18β-glycyrrhetinic acid-amantadine hybrid: Synthesis and anti-EMCV activity via NF-κB modulation

Encephalomyocarditis virus (EMCV) is an important pathogen; however, current prevention and treatment methods and drugs are limited. 18β-Glycyrrhetinic acid (GA) is widely used in traditional Chinese medicine as a key active ingredient in Glycyrrhiza glabra and has anti-inflammatory, antiviral, antioxidant, and hepatoprotective pharmacological effects. However, the bioavailability of GA is significantly reduced by its low water solubility and high toxicity. Amantadine (AM) is a versatile antiviral drug. We reduced the toxicity and increased the bioavailability of GA by introducing AM into the GA backbone, and further explored its antiviral mechanism. The chemical structures of the new 18β-glycyrrhetinic acid derivative grafted with amantadine (GA-AM) were confirmed by IR, ¹HNMR and ESI-MS. The cytotoxicity of GA-AM in BHK-21 and HEK-293 cells was determined using the CCK-8 assay. The antiviral effect and mechanism of action of the EMCV virus of GA-AM were detected in vitro and in vivo using TCID₅₀, RT-qPCR, Western blotting, IFA, and ELISA. Compared to GA, GA-AM exhibited lower cytotoxicity in BHK-21 and HEK-293T cells and demonstrated more significant antiviral effects against EMCV. GA-AM can also protect EMCV-infected cells and reduce the amount of the VP1 capsid protein in cells. Mechanistic investigations revealed that GA-AM might exert antiviral effects by regulating the NF-κB signaling pathway and modulating downstream cytokines, including TNF-α and IL-6. Furthermore, GA-AM alleviated these signs in EMCV-infected mice. The novel drug GA-AM has lower cytotoxicity and more significant antiviral effects. It may therefore serve as a new, low-toxicity antiviral agent.