalytic domain of SARS-CoV-2 PLpro, it is of interest to see if GRL0617 would disrupt the interactions between ISG15 or Ub and PLpro. Solution state nuclear magnetic resonance (NMR) was employed to characterize the binding of ISG15 or Ub to PLpro as well as the perturbation of their bindings by GRL0617. 2-D NMR 1 H, 15 N-HSQC spectrum of 15 N-ISG15 (0.1 mM) showed typical features for a wellfolded protein with well-dispersed cross peaks (Fig. 3a). The addition of 0.15 mM SARS-CoV-2 PLpro into 0.1 mM 15 N-ISG15 caused drastic peak broadening and peak intensity loss, which is a characteristic of PPIs in the intermediate chemical exchange regime (Fig. 3b). Increasing concentrations of GRL0617 were added into the mix of 0.1 mM 15 N-ISG15 and 0.15 mM SARS-CoV-2-PLpro, a dose-dependent response of peak intensity recovery was evident, which suggested that GRL0617 competes with ISG15 for the binding site in PLpro, and blocks the binding of ISG15 to PLpro (Supplementary Fig. 6a). The superposition of the HSQC spectra of 0.1 mM 15 N-ISG15 only and the 0.1 mM 15 N-ISG15/0.15 mM PLpro/0.25 mM GRL0617 mixture showed that these two spectra are essentially identical ( Supplementary  Fig. 6c), which indicated that GRL0617 is a potent binder to PLpro and almost completely abolished the binding of ISG15 to PLpro at a molar ratio of 1.67 (0.25 mM/0.15 mM). No peak shifting was observed in the superimposed HSQC (Supplementary Fig. 6b), suggesting that GRL0617 is a bona fide binder of PLpro rather than ISG15 because the HSQC spectrum of 15 N-ISG15 is not disturbed at all by 2.5 excess molar ratio (0.25 mM/ 0.10 mM) of GRL0617.
In comparison with the available complex structure of SARS-CoV-2 PLpro with Ub 32 (PDB ID: 6XAA [https://doi.org/ 10.2210/pdb6xaa/pdb]) (Fig. 3f) (Fig. 3e), GRL0617 binds in the S1 site (for binding the C-terminal lobe of ISG15) and blocks the access of the C-terminal tail of proximal Ub or ISG15 to the active site of SARS-CoV-2 PLpro, respectively. Titrations of PLpro into 15 N-Ub caused minimal peak shifting or peak broadening even at a high molar ratio of 3 ( Supplementary Fig. 7), showing much weaker binding for PLpro with monoUb compared with ISG15. Consequently, GRL0617 was not further titrated into the 15 N-Ub/ PLpro mixture. Taken together, our NMR and X-ray analysis indicate that GRL0617 is a potent PPI inhibitor for PLpro by blocking the binding of ISG15 to PLpro.
The C-terminus of ISG15 is dominant for ISG15/SARS-CoV-2 PLpro binding. As seen in Supplementary Fig. 6c, superimposed NMR spectra indicated an almost complete disruption of interactions of the N-and C-UBL domains of ISG15 with PLpro. It is intriguing that GRL0617 actually only blocked the binding of the C-terminal tail of ISG15, but it efficiently abolished the binding of both the N-and C-globular UBL domains of ISG15 with PLpro. Therefore, GRL0617, as a small-molecule compound (MW = 304.3), occupies a cleft near the active site and exerts a dominant negative effect for the rest of ISG15 binding to SARS-CoV-2 PLpro.
To further confirm the important role of the C-terminal tail of ISG15, we generated a truncated construct ISG15-ΔC6 (removing the C-terminal LRLRGG). As shown in the superimposed 1 H, 15 N-HSQC spectra of 15 N-ISG15-FL and 15 N-ISG15-ΔC6 ( Supplementary Fig. 8), ISG15-ΔC6 has a fold similar to the fulllength ISG15 (ISG15-FL). However, the superimposed 1 H, 15 N-HSQC also indicates that the interactions between ISG15 and SARS-CoV-2 PLpro were abolished by removal of the C-terminus of ISG15, as minimal peak shifting or peak broadening were observed for 15 N-ISG15-ΔC6/SARS-CoV-2 (molar ratio = 1:1.5) as compared with the massive peak broadening for 15 N-ISG15-FL/SARS-CoV-2 (molar ratio = 1:1.5) (Fig. 4a, b). Isothermal titration calorimetry (ITC) experiments confirmed that removal of the C-terminus of ISG15 is detrimental to the binding of ISG15 and SARS-CoV-2 PLpro (Fig. 4c) 32 , respectively. Further structural analysis of the human ISG15 C-UBL-PA/SARS-CoV-2 PLpro reveals that the PLpro backbone of L162, G163, Y268, C270, and G271 as well as sidechains of D164, R166, E167, and Y264 were involved in the interactions with the C-terminus of ISG15 through hydrogen bonds and electrostatic interactions. (Fig. 4d). Two mutants D164A and E167A were subsequently generated, and their enzymatic activities of cleaving Ub tags or ISG15 tags were examined using Ub-AMC or ISG15-AMC, respectively. Both D164A and E167A showed impaired activity on Ub and ISG15 cleaving (Fig. 4e). The cleavage of peptide substrate RLRGG-AMC also indicated diminished enzymatic activities for these mutants. These two mutants did not show observable activity on Ub-AMC at 1 μM substrate concentration, presumably because SARS-CoV-2 PLpro cleaves Ub-AMC almost 10-fold less efficiently than ISG15-AMC 30 .
The extensive interaction network between ISG15 C-terminus and SARS-CoV-2 PLpro revealed by complex structures. Previous studies reveal that the C-terminal UBL domain of ISG15 is sufficient for binding with MERS PLpro 38 or mammal USP18 39 . Based on a complex structure of full-length ISG15 and MERS PLpro, it was suggested that C-terminal tail of ISG15 plays an important role in the hydrogen-bond network formed by ISG15 and MERS PLpro 40 . Other studies also suggested that the Cterminal RLRGG of Ub is responsible for a major part of interaction between Ub and SARS-CoV 16 , MERS PLpro 16,41 , or human DUBs 42 .
Accordingly, we analyzed available human C-UBL-ISG15-PA/ SARS-CoV-2 PLpro and mouse ISG15-FL/SARS-CoV-2 PLpro complex structures using the PISA (proteins, interfaces, structures, and assemblies) program 43 (Fig. 5a, b and Supplementary  Fig. 10). Comparing these two structures, it is clear that the majority of the interactions between ISG15 and PLpro is from the C-UBL domain of ISG15. In two recent studies 31,32 , the PLpro residues V66, F69, Y171, and N156 were reported for interacting with the N-or C-globular domains of ISG15 31,32 . In addition, intermolecular interactions with the C-terminus of ISG15 involve PLpro residues G163, D164, E167, R166, Y264, Y268, and G271 (Fig. 5a, b and Supplementary Fig. 10). The backbone of G163, Y268, G271 and the sidechains of D164, R166, E167, Y264 formed hydrogen bonds and electrostatic interactions with PLpro ( Fig. 5b and Supplementary Fig. 10c). By comparing the structures of apo-and ISG15-bound SARS-CoV-2 PLpro, a shift of the BL2 loop of PLpro was observed (Fig. 5c-e). In apo-SARS-CoV-2 PLpro, the ISG15-C-terminus binding pocket takes an open conformation. Upon binding to ISG15, the PLpro BL2 loop shifts toward the substrate, i.e., the C-terminus of ISG15, and firmly holds the C-terminus through a hydrogenbond and electrostatic interaction network (Fig. 5). Using the sidechain OH of Y268 as a reference, the BL2 loop shifted 4.5 Å to encircle the C-terminus of ISG15 (Fig. 5e), which also happened to the binding of GRL0617 in the same pocket (Fig. 2d).

Discussion
Our biochemical, structural, and antiviral data of SARS-CoV-2 PLpro support the claim that PLpro is a promising drug target for COVID-19 treatment. Our co-crystal structure of PLpro C111S in complex with the potent inhibitor GRL0617 and its antiviral effect on Vero 6 cells validated that SARS-CoV-2 PLpro is a druggable target for SARS-CoV-2. In two recent studies, it was reported that the inhibition of deISGylating activity of PLpro is linked to the antiviral activity of PLpro inhibitors 31,44 . Our studies provided the mechanism of action for GRL0617. GRL0617 blocks the binding of ISG15 or Ub to PLpro, naturally it will also inhibit the processing of viral polyproteins of SARS-CoV-2 since these viral polyproteins share similar substrate cleavage site with Ub and ISG15.
The dominant role of the C-terminus of ISG15 in binding SARS-CoV-2 PLpro is intriguing. ISG15 contains N-and C-terminal globular domains and a short C-terminal tail (residues RLRGG), which is often considered part of the C-domain 45 . Recent structural analysis reported that the SARS-CoV-2 PLpro S1 site is mainly for high ISG15 activity while the S2 site determines substrate selectivity 31,32 . In our study, we show that the short and linear C-terminal tail of ISG15 dominates its binding with PLpro. Because the tail is short and flexible, it offers transient and reversible interactions with its binding partner, i.e., PLpro in this case, which ensures optimal enzyme efficiency and high enzymatic turnover. Since the C-terminus of Ub is also heavily involved in its interactions with MERS or SARS-CoV PLpro 16,41 , primarily interacting with the C-terminus of Ub or ISG15 could be a general strategy for viral DUBs to attack host immune system.
The ISG15 C-terminus binding cleft in PLpro contributes a disproportionately large portion of the binding energy compared with the rest of the protein, therefore this is a hot spot pocket for antiviral PPI drug discovery. Small-molecule drugs often occupy hot spots on PPI interfaces and inhibit target proteins 46 . Because PLpro is a viral DUB with Ub and ISG15 cleavage functions, we compared our co-crystal structure with the structures of known USP7 and USP14 inhibitors to see if they share the same binding sites. Indeed, several non-covalent USP7 or USP14 inhibitors occupy the same pocket in the USP domain (Fig. 6) and block the binding of the C-terminus of Ub [47][48][49] 50 and GRL0617 reside in the same pocket encircled by the BL2 loop (Fig. 6a, c). The superposition of all these DUB inhibitors on PLpro (Fig. 6d), shows that they are all at the same Ub or ISG15 C-terminus binding cleft, illustrating arguably the most important pocket in SARS-CoV-2 PLpro for the discovery of PPI inhibitors. In addition, analysis of available co-crystal structures of inhibitorbound DUBs suggests that stabilization of the inactive conformations of DUB 51 , and/or structural plasticity of the BL1/BL2 loops in DUB are also potential mechanisms of inhibition 52 . Since several independent high-throughput screening assays targeting SARS-CoV-2 using approved drugs have been unsuccessful 32,53 , structure-based drug discovery for this hot spot in PLpro and optimization of GRL0617 or its analogs with PLpro would be a promising approach for combating COVID-19. Recently reported covalent peptidic inhibitors targeting the active C111 also bind in this pocket in PLpro 33 .
Although the seven approved drugs obtained in our screening show low potency against PLpro, we cannot rule out the potentials of these drugs to therapeutically treat COVID-19 because they may have higher antiviral activities through other more complex mechanisms, e.g., the 6-TG 44 .
In summary, we report the co-crystal structure of SARS-CoV-2 PLpro and GRL0617. We also found that the C-terminus of ISG15 is dominant in its binding with PLpro. The ISG15 Cterminus binding cleft in PLpro is a hot spot for non-covalent PPI inhibitor discovery. Our study implicates that it may be an efficient approach to focus on this pocket for future efforts of drug discovery targeting SARS-CoV-2 PLpro.
The plasmids were subsequently transformed into E. coli BL21 (DE3) cells. Protein expression was carried out in LB medium. E. coli cells were grown in LB medium at 37°C until OD 600 reaches 0.8-1.0, 0.5 mM Isopropyl β-D-1thiogalactopyranoside (IPTG) and 100 mM ZnSO 4 were added, and cells were growing overnight at 18°C. For the production of 15 N-labeled ISG15 and Ub, protein samples were prepared by growing bacteria in M9 medium containing 15 NH 4 Cl.
Cell pellets were resuspended in buffer A (30 mM Tris, 400 mM NaCl, 30 mM imidazole, 2 mM β-ME, pH 8.5) with the addition of 1 mM phenylmethylsulfonyl The mUSP18 was prepared with the MultiBac system 54,55 as described previously 56 . It is briefly described as follows: pBac-His-SUMO-TEV-mUSP18 was transformed into DH10EmBacY cells and the positive clones were screened and selected with a blue-white screening protocol. The virus bacmid was transfected into Sf9 cells (Thermo Fisher Scientific) with lipofectamine 2000 (Thermo Fisher Scientific). Sf9 cells were grown in Sf-900™ II SFM media (Thermo Fisher Scientific). The initial virus was harvested and successful infection was monitored by the measurement of yellow fluorescent protein expression using a fluorescence spectrophotometer. Western blotting was used to test the expression of mUSP18. For large-scale expression of mUSP18, 100 mL of Sf9 cells at a density of 1 × 10 6 cells/mL were infected with 200-600 μL of the virus. The cells were kept at the same density until proliferation arrest. Then the cells were harvested for purification. The purification process is the same as for PLpro proteins.
In the assay of IFN-β (Sino Biological Inc., 10704-HNAS) induced ISGylation and the inhibition of PLpro by GRL0617, HEK293T cells were first treated with 500 U/mL IFN-β for 48 h and cell extracts were prepared in RIPA lysis buffer; 30 μg of extracts were mixed with purified recombinant PLpro (100 nM) and indicated concentrations of GRL0617 in the reaction buffer (50 mM Tris-HCl, 50 mM NaCl, 5 mM DTT, pH 7.5). Reactions were incubated at 37°C for 1 h and prepared for immunoblotting analysis as indicated.
PLpro activity assays and IC 50 determination. In this study, PLpro activity was monitored using the substrate peptide-AMC (Z-Arg-Leu-Arg-Gly-Gly-AMC, Cat. No. 4027158, Bachem Bioscience). Experiments were performed in 384-well black non-binding plates (Cat. No. 3575, Corning) with a final reaction volume of 50 μL. The assay buffer contained 50 mM HEPES, pH 7.4, 0.01% Triton X-100 (v/v), 0.1 mg/mL BSA, and 2 mM DTT. PLpro was added to the plates at a final concentration of 100 nM. Enzyme reactions were initiated with 5 μL of peptide-AMC (final 50 μM) dissolved in the above assay buffer. Upon addition of peptide substrate, the fluorescence signals were monitored at 340 nm (excitation) and 450 nm (emission) with 3 min intervals in a 2104 EnVision Multilabel Plate Reader (PerkinElmer).
An approved drug library (TargetMol, USA), of 2040 compounds including drugs approved by US FDA and CFDA, was used. The first round screening reaction mixture included 100 nM PLpro, 50 μM substrate, and 100 μM compounds. The top 1.5% compounds of each plate were selected with a minimum 50% inhibition using the reaction in DMSO as a control. Around 30 compounds were tested in the second round of screening using the same assay and 23 compounds were excluded due to their reactivities, insolubility, or fluorescence interference. To determine the IC 50 values of the remaining 7 compounds, a series of 8-point, 1 : 2 serial dilutions was performed from a highest starting concentration of 200 μM. Seven drugs used in this study were purchased from TargetMol (USA). The data were fitted using GraphPad Prism.
To test whether GRL0617 can inhibit mouse deISGylating enzyme USP18, its activity was assayed in the presence of an inhibitor with 250 nM ISG15-AMC (Boston Biochem) as substrate (excitation: 340 nm; emission: 450 nm).
Crystallization and data collection. The complex of GRL0617 (Cat. No. HY-117043, MCE) with PLpro C111S was crystallized by vapor diffusion in a sitting-drop format after a 20 h incubation of 9.5 mg/mL PLpro in the buffer (50 mM Tris, pH 8.5,100 mM NaCl) with 2 mM inhibitor at 4°C. Immediately before crystallization, the sample was clarified by centrifugation. A 0.75 μL volume of the enzymeinhibitor solution was then mixed with an equal volume of well solution containing 5 mM Cobalt (II) chloride hexahydrate; 5 mM Cadmium chloride hemi (pentahydrate); 5 mM Magnesium chloride hexahydrate; 5 mM Nickel (II) chloride hexahydrate; 0.1 M HEPES, pH 7.5; and 12% PEG 3350 and equilibrated against well solution at 12°C. Before data collection, crystals were soaked in a cryo solution containing well solution, 400 μM inhibitor, and 20% glycerol. Crystals were flashfrozen in liquid N 2 . All diffraction data were collected at an in-house light source Rigaku MicroMax-007 HF and indexed, integrated, and scaled using CrysAlisPro and XDS 57 . Isothermal titration calorimetry of ISG15 and its truncated mutants with PLpro proteins. ITC was performed using a Microcal-ITC200. One injection with 0.4 μL and subsequent 19 injections of 2 μL were performed at 25°C with a reference power of 5 μcal/s. The ISG15 and its truncated mutants with SARS-CoV-2/SARS-CoV/MERS PLpro proteins were all in PBS buffer. For binding experiment of SARS-CoV-2 PLpro with ISG15 or ISG15-△C6, 92 μM PLpro was placed in the cell with 0.95 mM of ISG15 or 1 mM ISG15-△C6 in the syringe. For binding experiment of SARS-CoV or MERS PLpro with ISG15-△C6, 100 μM of SARS-CoV or MERS PLpro was placed in the cell with 1.2 mM or 1 mM ISG15-△C6 in the syringe. For binding experiment of SARS-CoV-2 PLpro with ISG15 truncated mutants (△C5, △C4), 100 μM of SARS-CoV-2 PLpro was placed in cell with 1-2 mM various ISG15 mutants in the syringe. The data were processed using Microcal-ITC200 analysis Software.
Antiviral and cytotoxicity assay. For the assay, 1 × 10 4 Vero E6 cells were seeded in triplicates in 96-well plates. After 20-24 h, fresh medium with different concentrations (100, 50, 25, 12.5, 6.25, 3.125, and 1.56 μM) of inhibitors, DMSO as a control, was replaced. For measuring the cytotoxicity, cells were incubated for 48 h followed by the cell viability test with CCK8 reagent. For measuring the antiviral activity, cells were kept in the medium with inhibitors for 1 h, followed by infection with SARS-CoV-2 virus strain BetaCoV/Shenzhen/SZTH-003/2020, which was clinically isolated from local patients, at MOI = 0.01. After a 2 h incubation, the virus-compound mixture was subsequently removed, and fresh medium containing candidate compounds (100, 50, 25, 12.5, 6.25, 3.125, and 1.56 μM) or DMSO was added, and cell growth was continued for 48 h. The viral RNA was extracted from the supernatant medium and the qRT-PCR assay was performed. The linearized plasmid containing the Spike gene of the SARS-CoV-2 virus was transcribed in vitro and was used to prepare a standard curve to quantify the copy number of the virus as previously described 11 . Briefly, the SARS-CoV-2 Spike gene were synthetized in pcDNA3.1 (Sangon, China). This vector was then linearized by single locus restriction endonuclease and subjected to in vitro transcription. The concentration of resulting RNA transcripts was measured by NanoDrop 2000. Microgram concentration of the plasmid can be transformed to the RNA copies. The first dilution is 10 7 copies, then diluted to 10 2 copies (6 diluted concentrations: 10 7 , 10 6 ,10 5 ,10 4 ,10 3 ,10 2 ). The qRT-PCR assay was performed with the above template and the probe, and primers of Spike gene. Standard curves were prepared according to the results of PCR amplification. The standard curve formula is y = −3.2998x + 37.758, R 2 = 0.9997. Primer and probe information: TaqMan primers for COVID-19 virus: 5′TCCTGGTGATTCTTCTTCAGG-3′ and 5′-TCTGAGA GAGGGTCAAGTGC-3′ and COVID-19 virus probe 5′-FAM-AGCTGCAGCAC CAGCTGTCCA-BHQ1-3′. Data analysis was done with GraphPad Prism.
SARS-CoV-2-induced CPE on Vero E6 cells was observed and analyzed using reverse-phase light microscope. The inhibition effect of GRL0617 against SARS-CoV-2-induced cytopathogenic effect was measured under different concentrations. The cytopathic effect was examined for 3 days post-infection. The complete absence of cytopathic effect in an individual culture well was defined as protection. The value of EC 50 was calculated using GraphPad prism software.
NMR spectroscopy. NMR data were acquired at 25°C on a 600 MHz Bruker AVANCE III spectrometer. The 600 MHz spectrometer was equipped with a 5 mm TCI Cryoprobe. In NMR titrations, the samples of 0.1 mM 15 N-labeled ISG15 were incubated in the presence or absence of 0.15 mM PLpro with or without the indicated concentration of GRL0617 (0.05 mM, 0.15 mM, and 0.25 mM) were investigated in assay buffer containing 30 mM Tris, 100 mM NaCl, pH 7.4, 5% DMSO, and 10% D 2 O. For Ub titrations, the samples of 0.1 mM 15 N-labeled Ub were incubated with PLpro (0.1 mM, 0.2 mM, and 0.3 mM). 1 H, 15 N-HSQC titration spectra were collected for all the samples. All of the NMR spectra were processed using NMRPipe/NMRDraw and further analyzed using NMRView.