High-Throughput Discovery of Inhibitors Targeting Monkeypox Virus H1 Phosphatase.

Mpox, caused by monkeypox virus (MPXV), remains a Public Health Emergency of International Concern (PHEIC) and poses a serious global health threat. Current therapeutic options for MPXV infection are limited. The orthopoxvirus dual-specificity phosphatase H1 plays critical roles in suppressing interferon signaling, regulating early viral transcription, and modulating viral core protease activity. Suppressing H1 expression markedly reduces the production of infectious viral particles, highlighting it as a promising antiviral target. Here, we developed a high-throughput enzymatic assay using p-nitrophenyl phosphate as a substrate to discover MPXV H1 inhibitors. We demonstrated that both the N-terminal helix α1, which mediates H1 dimerization, and the catalytic residue Cys110 are indispensable for enzymatic activity, validating them as potential "hot spots" for drug design. Screening identified 17 potent inhibitors with nanomolar IC50 values and minimal cytotoxicity. Molecular docking revealed that these inhibitors bind within the active site of MPXV H1, interacting with residues in the P-loop and WPD-loop, thereby restricting substrate access and suppressing activity. This study identifies several potent inhibitors of MPXV H1, establishing a foundation for the development of novel antivirals against MPXV infection.