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 EC₅₀ 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.

Figure 1

(a) Structure and domains of PLpro from SARS-CoV-2 (PDB entry 7JIR). Selected features are labeled. (b) Interactions between PLpro and the noncovalent inhibitor GRL0617.

Figure 2

Left to right: Docked poses of compound 3, compound 5, and compound 7 docked both noncovalently and covalently. Structures of compounds are shown in Figure 3. Polar hydrogens have been added. Docked poses for additional inhibitor candidates are shown in Figure S2. Ligand carbons are shown in gray and predicted protein-ligand interactions are shown as dashed yellow lines.

Figure 3

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.

Figure 4. Characterization of a designed covalent PLpro inhibitor, compound 7.

(a) Fluorogenic peptide activity assay after 30-min preincubation with compound 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% confidence interval. 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 values were determined from n = 2 independent experiments with n = 2 independent samples each ± 95% confidence interval of the nonlinear regression. 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 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% confidence interval. Curves are nonlinear regressions to the normalized dose response equation.

Figure 5

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 and whole cell lysates probed with anti-HA antibody. The asterisk indicates IgG heavy and light chains. 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.

Figure 6. 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 UCHL1 (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 30). (d) 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.