Nanobodies targeting SARS-CoV-2 papain-like protease exert dual antiviral and anti-inflammatory effects

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.

Identification of a Potent Pan-Coronaviral Main Protease Inhibitor.

The global impact of SARS-CoV-2 and the continued emergence of zoonotic coronaviruses underscore the urgent need for broad-spectrum antivirals for pandemic preparedness. Herein, we report AVI8122, a covalent pan-coronaviral inhibitor that targets 19 Mpros across the α, β, γ, and δ genera, encompassing bat, human, and other animal coronaviruses. AVI8122 exhibits low nanomolar potency and favorable pharmacokinetics in mice. Structural studies reveal that AVI8122 forms a covalent bond with the catalytic cysteine and maintains conserved interactions within the active sites of these Mpros. In cellulo, AVI8122 efficiently inhibited the replication of SARS-CoV-2 and its variants of concern as well as the activity of Mpros from all four genera. Furthermore, in mouse models, AVI8122 conferred dose-dependent protection against a lethal SARS-CoV-2 infection. Our findings position AVI8122 as an early lead compound and a tractable structural starting point for the development of broad-spectrum antivirals against future coronavirus spillover threats.

PROTEASE AND ITS MODULATION BY SMALL MOLECULES”

Targeted degradation of Influenza a virus nucleoprotein via aptamer-based PROTACs for antiviral therapy

ABSTRACT Influenza A virus (IAV) poses a serious threat to public health due to its high mutability and rapid transmissibility. The viral nucleoprotein (NP), a highly conserved and essential component, has emerged as an ideal target for antiviral therapies. However, its biological function has proven challenging to modulate with conventional drugs, and no NP-targeting therapeutics have reached the market so far. Here, we report the development and application of an aptamer-based proteolysis-targeting chimera (PROTAC) for the targeted degradation of IAV NP. Utilizing the Direct-to-Biology (D2B) platform, we efficiently screened and identified NP-PROTAC#4 as a functional candidate. Subsequently, we developed a lipid nanoparticle (LNP) formulation of NP‑PROTAC#4 (LNP@NP‑PROTAC#4) for effective intracellular delivery. Importantly, LNP@NP-PROTAC#4 demonstrated potent antiviral activity both in vitro and in vivo. Mechanistically, NP-PROTAC#4 exerts its antiviral effects by targeting and degrading NP via the ubiquitin-proteasome system. In conclusion, our findings provide the first evidence that NP-targeted PROTAC degrader exhibits therapeutic effects, proposing a novel therapeutic strategy for IAV.

Targeted Degradation of Picornaviral 3C Protease via PROTACs Confers High Barrier to Viral Resistance and Broad‐Spectrum Antiviral Activity

ABSTRACT Diseases caused by picornaviruses pose a serious threat to society due to their high contagiousness and widespread prevalence. Beyond the poliovirus vaccine, antiviral therapeutics remain unavailable for most picornaviral infections. Moreover, existing inhibitors under development generally exhibit narrow‐spectrum activity and low barriers to resistance owing to the high mutability of RNA viral proteins. To address these issues, this study constructed a proteolysis‐targeting chimera (PROTAC) targeting the 3C protease (3CPro) — a key viral enzyme essential for picornavirus protein processing, host shutoff, and immune suppression. In enterovirus 71 (EV71), a representative member of the picornavirus family, experimental results showed that PROTAC molecule effectively inhibits viral replication through a dual mechanism of action: directly inhibiting the catalytic function of the 3CPro and inducing its degradation via the ubiquitin‐proteasome pathway. Moreover, this PROTAC molecule not only demonstrated effective degradation of previously reported and AI‐predicted potential drug‐resistant EV71 mutants, but also exhibited degradation activity against 3CPro from multiple picornaviruses, indicating its potential for broad‐spectrum antiviral activity. By integrating multidisciplinary approaches, this work demonstrates that targeted protein degradation can effectively induce the degradation of 3CPro in picornaviruses, supporting the development of resistance‐resistant, broad‐spectrum antiviral agents and highlighting their potential to address evolving viral threats.

ACE2-PNA conjugates exploit viral endocytosis for targeted intracellular delivery and exhibit dual antiviral efficacy against SARS-CoV-2

The COVID-19 pandemic and its protracted consequences underscore the urgent need for more effective antiviral strategies. Current antiviral strategies face a persistent challenge achieving sufficient viral suppression while minimizing off-target toxicity, particularly against such highly mutable viruses. Here, we describe a Receptor-Drug Conjugate (RDC) strategy, in which a therapeutic payload is covalently linked to a decoy receptor, enabling virus-triggered targeted intracellular delivery. Angiotensin-converting enzyme 2 (ACE2), as the essential receptor for SARS-CoV-2 entry, has been widely exploited for virus-neutralizing strategies. Targeting SARS-CoV-2 as a proof-of-concept, we conjugated peptide nucleic acids (PNAs) designed to target the viral ORF1ab region (±30 bp) to ACE2-Fc. The ACE2-PNA conjugate demonstrated superior inhibitory efficacy against multiple SARS-CoV-2 variants relative to ACE2-Fc alone. We confirmed that ACE2-PNA retains the extracellular neutralization activity of soluble ACE2, while being selectively internalized into virus-infected cells via virus-mediated endocytosis. Owing to its inherent protease resistance, the PNA component remains intact upon cytoplasmic entry and subsequently exerts antisense inhibitory activity against viral RNA. The mechanistic feasibility of ACE2-PNA was further validated in a mouse model. Collectively, RDC represents a novel virus-triggered targeted delivery platform that confers dual antiviral efficacy through extracellular virion neutralization and intracellular inhibition of viral replication. It has significant implications for reducing off-target toxicity and enhancing antiviral potency, and is furthermore readily adaptable to diverse viral pathogens and therapeutic payloads.