A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication

Abstract

We report the discovery and optimization of a potent inhibitor against the papain-like protease (PLpro) from the coronavirus that causes severe acute respiratory syndrome (SARS-CoV). This unique protease is not only responsible for processing the viral polyprotein into its functional units but is also capable of cleaving ubiquitin and ISG15 conjugates and plays a significant role in helping SARS-CoV evade the human immune system. We screened a structurally diverse library of 50,080 compounds for inhibitors of PLpro and discovered a noncovalent lead inhibitor with an IC(50) value of 20 microM, which was improved to 600 nM via synthetic optimization. The resulting compound, GRL0617, inhibited SARS-CoV viral replication in Vero E6 cells with an EC(50) of 15 microM and had no associated cytotoxicity. The X-ray structure of PLpro in complex with GRL0617 indicates that the compound has a unique mode of inhibition whereby it binds within the S4-S3 subsites of the enzyme and induces a loop closure that shuts down catalysis at the active site. These findings provide proof-of-principle that PLpro is a viable target for development of antivirals directed against SARS-CoV, and that potent noncovalent cysteine protease inhibitors can be developed with specificity directed toward pathogenic deubiquitinating enzymes without inhibiting host DUBs.

Fig. 1.

High-throughput screening using a ubiquitin-like peptide substrate identifies a PLpro inhibitor. (A) Schematic of the SARS-CoV polyprotein, with cleavage sites of the 2 proteases, PLpro (red) and 3CLpro (blue), indicated by color-coded arrows and dashed lines. Nsp numbering is indicated below the polyprotein. (B) The sequences of the 3 PLpro polyprotein cleavage sites are aligned with the peptide substrate used for high-throughput screening. Conserved residues are shown in bold. (C) Results from screening 50,080 compounds in duplicate for inhibition of PLpro activity. The replicate plot shows the percentage inhibition of PLpro by each compound. The structure of the lead compound 7724772 is shown, and its activity is plotted in red. The hit zone for the assay (>35% inhibition) is indicated by a white box.

Fig. 2.

PLpro inhibitors follow a structure–activity relationship and have antiviral activity against SARS coronavirus. (A) The structure–activity relationship of synthetic compounds based on the HTS lead compound 7724772 is shown in a table format. An asterisk indicates the position of the chiral carbon in the chemical structure at top. IC₅₀ values represent inhibitory activity of PLpro in vitro. EC₅₀ values represent antiviral activity of the compounds against SARS-CoV. NI, no inhibition; NA, not assayed. (B) SARS-CoV infected (red points) and mock-infected (blue points) Vero E6 cells were incubated in the presence of inhibitor compounds 7724772, 5, 6, or GRL0167 at the concentrations indicated for 48 h. Cell viability was measured 48 h after infection by using the CellTiter-Glo Luminescent Cell Viability Assay (Promega), and output was expressed as RLU. The error bars represent the standard deviation between triplicate samples. Structures of the tested inhibitors are included as Insets.

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.

Fig. 4.

Inhibitor GRL0617 is selective for SARS-CoV PLpro. (A) IC₅₀ values of compounds 5, 6, and GRL0617 are listed for PLpro and 5 other papain-like proteases. (B) Stereo representation of HAUSP (Protein Data Bank ID no. 1NBF, with ubiquitin ligand removed) superimposed with the PLpro-inhibitor complex. HAUSP is shown in green, PLpro in blue, and the inhibitor molecule in orange. HAUSP active site residues that clash with the inhibitor are labeled. (C) An α-HA Western blot of lysed Vero E6 cells treated with HA-Ub-VS in the presence of N-ethyl-maleimide (NEM) or varying concentrations of GRL0617. (D) An α-HA Western blot of lysed Vero E6 cells mixed with 0.1 μg of PLpro and HA-Ub-VS, both in the absence and presence of 40 μM GRL0617.