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 from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design 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 inhibits PLpro with k_inact/K_I = 9,600 M^-1 s^-1, achieves sub-μM EC₅₀ values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 μM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes 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.

Fig. 1. Structure of PLpro from SARS-CoV-2.

a Overall structure (PDB entry 7JIR) colored by domain and selected features labeled. b Interactions between PLpro and the noncovalent inhibitor GRL0617. Selected residues and substrate binding subsites are labeled.

Fig. 2. Design strategy for covalent PLpro inhibition.

a X-ray co-crystal structure of ubiquitin-propargylamine (cyan) covalently bound to Cys111 in PLpro (tan) from PDB entry 6XAA. Selected residues from PLpro and the LRGG motif of ubiquitin (cyan) are labeled and shown in stick representation. b Crystal structure of GRL0617 (cyan) bound to PLpro (PDB entry 7CMD). The distance between Sγ of Cys111 and the tolyl methyl of GRL0617 is labeled. c Components of covalent PLpro inhibitor candidates consisting of various electrophiles, a Gly-Gly mimetic linker, and the GRL0617 core. Reactive carbons on electrophiles are labeled with asterisks. d Mechanism of covalent bond formation between Cys111 and an inhibitor candidate with a fumarate ester electrophile.

Fig. 3. Docked poses of compounds 3, 5, and 7.

Compound 7 was docked both noncovalently and covalently. Structures of compounds are shown in Fig. 4. Docked poses for additional inhibitor candidates are shown in Supplementary Fig. 2. Ligand carbons are shown in cyan. Hydrogens were omitted for clarity.

Fig. 4. Synthesis of compounds 2-15.

Reaction conditions with yields in parentheses: I. Ac₂O, AcOH, DCM, 55%; II. HATU, DIPEA, DCM (3, 83%; 4, 91%); III. N₂H₄•H₂O, EtOH (5 and 6, 97%); IV. methyl (E)-4-chloro-4-oxobut-2-enoate, DIPEA, DCM for 7 (56%), and K₂CO₃, DMF for 8 (34%). Compounds 9 (50%), 10 (37%), 11 (56%), 12 (23%), and 13 (60%) were prepared with the corresponding acid chlorides under conditions described for step IV. Compounds 14 (89%) and 15 (83%) were prepared analogously to step II with 2-methylbenzoic acid and 5-acetamido-2-methylbenzoic acid, respectively.

Fig. 5. Characterization of a designed covalent PLpro inhibitor, compound 7.

a Fluorogenic peptide activity assay after 30-min preincubation with compound 7. Data are plotted for each of n = 2 independent samples. IC₅₀ is the concentration at which 50% inhibition was observed. Curve is the nonlinear regression to the normalized inhibitor dose response equation. b Time-dependent characterization with a fluorogenic peptide assay. Data points are k_obs values determined by fitting the exponential decay equation to initial rates determined at various inhibitor concentrations and preincubation times, normalized to no preincubation. k_obs data are presented as mean values determined from n = 2 independent samples. Line represents the linear regression yielding as its slope the second-order rate constant (k_inact/K_I). 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 Percent viability of Vero E6 cells after 48 h following pretreatment with 7 (black squares), pretreatment with 7 and infection with SARS-CoV-2 (red circles), or pretreatment with remdesivir and infected with SARS-CoV-2 (blue triangles). Data are plotted as the mean of n = 2 independent samples. EC₅₀ is the concentration at which 50% effect was observed. Curves are nonlinear regressions to the normalized dose response equation. Source data are provided as a Source Data file.

Fig. 6. Inhibition of the deISGylase activity of full-length SARS-CoV-2 hemagglutinin (HA)-Nsp3 transiently expressed in HEK293T cells.

A Anti-HA beads after immunoprecipitation (IP) and whole cell lysates probed with anti-HA antibody. The asterisks indicate immunoglobulin G (IgG) heavy chain (HC) and light chain (LC). Anti-HA beads were assayed for Nsp3 deISGylase activity using an ISG15-CHOP2 assay in the presence of the dose range of B compound 7 or C GRL0617. Data are presented as mean values for n = 2 independent experiments for compound 7 and n = 3 independent samples for GRL0617. Curves are nonlinear regressions to the normalized dose response equation. Source data are provided as a Source Data file.

Fig. 7. Crystal structure of SARS-CoV-2 PLpro in complex with covalent inhibitor 7.

a Overall structure. The electron density for 7 is shown in blue mesh (Fo - Fc omit map contoured at 1.5 σ). b Interactions between binding site residues (green sticks) and 7 (cyan sticks). c Composite omit map (σ = 1.0) showing the electron density for the covalent bond between Cys111 and 7. d Superposition of selected structures highlighting the positions of the side chain of Leu162 (sticks) and the BL2 loop (cartoon) in the absence and presence of selected inhibitors: Ligand-free (PDB entry 6W9C, light green), glycerol-bound (PDB entry 6WZU, purple), GRL0617-bound (PDB entry 7CMD, light purple), and compound 7-bound (this work; cyan). Additional structures are shown in Supplementary Fig. 11. e Structural basis for selectivity toward PLpro. Superposition of 7 bound to PLpro onto human carboxy terminal hydrolase UCH-L1 (PDB entry 3KW5). The crossover loop of UCH-L1 (residues 153–157) covers the narrow groove and likely blocks the naphthylmethylamine core of 7 from binding. f Superposition of 7 bound to PLpro onto human USP4 (PDB entry 2Y6E). Severe steric clashes are present between the naphthyl ring of 7 and Phe828 and Lys838 of USP4 (light pink sticks), both of which are conserved in 80% of human USPs.