Reporter Virus-Based High-Throughput Screening Identifies LM-10 as a Novel Inhibitor Targeting KRM1 for Coxsackievirus A10 Blockade

Enteroviruses causing hand, foot, and mouth disease, including currently circulating Coxsackievirus A10 (CVA10) and Coxsackievirus A6 (CVA6), rely on KREMEN1 (KRM1) for cell entry, making the virus–KRM1 interface a potential target for antiviral intervention. We screened GLMarNP, an in-house library of about 3000 natural products and derivatives. Candidates targeting the KRM1 ectodomain were selected by blind docking, drug-likeness assessment, and scaffold diversity analysis. To enable high-throughput antiviral screening, we inserted eGFP into a full-length infectious CVA10 cDNA clone to generate a reporter virus designated eGFP-A10. The virus was rescued in HEK293T cells and produced robust fluorescence in RD cells over serial passages. Among 29 candidate compounds, LM-10 showed the strongest antiviral activity and exhibited low cytotoxicity. Mechanistically, LM-10 bound KRM1 and acted early in the viral life cycle. Molecular docking suggested that LM-10 binds the KR domain at a site overlapping the VP2 EF-loop contact region. KRM1 also acts as a DKK1 receptor to regulate Wnt/β-catenin signaling. LM-10 did not significantly affect this pathway in the TOP-GFP reporter assay or in transgenic zebrafish embryos. These findings establish a practical CVA10 reporter-virus platform and support steric blockade of the receptor interface as a potential antiviral strategy.