Structure-Based Design of Covalent SARS-CoV-2 Papain-like Protease Inhibitors

Abstract

The COVID-19 pandemic is caused by SARS-CoV-2, a highly transmissible and pathogenic RNA betacoronavirus. Like other RNA viruses, SARS-CoV-2 continues to evolve with or without drug selection pressure, and many variants have emerged since the beginning of the pandemic. The papain-like protease, PL^pro, is a cysteine protease that cleaves viral polyproteins as well as ubiquitin and ISG15 modifications from host proteins. Leveraging our recently discovered Val70^Ub binding site in PL^pro, we designed covalent PL^pro inhibitors by connecting cysteine reactive warheads to the biarylphenyl PL^pro inhibitors via flexible linkers. Several leads displayed potent enzymatic inhibition (IC₅₀ = 0.1-0.3 μM) and antiviral activity (EC₅₀ = 0.09-0.96 μM). Fumaramide inhibitors Jun13567 (15), Jun13728 (16), and Jun13714 (18) showed favorable in vivo pharmacokinetic properties with intraperitoneal injection. The X-ray crystal structure of PL^pro with Jun13567 (15) validated our design strategy, revealing covalent conjugation between the catalytic Cys111 and the fumaramide warhead. The results suggest these covalent PL^pro inhibitors are promising SARS-CoV-2 antiviral drug candidates.

Figure 1.

Structures and activities of representative SARS-CoV-2 PL^pro inhibitors. The enzymatic inhibitory IC₅₀, K_i, and antiviral EC₅₀ values were from references: GRL0617, Jun9722, Jun9843, XR8–23, compound 7 (cmp7), Jun11165, Jun11313, Jun12682, and PF-07957472. It is worth noting that the enzymatic assay and antiviral assay were performed under different conditions. Therefore, comparing the IC₅₀, K_i, and EC₅₀ values from different studies is not meaningful.

Figure 2.

Design of covalent SARS-CoV-2 PL^pro inhibitors. (A) Superposition of the X-ray crystal structures of SARS-CoV-2 PL^pro with Jun11313 (PDB: 8UVM, green) and Jun12682 (PDB: 8UOB, yellow). (B) Design of biarylphenyl covalent PL^pro inhibitors based on Jun11313 and Jun12682.

Figure 3.

Structure-activity relationship (SAR) studies of covalent SARS-CoV-2 PL^pro inhibitors with diverse warheads. (A) PL^pro inhibitors with the diacylhydrazine linker. (B) PL^pro inhibitors with the glycine-glycine linker. (C) Summary of SAR. IC₅₀ values were determined with the FRET-based enzymatic assay. CC₅₀ values were determined in Vero E6 cells with a 48-h incubation time using the neutral red method. Antiviral EC₅₀ values were determined in Caco2-AT cells infected with the icSARS-CoV-2-nLuc reporter virus. T_1/2 was determined in the mouse liver microsomal stability assay.

Figure. 4.

Mechanistic studies of Jun13728 (16). (A) Differential scanning fluorimetry assay of Jun13728 (16) in stabilizing SARS-CoV-2 PL^pro. Jun12682 was included as a positive control for comparison. (B) K_i plot of Jun13728 (16) in inhibiting SARS-CoV-2 PL^pro hydrolysis of Ub-AMC. (C) K_i plot of Jun13728 (16) in inhibiting SARS-CoV-2 PL^pro hydrolysis of ISG15-AMC. (D) Counter-screening of Jun13728 (16) against USP7 in hydrolyzing Ub-AMC. (E) Counter screening of Jun13728 (16) against USP14 in hydrolyzing Ub-AMC. Data in (A), (B), and (C) are presented as mean ± S.D. of three technical repeats. Data in (D) and (E) are presented as mean ± S.D. of two technical repeats.

Figure. 5.

Antiviral activity of PL^pro inhibitor Jun13728 (16) against SARS-CoV-2 variants and a drug-resistant mutant. (A) Plaque images of nirmatrelvir and Jun13728 (16) in inhibiting SARS-CoV-2 WT WA1, Omicron variants XBB.1.16 and JN.1, and nirmatrelvir-resistant mutant rL50F/E166A/L167F. (B) EC₅₀ curves for nirmatrelvir. (C) EC₅₀ curves for Jun13728 (16). The assays were performed in two technical duplicates and the EC₅₀ values are mean ± S.D.

Figure 6.

In vivo PK of SARS-CoV-2 PL^pro inhibitors. Plasma drug concentrations of PL^pro inhibitors following i.p. or p.o. administration of 50 mg/kg in C57BL/6J (6 to 8 weeks old, n=3 per group). Compounds were formulated in 10% DMSO/90% corn oil for i.p. injection or 0.5% methylcellulose/2% Tween 80 in water for p.o. administration.

Figure 7.

The overall structure of the PL^pro complex with Jun13567 (15). (A) A surface representation of PL^pro with ribbon backbone (green) is shown, with Jun13567 (15) depicted as orange ball-and-stick models. (B) The atomic model of the Jun13567 (15) (green ball-and-stick) binding site in PL^pro. Light gray sticks represent residues within 5 Å of the inhibitor, with hydrogen bonds indicated by black dashed lines. (C) The Jun13567 (15) polder map (an unbiased omit map that removes the bulk solvent contribution near the inhibitor) is displayed as a blue mesh with 3σ contours around the inhibitor and the covalently linked catalytic Cys111 residue (yellow) of PL^pro. (D) Surface representation of the PL^pro substrate-binding pocket occupied by Jun13567 (15) (orange sticks).

Figure 8.

(A) Superposed crystal structures of SARS-CoV-2 PL^pro with Jun13567 (15) (green) and cmp7 (orange) (PDB ID 8UEA). The PL^pro protein is shown with ribbon backbones and inhibitors are represented using stick models. (B) Jun13567 (15) superposed with Jun11313. (C) Structure of Jun13567 (15) PL^pro superposed onto the apo protein, showing secondary structural changes. A black arrow indicates movement of the BL2 loop. (D) Comparison of PL^pro side chain conformations with and without bound inhibitor. Solid black arrows highlight the the structural differences of the Leu162 and Gln269 side chains.

Scheme 1.

Synthesis of covalent SARS-CoV-2 PL^pro inhibitors with various reactive warheads

Scheme 2.

Synthesis of covalent SARS-CoV-2 PL^pro inhibitors Jun13891 (24), Jun13917 (23), and Jun13918 (22).