Design and biological evaluation of candidate drugs against zoonotic porcine deltacoronavirus (PDCoV)

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus with zoonotic potential. PDCoV spillovers were recently detected in Haitian children with acute undifferentiated febrile illness, underscoring the urgent need to develop anti-PDCoV therapeutics. Coronavirus 3C-like protease (CoV 3CL^pro) is essential for viral replication, and therefore provides an attractive target for drugs directed against CoV. Here, we initially evaluated the anti-PDCoV effect of Nirmatrelvir (PF-07321332), an FDA-approved anti-SARS-CoV-2 drug targeting viral 3CL^pro. Regrettably, a very limited anti-PDCoV effect was achieved. By analyzing the binding modes of Nirmatrelvir with PDCoV 3CL^pro and SARS-CoV-2 3CL^pro, we demonstrated that the S2 pocket of 3CL^pro is the primary factor underlying the differential inhibitory potency of Nirmatrelvir against different CoV 3CL^pros. Based on the specific characteristics of the S2 pocket of PDCoV 3CL^pro, four derivatives of Nirmatrelvir (compounds T1–T4) with substituted P2 moieties were synthesized. Compound T1, with an isobutyl at the P2 site, displayed improved anti-PDCoV activity in vitro (cell infection model) and in vivo (embryonated chicken egg infection model), and therefore is a potential candidate drug to combat PDCoV. Together, our results identify the substrate-binding mode and substrate specificity of PDCoV 3CL^pro, providing insight into the optimization of Nirmatrelvir as an antiviral therapeutic agent against PDCoV.