INTERVENTIONCovalent PLpro inhibitors (compounds 7–13) designed from GRL0617 scaffold with various electrophilic warheads (fumarate ester, chloroacetamide, propiolamide, cyanoacetamide, α-cyanoacrylamide)
COMPARISONNoncovalent inhibitor GRL0617; noncovalent analogs 14 and 15; remdesivir; EIDD-1931
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This study designed covalent inhibitors of the SARS-CoV-2 papain-like protease (PLpro) by adding a peptidomimetic linker and reactive electrophile to the noncovalent inhibitor GRL0617. The most potent compound (compound 7) achieved a second-order rate constant of 9,600 M⁻¹ s⁻¹, sub-μM EC₅₀ values against three SARS-CoV-2 variants, and did not inhibit a panel of seven human deubiquitinases at >30 μM. These findings demonstrate that covalent inhibition of PLpro is a viable antiviral strategy, though the lead compound exhibited poor oral bioavailability and rapid clearance requiring further optimization.
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**Background:** Direct-acting antivirals are needed for COVID-19, caused by SARS-CoV-2. The papain-like protease (PLpro) domain of Nsp3 is essential for viral replication and also dysregulates the host immune response by cleaving ubiquitin and ISG15 from host proteins. PLpro is a promising but challenging drug target due to its flexible binding pockets and structural similarity to human deubiquitinases (DUBs). The noncovalent inhibitor GRL0617 inhibits PLpro (IC₅₀ ~1.2 μM) but its tolyl methyl group is located >7 Å from the catalytic Cys111, limiting potency.
**Methods:** The authors designed covalent inhibitors by replacing the tolyl methyl of GRL0617 with an N,N'-acetylacetohydrazine linker (as a Gly-Gly peptidomimetic) connected to various electrophiles: fumarate methyl ester, chloroacetamide, propiolamide, cyanoacetamide, and α-cyanoacrylamide. Covalent docking using an ensemble of 50 structural models prioritized compounds for synthesis. Seven covalent inhibitors (7–13) and two noncovalent derivatives (14, 15) were synthesized. Biochemical inhibition was measured using fluorogenic peptide substrate Z-RLRGG-AMC. Time-dependent inhibition assays determined k_inact/K_I values. Covalent adduct formation was confirmed by electrospray ionization mass spectrometry. Antiviral activity was assessed in Vero E6 cells (cytopathic effect protection) and Caco-2 cells (virus yield reduction) against USA-WA1/2020, Delta (B.1.617.2), and Omicron (B.1.1.529) variants. Selectivity was tested against seven human DUBs. An X-ray co-crystal structure of compound 7 bound to PLpro was determined at 3.10 Å resolution. Metabolic stability was measured in human, rat, and mouse liver microsomes and S9 fractions. Pharmacokinetics were assessed in male ICR mice (3 mg/kg i.v., 10 mg/kg p.o.).
**Key Results:** The fumarate methyl ester compound 7 was the most potent, with IC₅₀ = 0.094 μM after 30-min incubation and k_inact/K_I = 9,600 M⁻¹ s⁻¹. Compound 7 inhibited PLpro deubiquitinase and de-ISG15ylase activities with IC₅₀ values of 0.076 μM and 0.039 μM, respectively. It inhibited full-length Nsp3 deISGylase activity with IC₅₀ = 0.049 μM, compared to 4.7 μM for GRL0617. Compound 7 showed EC₅₀ = 1.1 μM in Vero E6 cells (comparable to remdesivir at 0.74 μM) with CC₅₀ >30 μM. In the presence of P-gp inhibitor CP-100356, EC₅₀ values were 0.068 μM (USA-WA1/2020), 0.29 μM (Delta), and 0.68 μM (Omicron). In Caco-2 cells, EC₉₀ values were 0.26 μM (USA-WA1/2020), >10 μM (Delta), and 2.4 μM (Omicron). Compound 7 did not inhibit any of seven human DUBs tested (IC₅₀ >30 μM). The X-ray structure confirmed covalent bond formation between Cys111 Sγ and the β carbon of the fumarate ester, with hydrogen bonds to Trp106, Asn109, Gly163, Gly271, Gln269, and Asp164. The BL2 loop shifted inward upon binding, and Leu162 rotated outward to accommodate the electrophile. In human liver microsomes, compound 7 had a half-life of 50 min (S9: 60 min), similar to noncovalent analog 14 (41 min; >60 min). However, compound 7 showed no oral bioavailability in mice (10 mg/kg p.o.), and after i.v. dosing (3 mg/kg) the half-life was 0.06 h with clearance of 11,047 mL/min/kg.
**Clinical Implications:** This study demonstrates that covalent inhibition of SARS-CoV-2 PLpro is achievable with high potency and selectivity over human DUBs. Compound 7 represents a significant improvement over the parent noncovalent inhibitor GRL0617 (~100-fold improvement in IC₅₀ against full-length Nsp3). However, the lead compound has major pharmacokinetic liabilities—no oral bioavailability, extremely rapid clearance, and a naphthyl group that is a known toxicophore and metabolic liability. The authors identify specific strategies for next-generation compounds: replacing the naphthyl with substituted 2-phenylthiophenes, introducing fluorine or deuterium at benzylic positions, replacing the benzylic methylene with cyclopropyl, and using more stable electrophiles such as substituted acrylamides or t-butyl esters. The structural insights from the co-crystal structure provide a clear roadmap for rational optimization. If these ADME/PK limitations can be addressed, covalent PLpro inhibitors could potentially be developed for combination therapy with 3CLpro inhibitors (e.g., nirmatrelvir) or RNA-dependent RNA polymerase inhibitors.
Covalent PLpro inhibitors (compounds 7–13) designed from GRL0617 scaffold with various electrophilic warheads (fumarate ester, chloroacetamide, propiolamide, cyanoacetamide, α-cyanoacrylamide)