ZN002: A Novel Natural Product Small Molecule Inhibitor Targets the Coxsackie-Adenovirus Receptor (CAR) to Control Coxsackie B3 Viral Proliferation

Introduction: The Coxsackie Adenovirus Receptor (CAR), encoded by the CXADR gene, facilitates entry of Coxsackie group B virus (CVB) and adenoviruses into host cells. Infections caused by CVB, particularly CVB3, are associated with severe complications, such as meningoencephalitis and cardiomyopathy. Currently, no specific antiviral therapy is available. This study aimed to identify novel lead compounds targeting CVB3 using an integrated in silico and in vitro approach. Materials and Methods: A diversity-based High-Throughput Virtual Screening (HTVS) of a Zn-based natural compound library was conducted, followed by Molecular Dynamic Simulation (MDS) to assess binding stability. Top hits were evaluated using in vitro Micro Tissue Culture Antiviral Assays (MTCA), including Direct Pre-infection Incubation (DPI), Pre-Treatment Assay (PTA), and Viral Adsorption Assay (VAA) in HEp-2 cells. MTT assay was performed to assess cytotoxicity. Results: Among the top five hits, ZN-002 [2-(3,4-dimethylphenyl) cyclohexa-2,5-diene-1,4- dione] showed strong binding affinity (< -5.0 kcal/mol) with stable interactions spanning residues Pro25 to Gln50 of the CAR protein. MDS confirmed binding stability over 100 ns. ZN- 002 demonstrated dose-dependent antiviral activity at 10–15 μM concentrations, effectively inhibiting 1, 10, and 100 TCID50 of CVB3 in multiple assay formats without cytotoxicity. Discussion: ZN-002 interferes with the CAR-virus interaction interface, likely hindering viral entry into host cells. The compound’s robust binding and broad antiviral efficacy across varying viral loads underscore its potential. The absence of cytotoxicity further supports its candidacy for therapeutic development. This dual in silico-in vitro validation platform accelerates earlystage antiviral discovery. Conclusion: ZN-002 represents a promising lead molecule against CVB3, warranting further evaluation through in vivo studies to explore its potential as a novel antiviral agent.