Picornaviruses cause substantial human and veterinary disease, yet direct-acting antivirals remain limited. The nonstructural protein 2C is among the most conserved picornaviral proteins and has ATPase and helicase-like activities. 2C is involved in membrane remodeling, RNA replication, encapsidation, morphogenesis, and multiple virus–host interactions. In this review, we summarize the domain architecture and structural biology of 2C, highlighting the N-terminal membrane-binding region, the ATPase core, the C-terminal helical domain, and accessory motifs that regulate oligomerization and ligand recognition. We further review experimentally supported roles of 2C throughout the viral replication cycle and compare known inhibitors, resistance mutations, and emerging design strategies. Collectively, current evidence supports 2C as a tractable yet still underexploited antiviral target for medically important picornaviruses.
Kan Li, Guangjin Fan, Wenyi Zhang et al.· ACS Infectious Diseases· 0 citations
Chikungunya virus (CHIKV), an arthropod-borne alphavirus, has emerged as a global health threat due to its rapid transmission and the lack of effective antiviral therapies. The cysteine protease activity of the virus-encoded nonstructural protein 2 (nsP2) is critical for CHIKV replication, as it processes viral polyproteins and counteracts host antiviral defenses, establishing it as a highly attractive target for therapeutic intervention. In this study, we present a rapid drug development platform that integrates covalent docking with direct-to-biology (D2B) synthesis and screening to identify nsP2 inhibitors. Candidates prioritized by in silico docking were synthesized and directly tested in FRET enzymatic assays without purification. This approach led to the identification of several nsP2 inhibitors with diverse chemical scaffolds, potent enzymatic inhibition, and antiviral activity. Together, these findings establish a streamlined strategy for covalent inhibitor development and provide promising leads for CHIKV antiviral development.
Zhengjun Cai, Kan Li, Sainetra Sridhar et al.· Journal of Medicinal Chemist...· 0 citations
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.
Jun Wang, Kan Li, Bin Tan· Accounts of Chemical Researc...· 0 citations
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