SARS-CoV-2 Papain-Like Protease: Drug Design, Assay Development, and Drug Resistance

SARS-CoV-2 papain-like protease (PLpro) is a compelling but historically underdeveloped antiviral target. Unlike the viral main protease (Mpro), which rapidly became the focus of intensive drug-discovery efforts and yielded clinical candidates and approved drugs, PLpro posed a more challenging medicinal chemistry problem: a shallow, flexible substrate-recognition surface and a mobile BL2 loop. Nevertheless, PLpro is a high-profile drug target because it is vital for viral replication by processing viral polyproteins and suppresses host innate immunity through deubiquitinating and deISGylating activities. These dual functions make PLpro more than a viral protease; it is a multifunctional immune-evasion enzyme whose inhibition could both block virus replication and restore antiviral host responses. This Account summarizes our group's effort to convert PLpro from a challenging target into a tractable antiviral drug-discovery platform. We began by developing and applying orthogonal assays to identify specific PLpro inhibitors and triage false positives. High-throughput screening and drug-repurposing campaigns yielded early hits, including Jun9722, Jun9754, and tropifexor, but also revealed that biochemical inhibition alone was insufficient to predict cellular antiviral activity. This motivated us to develop a FlipGFP cell-based reporter assay as a BSL-2-compatible bridge between enzymology and live-virus studies. In addition, we later developed a fluorescence polarization assay using a fluorescein-labeled PLpro ligand to enable direct, high-throughput quantification of inhibitor binding. Together with FRET enzymatic assays, thermal shift experiments, cellular FlipGFP assays, and antiviral assays, these tools established a rigorous validation framework for PLpro medicinal chemistry. With this platform in place, we pursued structure-based PLpro inhibitor design. Early cocrystal structures showed that potent noncovalent inhibitors engage the BL2 groove and stabilize inhibitor-bound PLpro conformations. A major conceptual advance came from structural analysis of the Jun11313-bound PLpro complex, which revealed that an inhibitor substituent occupied a hydrophobic surface pocket corresponding to the Val70 position of ubiquitin. We designated this newly recognized region the Val70Ub pocket. Exploiting this pocket transformed PLpro inhibitor design by expanding ligand engagement beyond the canonical BL2 groove and enabling substantial gains in enzymatic inhibition and antiviral activity. This design principle led to orally active noncovalent inhibitors, including Jun12682 and the quinoline lead Jun13296, both of which showed potent enzymatic inhibition, cellular antiviral activity, favorable mouse pharmacokinetics, and protection in SARS-CoV-2 mouse infection models. We further extended the Val70Ub-centered recognition strategy to covalent inhibitor design by appending cysteine-reactive warheads (covalent electrophiles) to optimized noncovalent scaffolds, thereby generating compounds that retained BL2 groove and Val70Ub binding while engaging the catalytic Cys111. Finally, resistance studies identified E167, Y268, and Q269 as drug resistance hotspots, highlighting the need to design inhibitors that engage less mutation-sensitive binding sites. Overall, this Account illustrates how integrated assay development, structural biology, medicinal chemistry, pharmacology, virology, and resistance analysis can transform a challenging viral deubiquitinase into a credible antiviral target. The lessons from PLpro should inform future efforts to design broad-spectrum coronavirus PLpro inhibitors and to target other viral protease-deubiquitinase enzymes with shallow, flexible binding surfaces.