Potent protease inhibitors of deadly lagoviruses: rabbit hemorrhagic disease virus and European brown hare syndrome virus

Rabbit hemorrhagic disease (RHD) and European brown hare syndrome (EBHS) are highly contagious diseases caused by lagoviruses in the Caliciviridae family and mainly affect rabbits and hares, respectively. These infectious diseases are associated with high mortality and a serious threat to domesticated and wild rabbits and hares, including endangered species such as Riparian brush rabbits. In the US, only isolated cases of RHD had been reported until Spring 2020. However, RHD caused by RHD type 2 virus (RHDV2) was unexpectedly reported in April 2020 in New Mexico and has subsequently spread to several US states infecting wild rabbits and hares, making it highly likely that RHD will become endemic in the US. Vaccines are available for RHD, however, there is no specific treatment for these diseases. RHDV and EBHSV encode a 3C-like protease (3CLpro), which is essential for virus replication and a promising target for antiviral drug development. We have previously generated focused small molecule libraries of 3CLpro inhibitors and demonstrated the in vitro potency and in vivo efficacy of some protease inhibitors against viruses that encode 3CLpro including caliciviruses and coronaviruses. Here we established the enzyme and cell-based assays for these uncultivable viruses to determine the in vitro activity of 3CLpro inhibitors, including GC376, a protease inhibitor being developed for feline infectious peritonitis, and identified potent inhibitors of RHDV1 and 2 and EBHSV. In addition, structure-activity relationship study and homology modelling of the 3CLpros and inhibitors revealed that lagoviruses share similar structural requirements for 3CLpro inhibition with other caliciviruses.