Introduction
Mpox, caused by the monkeypox virus (MPXV), continues to pose a profound global health concern as it remains classified as a Public Health Emergency of International Concern (PHEIC). Despite its expanding burden, therapeutic strategies targeting MPXV remain limited, underscoring the urgent need for novel antiviral approaches. Recent research has highlighted the orthopoxvirus dual-specificity phosphatase H1 as a critical regulator of MPXV pathogenesis, making it an attractive antiviral target. H1 contributes to viral immune evasion, early transcriptional regulation, and viral particle maturation, and its suppression significantly decreases viral infectivity. These insights drive scientific efforts to explore H1 inhibition as a pathway toward next-generation therapeutic development. This study advances such efforts by establishing a high-throughput platform to identify potent small-molecule inhibitors of MPXV H1.
H1 Phosphatase as a Therapeutic Target
The H1 phosphatase is a multifunctional viral enzyme that plays a central role in MPXV immune evasion and replication, making it a prime candidate for targeted antiviral intervention. By suppressing interferon signaling and regulating critical stages of viral transcription and core protease activity, H1 ensures efficient viral propagation within host cells. Research reveals that downregulating H1 dramatically reduces the number of infectious viral particles produced, confirming the enzyme's indispensability in MPXV's life cycle. These findings identify H1 not just as a structural component, but as a cornerstone of viral pathogenicity, making it one of the most promising molecular targets for therapeutic innovation.
High-Throughput Screening Assay Development
To accelerate the discovery of MPXV H1 inhibitors, a robust and efficient high-throughput enzymatic assay was developed using p-nitrophenyl phosphate (pNPP) as a substrate. This assay allows rapid quantification of H1 phosphatase activity and provides a scalable platform for screening large chemical libraries. Key structural elements—including the N-terminal helix α1 and the catalytic residue Cys110—were validated as essential contributors to enzyme functionality, reinforcing their importance in inhibitor design. The assay’s sensitivity and reproducibility make it suitable for identifying potent compounds capable of disrupting H1 function.
Structural Hot Spots: Helix α1 and Cys110
Detailed functional analyses confirmed that both helix α1, which mediates H1 dimerization, and the catalytic cysteine residue Cys110 are indispensable for enzymatic activity. Mutational studies demonstrated that disrupting either structural region leads to significant loss of enzymatic function, emphasizing their relevance as strategic “hot spots” for rational antiviral design. These structural determinants not only govern catalytic efficiency but also influence substrate accessibility and protein stability. This knowledge enhances structure-guided drug design efforts aimed at generating inhibitors that can effectively disrupt key interactions or catalytic mechanisms within H1.
Discovery of Potent MPXV H1 Inhibitors
Using the high-throughput assay, researchers identified 17 potent small molecules with nanomolar IC50 values and minimal cytotoxicity. These inhibitors demonstrated robust suppression of H1 enzymatic activity, highlighting their potential as lead compounds for further antiviral development. The discovery of molecules that exhibit both potency and safety underscores the promise of targeting H1 as a therapeutic strategy. Moreover, the chemical diversity of these inhibitors expands the landscape of potential antiviral scaffolds that can be further optimized through medicinal chemistry approaches.
Molecular Docking and Mechanistic Insights
Computational molecular docking studies revealed that the newly discovered inhibitors bind tightly within the active site of MPXV H1, interacting with residues in the P-loop and WPD-loop. These interactions effectively restrict substrate entry, thereby suppressing phosphatase activity. The structural alignment of inhibitor binding modes offers valuable insights into the molecular mechanisms underlying H1 inhibition and provides a blueprint for structure-based drug optimization. These findings collectively strengthen the rationale for advancing H1-targeted antivirals into preclinical development pipelines.
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