Structure-activity relationship of lycorine scaffolds reveals translation inhibition as a determinant of broad-spectrum antiviral activity.

Viral infections caused by Lentiviruses, Betacoronaviruses, and Orthoflaviviruses result in substantial global morbidity and mortality, underscoring the need for additional antiviral agents. The Amaryllidaceae alkaloid lycorine exhibits potent broad-spectrum antiviral activity but is limited by cytotoxicity. Although prior structure-activity relationship (SAR) studies have explored lycorine modifications against individual viruses, no comparative evaluation across distinct viral families has been reported. Here, we evaluated 12 lycorine derivatives against three representative RNA viruses, HIV-1, HCoV-OC43, and DENV-2, alongside mechanistic and safety profiling. Lycorine, evaluated as both free base and hydrochloride salt, did not inhibit HIV-1 at non-cytotoxic concentrations, whereas anhydrolycorine, not previously described as an antiviral agent, selectively inhibited HIV-1 (EC₅₀ = 11 μM, selectivity index (SI) > 10). Lycorene (EC₅₀ = 0.3 μM), lycorine-2-one (EC₅₀ = 1.4 μM), and 1-monoacetyllycorine (EC₅₀ = 1.4 μM) inhibited HCoV-OC43 with lower cytotoxicity than lycorine (EC₅₀ = 1 μM, SI = 78). Against DENV-2, lycorine and lycorene retained submicromolar potency. A cell-free translation assay revealed that lycorine and lycorene potently inhibited both general eukaryotic and DENV-2 viral RNA translation at concentrations consistent with their antiviral EC₅₀ values, whereas RNA-dependent RNA polymerase (RdRp) inhibition of IFN-type I production did not correlate with cellular antiviral potency. Metabolic profiling indicated that the derivatives decreased the glycolysis-to-oxidative phosphorylation ratio in primary macrophages, consistent with a host-directed metabolic mechanism. SAR analysis identified C1/C2 hydroxyl modifications, C-ring flexibility, and protonation state as key determinants of both potency and selectivity across viral targets. These findings provide a SAR framework across three viral families for optimizing lycorine-based broad-spectrum antivirals.