A clinical SARS-CoV-2 Mpro inhibitor blocks replication of multiple enteroviruses and confers oral in vivo protection in animal models

Figures

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  Fig 1. High-throughput screening identifies potent inhibitors of EV71 3Cpro..A. Composition of the protease inhibitor library comprising 104 compounds, categorized into cysteine protease inhibitors (20), aspartic protease inhibitors (9), serine protease inhibitors (10), proteasome inhibitors (4), Factor Xa inhibitors (5), ACE (angiotensin-converting enzyme) inhibitors (17), and others. B. Primary screening of the compound library based on EV71 3Cpro enzymatic activity. Exclude rupintrivir five compounds exhibiting >90% inhibition at 100 μM were identified as primary hits. C. Chemical structures and dose-response curves of selected inhibitors with significant EV71 3Cpro inhibitory activity. The chemical structures are shown above each dose-response curve. Red circles highlight the electrophilic warheads (e.g., aldehyde, fluoromethyl ketone) which are predicted to form covalent interactions with the catalytic cysteine residue of the 3Cpro. Inhibition was quantified using fluorescence-based enzymatic assays, and IC50 values were calculated accordingly. D. Antiviral activity and cytotoxicity of selected hits in RD cells. Black curves represent antiviral efficacy by using RT-qPCR assay…

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  Fig 2. Antiviral efficacy of bofutrelvir against EV71 and other enterovirus in vitro. A. Bofutrelvir inhibited EV71 replication in a dose-dependent manner across three different cell lines (U251, Vero, and Caco-2), as determined by antiviral activity with different MOI (U251 MOI = 0.005, Vero MOI = 0.01, Caco-2 MOI = 1) and cytotoxicity assays. Black curves represent antiviral activity determined by RT‒qPCR, while blue curves indicate cell viability. B. Dose-dependent effects of bofutrelvir inhibited EV71 replication in Vero cells as determined by viral TCID50 assay. C. Immunofluorescence assay (IFA) to visualize the antiviral effect of bofutrelvir in EV71-infected RD cells. Viral capsid protein VP1 was detected using an anti-VP1 antibody (green), and nuclei were counterstained with DAPI (blue). Treatment with bofutrelvir resulted in a marked decrease in VP1 signal in a concentration dependent. D. Broad-spectrum antiviral activity of bofutrelvir against other enteroviruses, including CVB5, CA16, CA10, CA6, and Echo11. Dose-dependent effects of bofutrelvir on virus (MOI = 0.005-0.05) replication in RD cells as determined by RT‒qPCR.

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  Fig 3. Interaction between EV71 3Cpro and bofutrelvir. A. Chemical structure of bofutrelvir (left) and its binding conformation within the active site of EV71 3Cpro (right). The three subsite-binding moieties (S1, S2, and S4) are indicated. B. Overall crystal structure of EV71 3Cpro in complex with bofutrelvir, shown as a ribbon model with partial surface representation. The black boxes highlight the S1, S2, and S3 subsite interactions between the inhibitor and the protease. (C–E) Close-up views of the S1 (C), S2 (D), and S4 (E) binding pockets, showing detailed interactions between bofutrelvir and key residues of EV71 3Cpro. In the S1 site, hydrogen bonds are formed with His161 and Thr142. The Gly 145 and Cys 147 chain interact with water; in the S2 site, interactions involve Glu71, His40 and Lys130; and in the S4 site, Gly163, Gly164 contribute to inhibitor binding. Hydrogen bonds are shown as yellow dashed lines. Water molecules are depicted as red solid circles.

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  Fig 4. Bofutrelvir suppresses EV71 replication and pathogenesis in a neonatal mouse model. A. Schematic of the in vivo experimental design.6 days old neonatal ICR mice were infected with EV71 via intraperitoneal (i.p.) injection 106 pfu on Day 0 and treated twice daily with bofutrelvir (12.5, 25, or 50 mpk, i.p.) or the positive control compound NITD008 (5 mpk, p.o.) from Day 1 to Day 6. Samples were collected on Day 6. B. Clinical scores of infected mice over time, showing treatment-dependent improvement in disease symptoms. (Clinical illness was scored as follows: 0, normalcy; 1, ruffled hair and hunchbacked appearance; 2, limb weakness; 3, paralysis in one limb; 4, paralysis in two limbs; 5, lose of the ability to move and ingest; 6, death. As mice with a clinical score of 5 usually die in 1 day, they were euthanized with carbon dioxide to reduce their suffering and their death days were accounted on the next day). Figure Created in BioRender. Cao, J. (2026) https://BioRender.com/yo37i97. C–E. Quantification of EV71 viral RNA copies numbers in brain, spinal cord, hind foot muscle, and hind foot tissues by RT-qPCR. F–H. Infectious viral titers measured in corresponding tissues by…

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  Fig 5. Bofutrelvir attenuates EV71-induced neuropathology and inflammation in brain and muscle tissues. A. Representative H&E-stained brain sections from EV71-infected neonatal mice treated with vehicle, bofutrelvir (12.5, 25, or 50 mpk, i.p.), or NITD008 (5 mpk, p.o.). Vehicle-treated mice exhibited typical histopathological signs of neuroinflammation, including neuronal degeneration and immune cell infiltration (arrowheads), which were alleviated by bofutrelvir. B–D. Expression levels of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 in brain tissue, as measured by RT-qPCR. Bofutrelvir treatment significantly reduced cytokine levels compared to the vehicle group. E. Representative H&E-stained hind limb muscle tissue sections from EV71-infected neonatal mice treated with vehicle, bofutrelvir (12.5, 25, or 50 mpk, i.p.), or NITD008 (5 mpk, p.o.). Histopathological analysis of hind limb muscle tissue. Vehicle-treated mice displayed severe muscle fiber damage and inflammatory infiltration. F–H. Expression levels of IL-1β, TNF-α, and IL-6 in muscle tissue, showing dose-dependent suppression of local inflammation following bofutrelvir administration. Data are shown as mean ± SD from…

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  Fig 6. Efficacy of orally administered bofutrelvir confirms its potential as a viable therapeutic against EV71. A. Pharmacokinetic profile of bofutrelvir following a single oral dose (25 mpk) in beagle dogs. Plasma concentrations over time show favorable oral exposure and support dosing strategies for subsequent in vivo evaluation, with calculated parameters (including Cₘₐₓ and T₁/₂) presented as mean ± SD. Dashed horizontal lines indicate the in vitro EC₅₀ and EC₉₀ values of bofutrelvir against EV71, highlighting that plasma levels remain above the therapeutic threshold for an extended period. B. Experimental Flow Chart. A schematic diagram illustrating the design of the oral efficacy study in neonatal mice. Six‑day‑old ICR mice were orally infected with EV71 (1 × 10⁶ PFU) and subsequently treated by oral gavage with bofutrelvir at varying doses from day 0 to day 5. Mice were monitored daily for clinical symptoms, and samples were collected at the designated endpoints. Figure Created in BioRender. Cao, J. (2026) https://BioRender.com/yo37i97. C-E. EV71 Viral RNA Copy Numbers in Brain, Spinal Cord, and Hind Foot Muscle. RT‑qPCR quantification demonstrated a marked reduction in viral RNA…