Synthetic Optimization of Lead Increases Potency.

The preference for a methyl group over hydrogen at the R1 position of 7724772 was found to be required for potency based on a structure–activity analysis of the primary high-throughput screen (HTS) data and on the follow-up studies of the primary screen. Therefore, other substituents at the R1 position were explored. The substitution of a chlorine atom at R1, 3, resulted in a 2-fold decrease in inhibitory potency (IC₅₀ = 14.5 ± 0.9 μM) compared with 2, and the substitution of a larger ethyl group, 4, almost abolished activity (IC₅₀ > 100 μM), suggesting that the optimum size of a substituent at the R1 position is a methyl group. Compound 2 was further optimized by probing the effect of changing the orientation of the relatively bulky naphthalene group (R2 in Fig. 2A). Replacing the 2-naphthyl group of 2 with a 1-naphthyl to form compound 5 resulted in a 4-fold increase in inhibitory potency (IC₅₀ = 2.3 μM). Finally, the addition of a second functional group to the R3 position of the ortho-methyl benzene ring of 5 was explored. Addition of an NHAc group, 6, was found to have little effect on the IC₅₀ value (2.6 ± 0.1 μM) compared with 5, whereas the addition of a nitro group, 7, decreased activity nearly 3-fold. In contrast, the addition of an amino group at the same position increased the inhibitory potency almost 4-fold (IC₅₀ = 0.6 ± 0.1 μM), suggesting that an additional hydrogen bond may be formed in the enzyme–inhibitor complex. The resulting compound, designated GRL0617, is the most potent inhibitor in the series (Fig. 2A) and was therefore subjected to more extensive mechanistic and structural analyses.

Mechanism of Inhibition of Most Potent Lead-GRL0617.

To characterize the mechanism of inhibition by the GRL0617 class of compounds, kinetic and biochemical studies of the enzyme–inhibitor complexes were performed. A kinetic study of PLpro activity, in which the concentration of its optimal substrate, ISG15-AMC, was varied relative to fixed concentrations of inhibitor, reveals that GRL0617 is a potent competitive inhibitor of PLpro with a K_i value of 0.49 ± 0.08 μM (Fig. S2A). Progress curve analysis also suggested that the inhibitor is noncovalent. To further probe for noncovalent inhibition of PLpro by GRL0617, PLpro was incubated for 1 h with 12 μM GRL0617 (>20-fold the K_i value), and the resulting complex was dialyzed to allow the inhibitor to diffuse away and therefore restore enzymatic activity (Fig. S2B). Approximately 25% of the PLpro activity was recovered after 3 h of dialysis compared with a recovery of 100% for the enzyme without inhibitor. The inability to fully recover PLpro activity after 3 h could be either a result of a slow off-rate of the inhibitor from the PLpro-GRL0617 complex or a result of covalent modification of the active-site cysteine by a direct reaction with the inhibitor or by indirect oxidation. Because GRL0617 has no apparent thiol-reactive groups, the inability to recover enzymatic activity is likely a result of both mechanisms, a slow off-rate of inhibitor and oxidation of the cysteine, despite the use of reducing agents throughout all studies. Evidence for cysteine oxidation is revealed in the structural studies described below.

SARS-CoV Antiviral Activity.

To probe the antiviral activity of the PLpro inhibitors, several compounds were assayed for their ability to rescue cell culture from SARS-CoV infection. The viability of virus-infected Vero E6 cells as a function of inhibitor concentration was measured relative to mock-infected cells by using a luminescence assay, which allows for the evaluation of both inhibitor efficacy and cytotoxicity (Fig. 2B). Most significantly, GRL0617 and compounds 5 and 6 display significant antiviral activity with EC₅₀ values ranging from 10 to 15 μM without toxicity up to the highest concentration tested (Fig. 2 A and B). Notably, the increasing antiviral potency correlates with the in vitro inhibition of PLpro, suggesting that the compounds work directly on the enzyme in cells.

Structural Basis for Potent Inhibition of SARS-CoV PLpro Revealed by X-Ray Crystallography.

To better understand the molecular basis for inhibition of PLpro by GRL0617 and the structure–activity relationship described above, we determined the X-ray structure of the PLpro-GRL0617 complex to a resolution of 2.5 Å (Table S1). The structure reveals unambiguous electron density for the inhibitor, which binds in a cleft leading to the active site (Fig. 3A). The inhibitor is well-removed from the catalytic triad and instead bound within the S3 and S4 subsites of PLpro (Fig. 3B). The interaction between GRL0617 and PLpro is stabilized through a pair of hydrogen bonds and a series of hydrophobic interactions stemming from residues lining the pocket. Specifically, the amide group of the inhibitor forms hydrogen bonds with the side chain of D165 and the backbone nitrogen of Q270 (Fig. 3C). D165 is highly conserved among the ubiquitin-specific protease (USP) family of deubiquitinating enzymes (17) and among most coronaviral papain-like proteases (11, 18). Several structural studies of USPs have revealed that this aspartic acid residue hydrogen bonds with the backbone of ubiquitin molecules at the P4 position, an interaction presumed to be important for ligand stabilization (19–21).

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Fig. 3.

Inhibitor GRL0617 binds within the S3 and S4 subsites of PLpro and induces loop closure over the inhibitor. (A) An F_o-F_c electron density omit map, generated with the inhibitor excluded from the phase calculation, is shown contoured at 4σ (orange mesh). The inhibitor is shown as orange sticks. The location of the catalytic triad is indicated, as is the oxidized active site cysteine residue. (B) Stereoview of PLpro (white surface) bound to the inhibitor (orange), which lies within a narrow groove proposed to bind to the P3 and P4 positions of substrate (cyan). The structure of the C-terminal residues of ubiquitin, which were modeled into the PLpro apoenzyme active site (10) is shown superimposed on the inhibitor-bound complex. For clarity, only the P3 (Arg) and P4 (Leu) residues of substrate are shown. (C) Stereoview of the hydrogen bonds and hydrophobic interactions between the inhibitor (orange) and PLpro (blue). Hydrogen bonds are indicated by dashed lines. (D) A ribbon diagram of apo PLpro (blue) superimposed on the inhibitor-bound enzyme (orange). The inhibitor is shown as spheres representing van der Waals radii. Catalytic triad residues are designated by asterisks.

Aside from the 2 aforementioned hydrogen bonds, the majority of contacts between PLpro and inhibitor GRL0617 are hydrophobic in nature. The 1-naphthyl group is partly solvent-exposed but forms hydrophobic interactions with the aromatic rings of Y265 and Y269 and with the side chains of P248 and P249 (Fig. 3C). These residues line the pocket and accommodate the leucine at the P4 position of PLpro substrates (10) (Fig. 3B). The (R)-methyl group, attached to the stereocenter of the inhibitor, points directly into the interior of the protein between Y265 and T302, where it is accommodated by a cavity that is mostly polar in nature. The positions of 3 bound water molecules in this cleft suggest the potential for extending the (R)-methyl group further into the pocket by the addition of polar substituents.

The di-substituted benzene ring at the opposite end of the inhibitor occupies the putative P3 position of bound substrate. The benzene ring stacks against the aliphatic portions of G164, D165 and Q270, whereas the ortho-methyl substituent at the R1 position points into the floor of the cavity, which is lined by the side chains of Y265, Y274 and L163 (Fig. 3C). The other ring substituent, -NH₂ at the R3 position of GRL0617, extends from the opening of the cleft where it is surrounded by a series of polar groups, including the side chain oxygens of Q270 and E168 and the hydroxyl of Y269, any of which could serve as a hydrogen bond acceptor (Fig. 3 C and D).

Comparison of the unbound and inhibitor-bound structures reveals 2 significant conformational differences, both presumed to be induced by inhibitor binding. In the apoenzyme structure, a highly mobile loop hinged by 2 glycine residues (G267-G272) is positioned in different conformations in each of the 3 monomers of the asymmetric unit (10). Movements of homologous loops in the deubiquitinating enzymes USP14 and HAUSP upon substrate binding have been observed (19, 20). With PLpro, inhibitor-binding induces closure of this loop such that it clamps the inhibitor to the body of the protein (Fig. 3D). The side chains of Y269 and Q270 become well-defined and reorient to close over the inhibitor, whereas the mainchain of the loop moves to within hydrogen bonding distance of the carbonyl at the center of the inhibitor. Additional movements are observed upon inhibitor binding whereby the side chain of L163 moves to cradle the ortho-methyl of the benzene ring while simultaneously blocking access to the catalytic triad. The plasticity of this region, especially the G267-G272 loop, which is a highly variable region both in length and sequence among papain-like proteases, may account for the range of substrates recognized by these enzymes.

In contrast to the motions observed outside of the catalytic center, the residues of the catalytic triad of PLpro (C112, H273, D287) undergo limited movement between the bound and unbound conformations. Interestingly, a significant amount of residual electron density surrounding the sulfur atom of the catalytic cysteine was observed. Modeling and refinement of this density against a fully oxidized sulfur atom was consistent with a sulfonic acid moiety, vs. sulfinic or sulfenic acids (Fig. S3). This observation likely explains the inability of PLpro to regain full activity after incubation with inhibitor over extended periods of time. The presence of reducing agents in solution most likely helps to maintain the active site cysteine of PLpro in a reduced state. However, upon inhibitor binding, loop closure may restrict access to the cysteine by reducing agents but still allow for oxidation, thereby generating an inactive enzyme. A similar mechanism has been proposed for protein tyrosine phosphatase 1B inhibitors (22).

Primary HTS Screening.

A compound library consisting of 50,080 structurally diverse small molecules was purchased from ChemBridge and maintained as 10 mM stock solutions dissolved in DMSO and stored desiccated at −20°C. The automated primary screen was performed on a Tecan Freedom EVO 200 robot equipped with a Tecan 3 × 3 mounted 96-well dispenser and a 384-pin stainless steel pin tool (V&P Scientific) with a 100-nL capillary capacity. Fluorescence values were measured on an integrated Tecan Genios Pro microplate reader. All assays were performed in duplicate at room temperature, in black flat-bottom 384-well plates (Matrix Technologies) containing a final reaction volume of 50 μL. The assays were assembled as follows: 40 μL of 142 nM PLpro in Buffer A (50 mM Hepes, pH 7.5; 0.1 mg/ml BSA; and 5 mM DTT) was dispensed into wells and then incubated with 100 nL of 10 mM inhibitor (20 μM final concentration) for ≈5 min. Reactions were then initiated with 10 μL of 250 μM RLRGG-AMC in Buffer A, shaken vigorously for 30 s, and then incubated for 6 min. Reactions were subsequently quenched with 10 μL of 0.5 M acetic acid, shaken for 30 s, and measured for fluorescence emission intensity (excitation λ: 360 nm; emission λ: 460 nm). Each 384-well plate contained 32 positive control wells (100 nL of DMSO replacing 100 nL of inhibitor in DMSO) and 32 negative control wells (assay components lacking PLpro). Because of the low hit rate of compounds displaying significant PLpro inhibition, compounds that showed >35% inhibition were selected for further analysis. Secondary screening methods are included in SI Text.

Synthesis of GRL0617 and Related Compounds.

¹H-NMR and ¹³C-NMR spectra were recorded on Varian Oxford 300, Bruker Avance 400 spectrometers. Optical rotations were recorded on Perkin-Elmer 341 polarimeter. Anhydrous solvent was obtained by distillation of dichloromethane from CaH₂. All other solvents were reagent grade. Column chromatography was performed with Whatman 240–400 mesh silica gel under low pressure of 3–5 psi. TLC was carried out with E. Merck silica gel 60-F-254 plates. The detailed protocols for synthesis of compounds 1–7 are included in SI Text.

IC₅₀ Value Determination.

IC₅₀ measurements were performed by hand, in duplicate, in a 96-well plate format. Buffer, enzyme, and substrate conditions matched those of the primary screen. Reactions containing 50 μM substrate, 2% DMSO, and varying concentrations of inhibitor (0–200 μM) were initiated with the addition of enzyme. Reaction progress was monitored continuously on a Tecan Genios Pro microplate reader (excitation λ: 360 nm; emission λ: 460 nm). Data were fit to the equation : v_i = v_o/(1 + [I]/IC₅₀) by using the Enzyme Kinetics module of SigmaPlot (v. 9.01 Systat) where v_i is the reaction rate in the presence of inhibitor, v_o is the reaction rate in the absence of inhibitor, and [I] is the inhibitor concentration.

Reversibility of Inhibition.

To test the reversibility of inhibition, 50 nM PLpro was incubated with and without inhibitor (at 20-fold the inhibitor IC₅₀ concentration) in buffer containing 0.05 mg/ml BSA; 50 mM Hepes, pH 7.5; 5 mM DTT; and 1% DMSO in a final volume of 3 mL, for 1 h at room temperature; 1.5 mL of each sample was then dialyzed against 1 L of dialysis buffer (50 mM Hepes, pH 7.5; 5 mM DTT) for 3 h at room temperature by using 10,000 molecular weight cutoff Slide-A-Lyzer dialysis cassettes (Pierce). Samples were transferred to 1 L of fresh dialysis buffer each hour. The other 1.5 mL of each sample (undialyzed samples) were excluded from dialysis but remained at room temperature for the 3-h time period. All samples were assayed for activity after the 3-h incubation in the same manner as used for IC₅₀ measurements.

PLpro de-ISGylating Assays.

PLpro activity with ISG15-AMC (Boston Biochem) was measured in 96-well half-volume plates at 25°C in buffer containing 50 mM Hepes, pH 7.5; 0.1 mg/ml BSA; 5 mM DTT; 2% DMSO; and fixed inhibitor concentrations of 0, 0.1, 1, and 3 μM. Substrate concentration was varied from 0 to 16 μM, and release of AMC was measured in the same manner as for the IC₅₀ measurements described above. The K_i and mode of inhibition of inhibitor GRL0617 were determined through Lineweaver–Burk analysis of the above data by using the Enzyme Kinetics module of SigmaPlot.

Inhibitor Specificity Assays.

The specificity of inhibitors 2, 5, and GRL0617 was tested against 2 human ubiquitin C-terminal hydrolases, UCH-L1 and UCH-L3, the human deubiquitinating enzyme HAUSP, the human de-ISGylating enzyme USP-18, and a coronaviral papain-like protease from HCoV NL63, PLP2. Full-length UCH-L1 and full-length UCH-L3 were purchased from Biomol International; the catalytic domain of HAUSP (residues 213–548) and full-length USP-18 were purchased from Boston Biochem; and PLP2 was purified as described (32). All kinetic assays were performed at 25°C in 50 mM Hepes, pH 7.5; 0.1 mg/ml BSA; and 5–10 mM DTT in a 96-well plate format. Enzymes were assayed in the absence and presence of 100 μM inhibitor, with 100 nM ubiquitin-AMC (Boston Biochem) as substrate (excitation λ: 360 nm; emission λ: 460 nm), with the exception of USP-18, which was assayed with 1 μM ISG15-AMC (Boston Biochem) as substrate. PLpro was assayed under the same conditions, as a control.

Electrophilic Labeling and Detection of DUB Profile in Vero E6 Cells.

For each labeling reaction, 50 μg of Vero E6 lysate was preincubated with 0–40 μM GRL0617 for 30 min in 60 μL of homogenization buffer (50 mM Tris, pH 7.5; 5 mM MgCl₂; 0.5 mM EDTA; 2 mM DTT; 2 mM ATP; and 250 mM sucrose). After the incubation, 0.8 μg of the probe HA-Ub-VS (Boston Biochem) was added and then incubated for an additional 30 min at room temperature. Samples were subjected to 10% SDS/PAGE, and the proteins were then transferred to nylon membrane and immunoblotted with mouse monoclonal antibody specific to HA followed by goat anti-mouse IgG-HRP. For samples containing PLpro, 0.1 μg of purified enzyme was added to cell lysate samples and analyzed as above. The DUB profile approach was modeled from the experiments of Kattenhorn et al. (33) and detailed materials and methods of lysis, labeling, and detection are included in SI Text.

SARS-CoVAntiviral Activity Assays.

Vero E6 cells were maintained in Minimal Essential Media (MEM) (Gibco) supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin (Gibco), and 10% FCS (Gemini Bio-Products). The SARS-CoV Urbani strain used in this study was provided by the Centers for Disease Control and Prevention (34). All experiments using SARS-CoV were carried out in a Biosafety Level 3 facility by using approved biosafety protocols.

Vero E6 cells were seeded onto flat-bottom, 96-well plates at a density of 9 × 10³ cells per well. Cells were either mock-infected with serum-free MEM or infected with 100-fold the median tissue culture infective dose of SARS-CoV Urbani per well in 100 μL of serum-free MEM and incubated for 1 h at 37°C with 5% CO₂. After the 1-h incubation period, the viral inoculum was removed and, 100 μL of MEM supplemented with 2% FCS and containing the inhibitor compound of interest at the desired concentration (serial 2-fold dilutions from 50 to 0.1 μM) was added. Cells were incubated for a period of 48 h at 37°C with 5% CO₂. Each condition was set up in triplicate, and antiviral assays were performed independently on at least 2 separate occasions. Cell viability was measured 48 h after infection by using the CellTiter-Glo Luminescent Cell Viability Assay (Promega), according to the manufacturer's recommendations. Cell viability for the CellTiter-Glo Luminescent Cell Viability Assay was measured as luminescence and output expressed as relative luciferase units (RLU).

We thank S. Forrester for help with crystallization condition screening, Y. M. Baez for assistance with kinetic specificity assays, B. D. Santarsiero for crystal structure data collection support, D. C. Mulhearn for computational modeling support, and T. E. O'Brien for assistance with statistical analysis of antiviral activity. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. This work was supported by Public Health Service Research Grant P01 AI060915 (“Development of Novel Protease Inhibitors as SARS Therapeutics”).