Design of a SARS-CoV-2 papain-like protease inhibitor with antiviral efficacy in a mouse model

Abstract

The emergence of SARS-CoV-2 variants and drug-resistant mutants calls for additional oral antivirals. The SARS-CoV-2 papain-like protease (PL^pro) is a promising but challenging drug target. We designed and synthesized 85 noncovalent PL^pro inhibitors that bind to a recently discovered ubiquitin binding site and the known BL2 groove pocket near the S4 subsite. Leads inhibited PL^pro with the inhibitory constant K_i values from 13.2 to 88.2 nanomolar. The co-crystal structures of PL^pro with eight leads revealed their interaction modes. The in vivo lead Jun12682 inhibited SARS-CoV-2 and its variants, including nirmatrelvir-resistant strains with EC₅₀ from 0.44 to 2.02 micromolar. Oral treatment with Jun12682 improved survival and reduced lung viral loads and lesions in a SARS-CoV-2 infection mouse model, suggesting that PL^pro inhibitors are promising oral SARS-CoV-2 antiviral candidates.

Fig. 1.. X-ray crystal structure of the covalent inhibitor Jun11313 with SARS-CoV-2 PL^pro and structure-based design of biarylphenyl SARS-CoV-2 PL^pro inhibitors.

(A) Design of the hybrid covalent inhibitor Jun11313 based on XR8–24 and Cp7. (B) Atomic model of the Jun11313 (in green balls and sticks) binding site in PL^pro (light gray sticks, residues within a 5 Å distance of the inhibitor), with hydrogen bonds indicated with black dashed lines. The Jun11313 polder map (an unbiased omit map that removes the bulk solvent contribution near the inhibitor (38)) is displayed as a gray mesh with 4σ contours. (C) Superposition of the PL^pro-Jun11313 structure onto the structure of the PL^pro-XR8–24 complex (PDB 7LBS), with XR8–24 in yellow sticks and spheres, with the relevant residues for binding of both compounds indicated. (D) Superimposed X-ray crystal structures of SARS-CoV-2 PL^pro with Jun11313 (green) (PDB: 8UVM), XR8–24 (yellow) (PDB: 7LBS), and ubiquitin (orange) (PDB: 6XAA). (E) Generic chemical structure of the designed biarylphenyl PL^pro inhibitor. Critical interactions are highlighted. (F) Flow chart for the lead optimziation of PL^pro inhibitors. Jun12682 was selected as the in vivo lead candidate.

Fig. 2.. Representative biarylbenzamide series of SARS-CoV-2 PL^pro inhibitors. (A) Positive control GRL0617.

(B) Thiophene-containing PL^pro inhibitors. (C-F) Pyrazole-containing PL^pro inhibitors. The FRET IC₅₀ and K_i values were determined in the enzymatic assay using the substrate Dabcyl-FTLRGG/APTKV-E(Edans). Cytotoxicity CC₅₀ was determined in Vero E6 cells with a 48 h incubation. FlipGFP PL^pro EC₅₀ values were determined in 293T cells transfected with PL^pro and FlipGFP-expressing plasmids. SARS-CoV-2 antiviral EC₅₀ values were determined using the icSARS-CoV-2-nLuc reporter virus in a Caco-2 line expressing hACE2 and hTMPRSS2 (Caco2-AT) with 2 μM p-gp inhibitor CP-100356. T_1/2 refers to the half-life of PL^pro inhibitors in mouse liver microsomes. Data are presented as mean ± s.d. of three technical replicates.

Fig. 3.. Antiviral activity of PL^pro inhibitors against SARS-CoV-2 variants and mechanistic studies of Jun12682.

Antiviral activity of nirmatrelvir (A), Jun11941 (B), and Jun12682 (C) against SARS-CoV-2 WA1 strain (WT) (USA-WA1/2020), Omicron strain (lineage B.1.1.529, BA.2; HCoV-19/USA/CO-CDPHE-2102544747/2021), Delta strain (Lineage B.1.617.2; hCoV-19/USA/MD-HP05647/2021), and nirmatrelvir-resistant strains rNsp5-S144M, rNsp5-L50F/E166V, and rNsp5-L50F/E166A/L167F was determined using a cell viability assay with Vero-AT cells. Data in A, B, and C are representative results of at least two independent experiments, and presented as mean ± s.d. of three technical replicates. (D) IC₅₀ curve of Jun12682 in inhibiting SARS-CoV-2 PL^pro hydrolysis of Ub-AMC. (E) K_i curve of Jun12682 in inhibiting SARS-CoV-2 PL^pro hydrolysis of Ub-AMC. (F) IC₅₀ curve of Jun12682 in inhibiting SARS-CoV-2 PL^pro hydrolysis of ISG15-AMC. (G) K_i curve of Jun12682 in inhibiting SARS-CoV-2 PL^pro hydrolysis of ISG15-AMC. Data in D, E, F, and G are presented as mean ± s.d. of three technical replicates. (H) Counter screening of Jun12682 in inhibiting USP7 hydrolysis of Ub-AMC. (I) Counter screening of Jun12682 in inhibiting USP14 hydrolysis of Ub-AMC. (J) Differential scanning fluorimetry assay of Jun12682 and GRL0617 in stabilizing the SARS-CoV-2 PL^pro. Data in H, I, and J are presented as mean ± s.d. of two technical replicates.

Fig. 4.. X-ray crystal structures of SARS-CoV-2 PL^pro with biarylphenyl PL^pro inhibitors.

Atomic model of the Jun12682 (in orange balls and sticks) binding site in SARS-CoV-2 PL^pro (residues within 5 Å of the inhibitor are shown in light gray sticks), with hydrogen bonds displayed in black dashed lines, van der Waals contacts as red dashed lines, and π–π interactions as light green dashed lines. Gallery of the polder maps (38) of the inhibitors, displayed as a gray mesh with contour levels between 2 and 4σ. The PL^pro inhibitory constant K_i in the FRET enzymatic assay and the SARS-CoV-2 antiviral activity EC₅₀ in Caco-2 cells were shown for each compound.

Fig. 5.. In vitro and in vivo PK profiling of PL^pro inhibitors, and in vivo antiviral efficacy of Jun12682.

(A) Plasma drug concentration of Jun12199, Jun12197, Jun12713, Jun12603, and Jun11941 in C57BL/6J mice (6 – 8 weeks old) following p.o. administration of 50 mg/kg of compound in 0.5% methylcellulose and 2% Tween 80 in water (n=3 per group). (B) Plasma drug concentration of Jun12682, Jun12763, Jun12395, Jun12602, and Jun12351 in C57BL/6J mice (6 – 8 weeks old) following p.o. administration of 50 mg/kg of compound in 0.5% methylcellulose and 2% Tween 80 in water. (C) Plasma drug concentration of Jun12682 in C57BL/6J mice (6 – 8 weeks old) following p.o. administration of 50 mg/kg and i.v. injection of 10 mg/kg (n=3 per group). (D) In vitro PK parameters of Jun12682. (E) Experimental design for the 5-day treatment experiment. Young BALB/c mice were intranasally (IN) inoculated with 5,600 PFU of SARS2-N501Y_MA30 and subsequently orally administered 500 mg/kg Jun12682 or vehicle twice a day (BID) for five days (BID_5). (F) Body weight loss and (G) survival rate of the BID_5 mice experiment. (H) Experimental design for the 3-day treatment experiment. Young BALB/c mice were intranasally (IN) inoculated with 5,600 PFU of SARS2-N501Y_MA30 and subsequently orally administered 125, 250 mg/kg Jun12682 or vehicle BID for three days (BID_3). (I) Body weight loss and (J) survival rate of the BID_3 mice experiment. Data in F, G, I, and J are pooled from two independent experiments (n=10 per group) and are shown as mean ± s.e.m. The p values in G and J were determined using a log-rank (Mantel–Cox) test. (K) Viral titers in lungs collected at 2 and 4 DPI from vehicle- or 250 mg/kg Jun12682-treated mice (n=5 per group). Data are mean ± s.e.m and analyzed with unpaired t test with Welch’s correction. *, p< 0.05. (L-M) Quantitative PCR analysis of viral nucleocapsid gene (L) and cellular cytokines (M) in lungs collected at 2 DPI from vehicle- or 250 mg/kg Jun12682-treated mice (n=5 per group). (N-Q) Lungs collected at 4 DPI from vehicle- or 250 mg/kg Jun12682-treated mice (n=5 per group) were stained with haematoxylin and eosin (H&E) (N) or immunostained for SARS-CoV-2 nucleocapsid (P), and the pathological lesions and immunostaining were quantified (O and Q, respectively). N, H&E stained lungs from vehicle-treated infected mice exhibited airway edema (asterisks), hyaline membranes (HM, arrowheads), and interstitial thickness (number sign). Scale bars, 100 μm (top) and 50 μm (bottom). O, Summary scores of lung lesions (n = 5 per group). P, Lungs from vehicle- or Jun12682-treated mice (n = 5 per group) were immunostained to detect SARS-CoV-2 nucleocapsid protein (brown color staining). Scale bars, 100 μm (top) and 50 μm (bottom). Q, Summary scores of nucleocapsid immunostaining of lungs. Data in L, M, O and Q are mean ± sem and analyzed with unpaired t test with Welch’s correction. ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001.