Design of SARS-CoV-2 PLpro Inhibitors for COVID-19 Antiviral Therapy Leveraging Binding Cooperativity

Abstract

Antiviral agents that complement vaccination are urgently needed to end the COVID-19 pandemic. The SARS-CoV-2 papain-like protease (PLpro), one of only two essential cysteine proteases that regulate viral replication, also dysregulates host immune sensing by binding and deubiquitination of host protein substrates. PLpro is a promising therapeutic target, albeit challenging owing to featureless P1 and P2 sites recognizing glycine. To overcome this challenge, we leveraged the cooperativity of multiple shallow binding sites on the PLpro surface, yielding novel 2-phenylthiophenes with nanomolar inhibitory potency. New cocrystal structures confirmed that ligand binding induces new interactions with PLpro: by closing of the BL2 loop of PLpro forming a novel "BL2 groove" and by mimicking the binding interaction of ubiquitin with Glu167 of PLpro. Together, this binding cooperativity translates to the most potent PLpro inhibitors reported to date, with slow off-rates, improved binding affinities, and low micromolar antiviral potency in SARS-CoV-2-infected human cells.

Figure 1.. Structure-guided design of SARS-CoV-2 PLpro inhibitors to explore druggable binding sites.

A) Identification of potential ligand binding Sites I-V (PDB: 3E9S). Key hydrogen bonds are shown as red, dashed lines, with distances (Å) labeled in italics. B) A superposition of GRL0617 (cyan; PDB 3E9S) onto the PLpro-ubiquitin structure (orange/magenta; PDB 4MM3) shows that Glu167 of PLpro (magenta) interacts with Arg72 of ubiquitin (orange) in Site I and Arg166 interacts with Gln49 of ubiquitin in Site II. New compounds were designed to mimic these two key interactions to improve binding affinity and to engage Sites I and II. C) Modeling of ZN-2-184 (5) (wheat) bound to PLpro, superimposed with PLpro-GRL0617 (cyan, PDB 3E9S), with the azetidine ring capturing the electrostatic interaction with Glu167 in Site I; D) Modeling of ZN-3-56 (13) (wheat) bound to PLpro, superimposed with PLpro-GRL0617 (cyan, PDB 3E9S; showing the glycine sidechain of ZN-3-56 (13) forming electrostatic interactions with Glu167 and Arg166. E) Summary of structure activity relationships of selected compounds designed to engage with Sites I-V of PLpro (Table 1 details potency and affinity for the selected compounds and full SAR is provided in Tables S1–S5).

Figure 2.. PLpro inhibition and binding affinity.

A) Chemical structures and dose response of the most potent PLpro inhibitors in enzymatic assays: GRL0617 (1), ZN-2-184 (5), ZN-3-80 (65), XR8-24 (73), XR8-23 (72). B) Comparison of K_D measured by SPR with IC₅₀ measured in enzyme inhibition assay. Also see Figure S1.

Figure 3.. Superposition of four novel SARS-CoV-2 PLpro:inhibitor crystal structures.

The chemical structures of inhibitors, their IDs, and associated pdb codes are listed at right, with colored boxes corresponding to the coloring used in the structures at left: XR8-24 (73), XR8-65 (86), XR8-69 (89), XR8-83 (92). The statistics for the crystal data processing and refinement, as well as Fo-Fc maps, are included in Supplemental Data 2.

Figure 4.. Structural characterization of SARS-CoV-2 PLpro inhibitors.

A) XR8-24 (73)-bound PLpro structure (yellow) superimposed with GRL-0617-bound (cyan) PLpro (PDB:7JRN). The extended structure of XR-8-24 (73) allows: 1) an additional electrostatic interaction with Glu167; and 2) occupies the BL2 groove. B) 2Fo-Fc electron density map of XR8-24 (73). The map is shown as blue mesh and is contoured at 1 sigma around the inhibitor (PDB: 7LBS). C) Details of the water-mediated interaction of XR8-24 (73) (yellow) with PLpro. D) Superposition of XR8-24 (73) (yellow) onto PLpro (blue) complexed with a covalent peptide-based inhibitor (cyan), VIR250 (PDB: 6WUU).

Figure 5.. PLpro inhibition by loop reorganization and distal blocking of substrate access to the active site.

A) An overlay of the XR8-24 (73):SARS CoV-2 PLpro structure with that of the apoenzyme structure (PDB: 7CJD), highlighting the BL2 loop reoragnization as the Gln269 mainchain residue in the BL2 loop is closed to form a hydrogen bond interaction with XR8-24 (73). B) The structure of the XR8-24(73)-bound PLpro structure superimposed with Ub-bound PLpro (PDB: 6XAA, orange) and ISG15-bound Plpro structures (PDB: 6YVA, teal). C) XR8-24 (73) extends into a novel binding site, the BL2 groove, which is positioned between the β8 and β9 strands, adjacent to the BL2 loop. The BL2 groove is approximately 15Å from the active site. Binding of XR8-24 (73) blocks the tails of ubiquitin (orange) or ISG15 (teal) from accessing the active site channel.

Figure 6.. Association and dissociation rates and binding cooperativity.

SPR was used to measure (A) association rates and (B) dissociation rates for PLpro inhibitors: GRL0617 (1), ZN-2-184 (5), ZN-3-80 (65), XR8-24 (73), XR8-23 (72), XR8-89 (94). (C) Binding affinity (K_D determined by SPR) was used to demonstrate potential cooperativity by engaging multiple weak interactions across multiple binding sites.

Figure 7.. Inhibition PLpro DUB activity.

Inhibition of (A) deubiquitinating and (B) de-ISGylating activities of Plpro inhibitors: GRL0617 (1), ZN-2-184 (5), ZN-3-80 (65), XR8-24 (73), XR8-23 (72).

Figure 8.. Improved PLpro inhibitors show potent antiviral efficacy.

To measure reduction in virus yield, A549-hACE2 cells were infected with MOI = 0.01 of SARS-CoV-2 cultured in Vero E6 cells with and without various concentrations GRL0617, XR8-23 (72), or XR8-24 (73) (cytotoxicity was not observed under the assay conditions at < 50 μM for XR8-24 (72) and < 10 μM for XR8-23 (73). After 48 hours, supernatants were harvested, and RNA was isolated and quantified by RT-qPCR. The data show mean ± S.D.

Scheme 1.

Reagents and conditions: I. (Amines, aldehydes or ketones), HOAc, NaBH₃CN, MeOH; II. (Amines or carboxylic acids), HATU, DMAP, DMF, rt; III. HCl (4M in dioxane), DCM; IV. XPhos Pd G2, K₃PO₄, DMF/EtOH/H₂O, 95 °C.

Scheme 2.

Reagents and conditions: I.Ti(OEt)₄, NaBH₄, THF, −78 °C to rt; II. HCl (conc. aq.), dioxane; III. HATU, DMAP, DMF, rt; IV. HCl (4M in dioxane), DCM