We first demonstrate that carrimycin, as an antibiotic, shows broad-spectrum anti-coronavirus activity by targeting frameshifting element (FSE) RNA. Herein, taking carrimycin as the lead, 26 new 16-membered macrolides were synthesized and evaluated for antiviral activity against coronavirus strains. Compound 2d exhibited the elevated antiviral efficacy against HCoV-OC43 and HCoV-229E with EC50 values of 0.85 μM and 1.45 μM by directly targeting coronaviral FSE RNA pseudoknot. Molecular simulations revealed that the introduction of a 4″-substituent transforms the macrocyclic core into U-shaped conformation, enabling the higher binding with FSE. Meanwhile, using thermal proteome profiling (TPP) technology, we identified DIS3L2 as a potential host target, which probably assisted 2d to exert the antiviral effect. Therefore, the 16-membered macrolides constituted a new class of RNA inhibitors against coronaviruses, and 2d owns a dual-target mechanism that acts on both viral FSE RNA and host DIS3L2.
