Potent and Selective Covalent Inhibition of the Papain-like Protease from SARS-CoV-2

Abstract Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we have designed a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibited PLpro with k inact /K I = 10,000 M − 1 s − 1 , achieved sub-µM EC 50 values against three SARS-CoV-2 variants in mammalian cell lines, and did not inhibit a panel of human deubiquitinases at > 30 µM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validated our design strategy and established the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.

the protein. Of all the cysteines in PLpro, Cys111 is the most prone to oxidation, 12 indicating that it is unique in its reactivity toward electrophiles.
Protein substrates of PLpro consist of a Leu-X-Gly-Gly peptide motif (X = Arg, Lys, or Asn) with proteolytic cleavage occurring after the second Gly residue. 4 Leu and X occupy the S4 and S3 subsites, respectively, and the two Gly residues occupy the S2 and S1 subsites, which are covered by a b-hairpin "blocking loop" (BL2 loop) that forms a narrow groove leading to the active site (Fig. 1b). 10 As a result, only extended peptide substrates with two Gly residues at the P1 and P2 positions can be accommodated in this space. 9,10 Several noncovalent inhibitors of PLpro have been developed that competitively inhibit PLpro. [12][13][14][15] The naphthylmethylamine compound GRL0617 inhibits SARS-CoV PLpro with an IC 50 of ~0.6 mM and inhibits viral replication in Vero E6 cells with EC 50 = 14.5 mM. 13 The desamino analog of GRL0617 exhibits similar inhibitory activity (IC 50 = 2.3 μM; EC 50 = 10 μM), as does the N-acetylated analog (IC 50 = 2.6 μM; EC 50 = 13.1 μM). GRL0617 exhibits similar inhibition activity against SARS-CoV-2 PLpro. 8,12,16 Importantly, GRL0617 does not inhibit the structurally similar human DUBs. The IC 50 values for GRL0617 toward HAUSP, the deISGylase USP18, or the ubiquitin C-terminal hydrolases UCH-L1 and UCH-L3 are all >100 mM. 13 In addition, GRL0617 does not display cytotoxicity at concentrations up to 50 mM in cell viability assays.
We designed a series of covalent PLpro inhibitors based on the noncovalent inhibitor GRL0617 ( Fig. 1 and 2). Crystal structures have revealed that the phenylmethyl group of GRL0617 points toward the active site but is located >7 Å from Sg of Cys111 (Fig. 1b). We reasoned that replacing the methyl substituent of GRL0617 with a hydrolytically stable linker connected to an electrophilic group capable of reacting with Cys111 would yield a potent covalent inhibitor of PLpro. We chose an N,N'-diacetylhydrazine linker as a linear Gly-Gly peptidomimetic that could reach through the narrow S2 and S1 groove to the active site while also preserving some of the hydrogen-bonding interactions (e.g., with Gly163 and Gly271) afforded by natural peptide substrates. To the resulting hydrazide linker we appended a series of electrophiles including a fumarate methyl ester, 17 chloroacetamide, 18 propiolamide, cyanoacetamide, and α-cyanoacrylamide.
To help prioritize designed molecules for synthesis and testing, we performed covalent docking of each candidate molecule to PLpro. We also docked each molecule noncovalently to assess the favorability of pre-covalent binding. We used an ensemble of 50 structural models derived from X-ray crystallographic data to account for protein exibility 12 and included selected crystallographic waters during docking, including those that are known to remain stably bound in the S4 subsite in the presence of noncovalent inhibitors. 12,13 Key interactions between PLpro and GRL0617 include (i) a hydrogen bond between the backbone N-H of Gln269 and the amide carbonyl of the inhibitor, (ii) a hydrogen bond between the N-H of the GRL0617 amide and the carboxylate side chain of Asp164, and (iii) an edge-to-face interaction of the naphthyl group of GRL0617 and Tyr268 (Fig. 1b). All candidate inhibitors contain the naphthylmethylamine core of GRL0617 (Fig. 2) and we aimed for our modi ed compounds to recapitulate its binding mode. To assess pose similarity, we measured the maximum common substructure RMSD (MCS-RMSD) between the docked poses of the candidate inhibitors and the crystallographic pose of GRL0617. In general, the core of the inhibitor designs reproduced the binding mode of GRL0617 to within 2 Å RMSD, maintaining interactions with Asp164, Tyr268, and Gln269 while the linker simultaneously occupied the S2 and S1 subsites to place the electrophilic group near the catalytic Cys111 nucleophile (Fig. 1e, f and Extended Data Fig. S2). Compounds were prioritized for synthesis based on low MCS-RMSD values (≤ 2 Å), favorable noncovalent and covalent docking scores (Extended Data Fig. S3 and Extended Data File 1), and synthetic tractability.
We synthesized compounds 2-13 and assessed their inhibition of SARS-CoV-2 PLpro using a plate-based assay with the ubiquitin C-terminus-derived Z-RLRGG-AMC uorogenic substrate 13,19,20 (Fig. 2 and Extended Data Fig. S4). IC 50 values were determined following a 30-minute incubation of PLpro with inhibitor (Extended Data Fig. S5). Of the noncovalent analogs of GRL0617, we found that both 14 and 15 had somewhat increased IC 50 values, with the N-acetylated compound 15 having an IC 50 more like that of GRL0617. We found that extension of the methyl group with a substantially larger peptidomimetic group could maintain potency. For example, addition of the linker alone without an electrophile to form 5 led to an IC 50 of 24 mM ( Fig. 2 and Extended Data Fig. S5). The introduction of ve different electrophilic warheads to produce compounds 7, 9, and 11-13 resulted in improved IC 50 values for all except αcyanoacrylamide 13. Time-dependent inhibition assays were performed as time-dependence is consistent with multiple mechanisms of slow-binding inhibition, including covalent inhibition via bond formation between Cys111 and the electrophile. Installation of a chloroacetamide electrophile to form 9 improved the IC 50 compared to 5 to 5.4 mM after 30-minute incubation and resulted in a k inact /K I of 100 M -1 s -1 , where k inact /K I is a second-order rate constant describing the e ciency of the overall conversion of free enzyme to the covalent enzyme-inhibitor complex (Extended Data Fig. S6). 21 Similarly, the IC 50 and k inact /K I for N-acetylated analog 10 are 4.4 mM and 120 M -1 s -1 , respectively.
A vinyl methyl ester electrophile was recently used in tetrapeptide-based, irreversible covalent inhibitors of PLpro. 9 We reasoned that a similar ester would occupy the oxyanion hole in the active site and engage in a hydrogen bond with Trp106. Fumarate methyl ester 7 had an IC 50 of 94 nM after 30-minute incubation and k inact /K I = 10,000 M -1 s -1 , indicating potent inhibition (Fig. 2, 3a, b, and Extended Data Fig. S6). Nacetylated analog 8 showed similar potency, with IC 50 and k inact /K I = 230 nM and 14,000 M -1 s -1 , respectively. To examine the inhibitory activity of other electrophiles, we synthesized and performed timeindependent inhibition assays with cyanoacetamide 11 (IC 50 , 8 mM), propiolamide 12 (98 nM), and acyanoacrylamide 13 (>200 mM). Time-dependent inhibition was observed for 12, but not for 11 or 13 (Extended Data Fig. S7). To provide additional evidence for a covalent mechanism of action, compounds 7-10 and 12 were incubated with PLpro, and the protein intact masses were determined by electrospray ionization mass spectrometry (ESI-MS). Covalent adduct formation with PLpro was con rmed for these ve compounds (Fig. 3c, Extended Data Fig. S8 and Extended Data Table 1).
Following the promising results from in vitro assays and mass spectrometry experiments, we used X-ray crystallography to obtain structural insight into covalent inhibition of PLpro. We determined a crystal structure of wild-type PLpro in complex with fumarate methyl ester 7, the most promising lead, at 3.10 Å resolution (Extended Data Table S2). The electron density maps show clear densities for PLpro, Zn cations, and 7, con rming the design concept of this compound and revealing key interactions with PLpro ( Fig. 4). A covalent bond is present between Sg of Cys111 and C1 of compound 7 (Fig. 4a). The carbonyl oxygen from the fumarate ester accepts hydrogen bonds from the indole side chain of Trp106, like that of the tetrapeptide-based covalent inhibitor VIR251, 9 as well as the side chain of Asn109. The N, N'diacetylhydrazine moiety was designed to link the electrophile and the naphthylmethylamine core while also hydrogen bonding with residues in the S1-S2 groove. Indeed, the crystal structure revealed that the proximal and distal carbonyl oxygens of the N, N'-diacetylhydrazine linker interact with the backbone N-H groups of Gly163 and Gly271, and the proximal and distal N-H groups of this moiety participate in hydrogen bonds with the carbonyl backbones of Gly271 and Gly163. As intended, the carbonyl oxygen and N-H group of the amide adjacent to the naphthyl group of 7 are hydrogen bonded with the N-H backbone of Gln269 and the carboxylate side chain of Asp164. Compound 7 makes ve main-chain and three side-chain hydrogen bonding interactions in the binding site. In addition, the side chains of Tyr268 and Gln269 interact with 7 similarly to GRL0617. Electron density for the methyl group of the ester of 7 was not visible. It is possible that the ester linkage is exible and adopts multiple conformations or that it could have been hydrolyzed. Encouragingly, the covalently docked pose for 7 agrees closely with the cocrystal structure (Fig. 4b).
The ability of the inhibitors to protect Vero E6 cells from viral infection-induced cell death, represented by EC 50 (Fig. 2, 3d and Extended Data Fig. S9), was assessed by incubating cells with and without compound and then infecting them with SARS-CoV-2. 25 Uninfected cells were used to assess the cytotoxicity of the compounds, represented by CC 50 (Fig. 2). Compound 7 displayed notable antiviral activity with an EC 50 of 1.1 μM, comparable to that of the remdesivir drug control (0.74 μM).
Chloroacetamide 9 also displayed antiviral activity, although with less potency (34 mM values of 1-5 μM, suggesting that propiolamide and α-cyanoacrylamide electrophiles may be too reactive, lack speci city, or both. In addition to its role in processing the replicase polyprotein, SARS-CoV-2 PLpro displays deubiquitinase and de-ISG15ylase activity. 10,26 To ensure that our most promising covalent inhibitors 7 and 9 can inhibit these physiologically relevant activities, IC 50  Because PLpro bears structural and functional similarity to human DUBs and related enzymes, inhibitor selectivity is an important consideration. Seven human DUBs, UCHL1, USP2, USP4, USP7, USP8, USP15, and USP30, were assayed to determine whether they were inhibited by 7 and 9. No inhibition of the human DUBs was observed for either compound at concentrations up to 30 μM (Extended Data File 2). In silico analysis of the superposed structures of human DUBs with the active site residues and the helix bearing Cys111 of the co-crystal structure of PLpro with 7 suggests that the naphthyl ring in 7 would experience severe clashes with the crossover loop (Arg153-Lys157) of UCHL1, and Phe828 and Lys838 of USP4 ( Fig. 4d and 4e, respectively), providing a structural basis for the selectivity of 7 against human DUBs. Thus, compounds 7 and 9 inhibit PLpro-mediated peptide cleavage, ubiquitin cleavage, and ISG15 cleavage, they have antiviral activity against SARS-CoV-2 and lack cytotoxicity in Vero E6 cells, and they do not inhibit a representative panel of human DUBs.
We next sought to determine the metabolic stability of our compounds in human, rat, and mouse liver microsomes and the corresponding S9 fractions (Extended Data Table S3 and S4). Chloroacetamide 9 demonstrated very short half-lives of 3 and 7 minutes in human liver S9 and microsomes, respectively, likely due to the highly reactive electrophile. Non-covalent inhibitor 14 exhibited a half-life >60 min in the S9 fraction, and 41 min in microsomes. Conversion to its covalent counterpart 7 maintained the half-life (60 min in S9, 50 min in microsomes). Analysis of 14 and 7 with MetaSite 6.0.1 27 suggested that successive oxidations of the tolyl methyl of 14 were the predominant metabolic liability, followed by the benzylic methylene (Extended Data Figure S10). Given that the linker and electrophile replaced the labile methyl group, it is unsurprising that the benzylic methylene is predicted to be the primary site of metabolism for 7. To address the benzylic liability several modi cations could be pursued, including substitution of the benzylic position with heavy atoms such as deuterium 29 or uorine 30 to increase steric hindrance, 28 or blocking the site of metabolism via replacement of the tolyl methyl with cyclopropane. 31 Numerous research efforts have focused on developing inhibitors of 3CLpro, but relatively few have focused on PLpro inhibition. A predominant reason for the emphasis on 3CLpro as an antiviral target is that there are no structural homologs in the human proteome whereas PLpro bears structural similarity to human DUBs and deISGylases. However, our ndings demonstrate that covalent inhibition of PLpro is a promising strategy for developing potent and selective therapeutics to combat SARS-CoV-2. Furthermore, a crystal structure of our most promising inhibitor covalently bound to PLpro provides insight that will facilitate the development of next-generation PLpro inhibitors with enhanced pharmacokinetic and pharmacodynamic properties.

Methods
Docking preparation. The 2.09 Å X-ray co-crystal structure of the C111S mutant of PLpro with GRL0617 (PDB entry 7JIR) 12 was used for the docking calculations. Rather than docking to a single structure, we used PHENIX 32 to generate an ensemble 33 of 50 conformations from the corresponding crystallographic data in which conformations were sampled to generate an ensemble that collectively t the data better than any single model. This approach provides valuable information about regions of high and low conformational variability in the protein, such as the BL2 loop, which is known to undergo large conformational changes upon substrate or inhibitor binding. Ser111 was converted back to Cys in all models.
Selected water molecules present in the models were retained during docking. Cys111 was modeled as a neutral thiol and His272 was protonated on Ne in accordance with its local hydrogen bonding environment and the proton transfer chemistry that is expected to occur during catalysis. Other histidines were protonated based on their inferred hydrogen bonding patterns. All other residues were protonated according to their canonical pH 7.0 protonation states. The program tleap from AmberTools20 34 was used to prepare the parameter and coordinate les for each structure. The ff14SB force eld 35 and TIP3P water model 36 were used to describe the protein and solvent, respectively. Energy minimization was performed using sander from AmberTools20 with 500 steps of steepest descent, followed by 2000 steps of conjugate gradient minimization. Harmonic restraints with force constants of 200 kcal mol -1 Å -1 were applied to all heavy atoms during energy minimization.
The peptide substrate binding cleft of PLpro spans ∼30 Å along the interface of the palm and thumb domains (Extended Data Fig. S1). Thus, we de ned a rectangular docking box spanning the entire binding cleft (S1-S4 sites) and the active site (catalytic triad). AutoGrid Flexible Receptor (AGFR) 37 was used to generate the receptor les for both noncovalent and covalent docking using a grid spacing of 0.25 Å. All docking calculations were performed with AutoDock Flexible Receptor (ADFR). 37 Compounds with electrophilic groups were docked both noncovalently (i.e., in the reactive form with an explicit electrophile present) and covalently (i.e., in the post-reactive Cys111 adduct form).
Ligand preparation. SMILES strings for candidate inhibitor designs were converted to PDB format using Open Babel 38 and custom Python/RDKit 39 scripts. Covalent docking with AutoDockFR requires that ligands be modi ed such that they include the covalent linkage to the side chain of the reactive residue, in this case Cys111, which then serves as an anchor to place the ligand approximately in the binding site. 37 Thus, the Ca and Cb atoms of Cys111 were used as anchors and the backbone N atom of Cys111 was used to de ne a torsional angle connecting the covalently bound ligand and the protein. MGLTools 1.5.6 40 was used to generate PDBQT les for ligands and receptors. Only polar hydrogens were retained during docking.
All candidate inhibitors considered in this work include the naphthylmethylamine core of GRL0617, for which co-crystal structures are available. 12 We expected that our covalent compounds would adopt a pose like GRL0617. Thus, to assess the similarity between the poses of docked candidate ligands and GRL0617 in the X-ray structure, we calculated the maximum common substructure (MCS) RMSD between them. MCS RMSDs were calculated for poses with docking energies within 3 kcal/mol of the overall most favorable pose for each candidate inhibitor. Compounds were prioritized for synthesis that had docked poses with MCS-RMSD values ≤2 Å and favorable noncovalent and covalent docking scores (Extended Data Fig. S2 and Extended Data File 1

(R)-N-(1-(naphthalen-1-yl)ethyl)-2-(3-oxo-3-(2-propioloylhydrazineyl)propyl)benzamide (12). Compound 12
was synthesized under the same conditions as compounds 7, 9, 11, and 13 except the initial coupling to the hydrazide of 5 was achieved with 3-(trimethylsilyl)propioloyl chloride. The DCM was removed under reduced pressure and the crude material was immediately dissolved in 1:1 THF:MeOH (6 mL total volume) and 10 mg of K 2 CO 3 was added. Protein expression and puri cation. PLpro from SARS-CoV-2 was produced using a previously described procedure with minor modi cations, 43 which we summarize here. First, the protein was expressed using E. coli BL21(DE3) cells that had been transformed with a pMCSG92 expression plasmid, which includes a T7 promoter and TEV protease-cleavable C-terminal 6xHis tag. Cells were plated on LB agar and cultivated in a shaking incubator (250 rpm) at 37°C in Lysogeny Broth medium (Lennox recipe) using 1 L per ba ed 2.8 L Fernbach ask. Carbenicillin was used for antibiotic selection throughout. Bacterial growth was monitored by measuring the absorbance at 600 nm (OD 600 ). Upon reaching an OD 600 of ∼0.7, the incubator temperature was set to 18 °C and isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to 0.2 mM. After approximately 18 hours, the culture was harvested by centrifugation at 6000×g for 30 minutes. After decanting off the supernatant, the pellets were stored at -80°C until needed for protein puri cation.
A cell pellet harvested from a 1 L culture was thawed and resuspended in 100 mL of lysis buffer containing 50 mM HEPES, 300 mM NaCl, 50 mM imidazole, 5% glycerol, and 1 mM TCEP at pH 7.4. Following resuspension, the cells were subjected to tip sonication on ice at 50% amplitude (2 seconds on and 10 seconds off) for a total sonication time of 5 minutes using a Branson 450D digital soni er. After clarifying the lysate by 38,500xg centrifugation for 35 minutes at 4°C, the decanted supernatant was passed through 1.6-and 0.45-micron syringe lters sequentially and kept on ice while loading a 5-mL HisTrap HP column (Cytiva) at 2 mL/min. After washing the column with 10 column volumes (CV) of lysis buffer, partially puri ed PLpro was eluted using a linear gradient (20 CVs) of lysis buffer with 500 mM imidazole. Elution fractions (2 mL) were collected and PLpro was identi ed using SDS-PAGE on a 4-20% Mini-Protean TGX Stain-Free protein gel (Bio-Rad). Pooled fractions containing PLpro were dialyzed overnight at 6°C in 50 mM HEPES pH 7.4 with 150 mM NaCl, 5% glycerol, 20 mM imidazole, and 1 mM TCEP in the presence of His-tagged TEV protease (1 mg TEV protease:100 mg PLpro). After con rming His-tag cleavage by SDS-PAGE, the dialyzed protein solution was passed over a 5-mL HisTrap HP column to remove His-tagged impurities. The column owthrough was collected, evaluated with SDS-PAGE, and concentrated with a 10-kDa molecular weight cutoff Amicon Ultra15 ultra ltration membrane. Upon concentration, partially puri ed protein was applied at 0.5 mL/min to a Superdex 75 10/300 GL sizeexclusion column (Cytiva) that had been equilibrated with 50 mM Tris HEPES pH 7.4 with 150 mM NaCl, 5% glycerol, and 1 mM TCEP. Fractions (0.5 mL) containing puri ed PLpro were collected, pooled, and concentrated for further use.
PLpro inhibition assays. The assays were performed in 40 μL total volume in black half area 96-well plates (Greiner PN 675076) at 25°C. The assay buffer contained 20 mM Tris-HCl pH 7.45, 0.1 mg/mL bovine serum albumin fraction V, and 2 mM reduced glutathione. The nal DMSO concentration in all assays was 2.5% v/v. PLpro initial rates were measured using a previously established uorogenic peptide substrate assay. 13,19,20 The substrates Z-LRGG-AMC and Z-RLRGG-AMC were purchased from Bachem (PN 4027157 and  μL substrate to 100 μM nal concentration. Initial rates were determined as described above and % residual activities were determined by normalizing to the average of no inhibitor controls (100% activity). Time-dependent inhibition assays were performed as described above, except that preincubation times were varied by adding the inhibitor to the enzyme at speci c time points. For each inhibitor concentration, initial rates were normalized such that 0 preincubation time is 100% and plotted against preincubation time. A nonlinear regression to a one phase decay model was performed to determine the rate constants k obs for each concentration and their 95% con dence intervals. These rate constants were then plotted against inhibitor concentration, and the data in the initial linear region was t to determine the slope, which is k inact /K I . All regressions were performed with GraphPad Prism 9.
Mass spectrometry to assess covalent adduct formation. A Waters Synapt HDMS QTOF mass spectrometer was used to measure the intact protein mass of PLpro with and without preincubation with inhibitors to detect covalent adduct formation. To prepare the samples, 2 μL of 20 mM inhibitor stocks in DMSO were added to 100 μL PLpro at 1 mg/mL concentration and incubated 1 h at room temperature.
Previously described protocols for ultra ltration and denaturing direct infusion 44 were implemented as follows. Samples were processed by ultra ltration with a Vivaspin 500 10 kDa PES membrane by diluting the sample to 0.5 mL with 10 mM LC-MS grade ammonium acetate and reducing volume to 50 μL twice, followed by the same procedure with 2.5 mM ammonium acetate. Protein concentrations were estimated by A280 with a NanoDrop 2000, and samples were diluted to 2 mg/mL in 2.5 mM ammonium acetate, and then 10 μL were further diluted into 90 μL 50:50 acetonitrile:water with 0.1% formic acid. Sample was introduced into the electrospray ionization source by syringe pump at a ow rate of 10 μL/min and MS1 spectra were collected for m/z 400-1500, 5 s/scan, for 1 min. The protein monoisotopic mass was determined from the averaged spectra using mMass 5.5. 45 Inhibition of PLpro deubiquitinase and de-ISG15ylase activities and deubiquitinase selectivity. Candidate inhibitors were assayed by LifeSensors, Inc. (Malvern, PA) in quadruplicate for inhibition of SARS-CoV-2 PLpro with Ub-rhodamine and ISG15-CHOP2 and with human deubiquitinase (DUB) enzymes, including USP30, 15,8,7,4, and 2C as well as UCHL1 with Ub-rhodamine, except for USP7, which was tested with Ub-CHOP2. The CHOP assay 46  Data collection and structure determination. The diffraction data were collected at 100 K at the BL12-2 beamline of the Stanford Synchrotron Radiation Light Source using Pilatus 6M detectors. Crystals for the complex were cryo-cooled using the well solution supplemented with 20% ethylene glycol. Diffraction data from two crystals were collected with 360 degrees of data per crystal and 0.2 degrees oscillation per image. For each crystal, diffraction data were merged and processed with the XDS suite of programs. 47 The structures were solved by molecular replacement with AMoRE 48 using the coordinates of SARS-CoV-2 PLpro complexed with the tetrapeptide-based inhibitor VIR251 (PDB 6WX4 9 ) as the search model. Iterative rounds of model building and re nement were performed with the programs COOT 49 and REFMAC. 50 The details of data collection and re nement for the higher resolution data (3.10 Å) are presented in Extended Data Table S2.
SARS-CoV-2 antiviral assays. Initial screening to measure cytopathic effect (CPE) protection for the 50% e cacy concentration (EC 50 ) and cytotoxicity (CC 50 ) was performed using an assay based on African green monkey kidney epithelial (Vero E6) cells in 384-well plates. 51     Characterization of designed covalent PLpro inhibitor, compound 7. a, Fluorogenic peptide activity assay after 30-min preincubation with fumarate methyl ester 7. Data points are the average of n = 2 independent samples ± range and are representative of n = 3 independent experiments. IC50 is the concentration at which 50% inhibition was observed, and bracketed values are the 95% con dence interval. Curve is the nonlinear regression to the normalized inhibitor dose response equation. b, Timedependent characterization with a uorogenic peptide assay. Data points are kobs values determined by tting the exponential decay equation to initial rates determined at various inhibitor concentrations and preincubation times, normalized to no preincubation. kobs values were determined from n = 2 independent experiments with n = 2 independent samples each ± 95% con dence interval of the nonlinear regression. Line represents the linear regression yielding as its slope the second-order rate constant (kinact/KI). c, Intact protein ESI-MS spectra of PLpro (black) and PLpro incubated with 7 (red); a.i., arbitrary intensity; m/z, mass-to-charge ratio. d, Cell viability in Vero E6 cells for uninfected cells pretreated with 7 (black squares), SARS-CoV-2-infected cells pretreated with 7 (red circles) and with remdesivir drug control (blue triangles). Data points are the average of n = 2 independent samples ± range and are representative of n = 2 independent experiments. EC50 is the concentration at which 50% effect was observed and bracketed values are the 95% con dence interval. Curves are nonlinear regressions to the normalized dose response equation.

Figure 4
Crystal structure of SARS-CoV-2 PLpro in complex with inhibitor 7. a, Overall structure and interactions between the active site residues and 7 (cyan sticks). The electron density for 7 is shown in blue mesh (Fo -Fc omit map contoured at 1.5 σ). b, Superposition of the covalently docked model of 7 (grey sticks) and the co-crystal structure of PLpro and 7 (cyan sticks). c, Structural basis for selectivity toward PLpro. Superposition of 7 bound to PLpro onto human deubiquitinase UCHL122 (PDB entry 3KW5). The crossover loop of UCHL1, 153-RVDDK-157, covers the narrow groove and blocks the naphthylmethylamine core of 7 from binding. The crossover loop is longer and, in some cases, more disordered in UCHL3 and UCHL5 (see for example ref 23