Aug 2026· Journal of Biological Chemistry· pp.
113453
· 0 citations· 32 references
Medicine
TL;DR
A fragment-based drug discovery strategy combined with crystallographic screening and structural similarities among the fragments provide a robust structural framework for the rational design of PLpro inhibitors and support the development of novel antiviral therapeutics.
Abstract
Coronaviruses are a large family of viruses capable of causing severe respiratory diseases, with recent outbreaks posing major threats to global public health. The SARS-CoV-2 papain-like protease (PLpro) is an essential viral enzyme involved in viral replication and host immune evasion, making it an attractive target for antiviral drug development. However, no PLpro-targeted drugs have yet been approved. Here, we employed a fragment-based drug discovery (FBDD) strategy combined with crystallographic screening and identified 23 fragment compounds with well-defined electron density. These fragments bind to seven distinct sites on PLpro and reveal multiple compound scaffolds and potential binding pockets through systematic structural analysis. Notably, Site 2 represents a newly identified pocket adjacent to the canonical substrate-binding site (Site 1) and shows strong potential for fragment linking to enable inhibitor optimization. Among them, Frag368 exhibited good enzyme inhibitory activity. Furthermore, based on structural similarities among the fragments, the FDA-approved drug 5-fluorouracil was identified as a ligand for Site 2. Biochemical assays confirmed that 5-fluorouracil exhibits inhibitory activity against PLpro. Collectively, these findings provide a robust structural framework for the rational design of PLpro inhibitors and support the development of novel antiviral therapeutics.
Despite advances in vaccines and supportive therapies, a targeted treatment for COVID-19 remains lacking, prompting researchers to explore specific pharmacological interventions to fill this gap. In this quest, we targeted the main protease (Mpro) of SARS-CoV-2, a well-established druggable target crucial for viral replication and transcription. 714 antiviral compounds were computationally screened against the active-site residues of Mpro, and 17 hits showed significantly better interactions with Mpro than the selected control (N3). The two top-scoring compounds, LY2784544 and Pimobendan, were further evaluated through 100-ns molecular dynamics (MD) simulations. Comprehensive post-simulation analyses, including root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration, solvent-accessible surface area, principal component analysis, free energy landscape (FEL) mapping, and MM/PBSA binding free energy calculations, demonstrated stable complex formation and persistent engagement of the catalytic dyad (His41–Cys145) and S1/S2 subsites. Molecular Mechanics/Poisson–Boltzmann Surface Area (MM/PBSA) results indicated favorable binding free energies, with Pimobendan showing comparatively stronger energetic stabilization. The study identifies LY2784544 and Pimobendan as potential antiviral agents for Mpro. Given their stability, strong binding energy (BE) profiles, and existing clinical data, these compounds warrant additional experimental validation to determine their potential efficacy against SARS-CoV-2.
M. Alam, Lina I. Alnajjar, T. K. Upadhyay et al.· Journal of King Saud Univers...· 0 citations
Structurally optimized triazine-based nucleoside analogues are identified as potential SARS-CoV-2 Mpro inhibitors and the utility of an integrated computational strategy for the rational design and prioritization of antiviral candidates for further experimental validation is demonstrated.
Madhu Sudan, Sarika Arora, Mohd Saeed et al.· Journal of the Iranian Chemi...· 0 citations
The emergence of SARS-CoV-2 variants poses a significant threat to public health, highlighting the urgent need for novel therapeutic agents to address gaps in current healthcare solutions. Although several preventive vaccines have been developed and approved, their long-term efficacy remains uncertain, and the spike protein targeted by these vaccines is highly susceptible to mutation. Consequently, targeting host proteases such as Cathepsins L and B, which are implicated in viral entry, could provide a potent antiviral strategy against SARS-CoV-2. These Cathepsins are appealing drug targets because of their well-defined substrate-binding pockets, which can be utilized as binding sites for drug enzyme inhibitors. In this study, we present an integrated computer-aided drug discovery approach for a selection of 14 pyrrole-imidazole alkaloids targeting Cathepsins L and B. This approach employs a combination of modern computational methodologies, including molecular docking and molecular dynamics (MD) simulations. Our molecular docking studies identified a promising marine alkaloid, Mauritiamine, which exhibited notable binding affinities for Cathepsin L (-9.35 kcal/mol) and Cathepsin B (-9.03 kcal/mol). Subsequently, MD simulations were conducted to investigate the interaction dynamics and structural stability of the docked complexes. The resulting analyses, including maps of structural deformability, b-factors, eigenvalues, variance and covariance matrices, along with elastic network models, confirmed the structural stability of both the Cathepsin L-Mauritiamine and Cathepsin B-Mauritiamine complexes. These results underscore the potential of Mauritiamine as a promising candidate for antiviral therapy against SARS-CoV-2. Further in vitro and in vivo studies will pave the way for the identification of clinically relevant antivirals inspired by marine pyrrole-imidazole alkaloids.
M. Kabrine, W. Tachoua, S. Abla· International Journal of Com...· 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
The main protease (MPro) of coronaviruses (CoVs) is an essential enzyme involved in viral replication and represents an attractive target for antiviral drug discovery. Based on the similar binding pocket residues within the MPro of different CoVs, this study aimed to identify potential inhibitors of SARS-CoV-2 MPro from PDB ID 6M2N using integrated computational approaches. Interaction-based pharmacophore modeling, virtual screening, molecular docking, MM-GBSA binding energy calculation, and molecular dynamics simulation (MDS) were performed using BIOVIA Discovery Studio. The validated pharmacophore model was utilized to screen the ZINC database, followed by docking and 100 ns MDS analyses of the top-ranked compounds. The pharmacophore model 01 demonstrated favorable predictive performance (AUC = 0.781). Virtual screening identified 483 compounds, from which 15 compounds were selected for docking studies. Among them, ZINC95473654 (Lig-1), ZINC95473725 (Lig-2), and ZINC08792368 (Lig-3) exhibited strong binding affinity toward MPro. Lig-1 demonstrated the best docking score and binding free energy, along with stable interactions with key catalytic residues HIS41, CYS145, and GLU166. MDS analyses further confirmed that Lig-1, Lig-2 and Lig-3 maintained stable conformations. The hydrogen bond distance monitoring and post MDS-MM-GBSA results suggest Lig-1 followed by Lig-3 as an inhibitor for MPro and persistent intermolecular interactions throughout the 100 ns simulation period. The findings suggest that Lig-1, followed by Lig-3, may serve as promising computational lead compounds targeting SARS-CoV-2 MPro, representing promising candidates for further experimental validation.
Mohd Yasir Khan, Farah Maarfi, A. Shah et al.· International Journal of Mol...· 0 citations
Molecular dynamics simulations showed that the S-adenosylmethionine (SAM)-binding site ligand formed a more stable complex with lower root-mean-square deviation (RMSD) and reduced flexibility, indicating a promising lead candidate for further optimization and experimental validation against dengue virus NS5.
Nabeel Haider, Abolfazl Zare, Yi Zhou et al.· Journal of Molecular Modelin...· 0 citations