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

Abstract

Introduction: 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 PL^pro and M^pro, which are crucial targets for inhibitors. Additionally, PL^pro plays a role in modulating the host's immune response, and the inhibition of PL^pro exhibits significant anti-inflammatory properties.

Objectives: A series of dual-targeted inhibitors of M^pro and PL^pro were designed and synthesized, and their antiviral and anti-inflammatory activities were subsequently evaluated in vitro and in vivo.

Methods: Dual-targeted inhibitors of M^pro and PL^pro were designed by merging two series of M^pro 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.

Results: Among the sixty-four synthesized compounds, seventeen potent dual-targeted inhibitors of PL^pro and M^pro 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.

Conclusions: In summary, a potent dual-targeted inhibitor of PL^pro and M^pro, f2, was designed, synthesized, and found to be effective in the inhibition of PEDV replication and inflammatory response in vitro and in vivo.

Keywords: Anti-inflammatory; Dual-target inhibitors; M(pro); PEDV; PL(pro).