Design, synthesis and activity evaluation of dual-target inhibitors against papain-like and main proteases of porcine epidemic diarrhea virus

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), threatens the global pig industry due to lack of drugs. PEDV replication relies on PLpro and Mpro, which are crucial targets for inhibitors. Additionally, PLpro plays a role in modulating the host's immune response, and the inhibition of PLpro exhibits significant anti-inflammatory properties. A series of dual-targeted inhibitors of Mpro and PLpro were designed and synthesized, and their antiviral and anti-inflammatory activities were subsequently evaluated in vitro and in vivo. Dual-targeted inhibitors of Mpro and PLpro were designed by merging two series of Mpro inhibitors and PLpro inhibitors. sixty-four compounds were synthesized and screened in vitro by FRET for inhibitory activities and by RT-qPCR for antiviral activity on Vero-E6 cells. The anti-PEDV activity of f2 on Vero-E6 cells and IPEC-J2 cells was further confirmed by immunofluorescence. The mechanism by which f2 inhibited PEDV-induced inflammation was investigated by Western blot and RT-qPCR. The anti-colitis activity of f2 was verified in vivo. Among the sixty-four synthesized compounds, seventeen potent dual-targeted inhibitors of PLpro and Mpro were identified with IC50 values less than 10 μM. Six compounds demonstrated excellent antiviral activity and safety in cell-based assays. The most potent compound f2 inhibited PEDV replication in Vero-E6 and IPEC-J2 cells with EC50 values of 1.17 ± 0.73 μM and 2.02 ± 0.56 μM, respectively, without cytotoxicity (CC50 > 800 μM). Moreover, f2 was found to inhibit the inflammatory response induced by PEDV infection via suppressing TLR2/PI3K/Akt/NF-κB signaling pathway. Oral f2 attenuated colitis by decreasing p65 phosphorylation, a major PEDV mortality cause. The in vivo acute toxicity test showed that oral administration of f2 did not affect the body weight and internal organs of mice. In summary, a potent dual-targeted inhibitor of PLpro and Mpro, f2, was designed, synthesized, and found to be effective in the inhibition of PEDV replication and inflammatory response in vitro and in vivo.