In Vivo Efficacy Evaluation and Mechanistic Study of Broad-Sprectrum Inhibitors against Enteroviruses

Recurring outbreaks of enterovirus infection impose substantial financial and social burdens each year in Taiwan. The current lack of effective vaccinations and approved therapeutic agents motivated us to develop new broad-spectrum antiviral compounds against enterovirus 71 (EV71) and enterovirus D68 (EV-D68). This study aims to identify the antiviral mechanisms of the active imidazolidinone compounds and to test the in vivo activities of these compounds in animal models. An inhibition of virus-induced cell death assay in RD cells was used to identify PR66 and PR55 by screening a compound library of about 60 imidazolidinone derivatives with novel entities. PR66 exhibited the most potent anti-EV71 activity, with an EC50 of ca 20 nM against EV71 and ca 187 nM against CVA16. Importantly, the selectivity index was more than 1000-fold, which indicates that PR66 is an ideal drug candidate. In addition, PR55 had potency against both EV71 and EV-D68, with an EC50 of ca 0.57 μM. PR66 had the highest potency and was thus used as a model compound for mechanistic study. PR66 inhibited EV71 at an early stage of the viral life cycle in a time-of-addition assay. We also demonstrated using a centrifugal filtration assay that PR66 inhibited virus replication by direct binding to viruses. The results suggest that PR66 was capable of inhibiting virus replication by targeting the virion directly. We established an EV71 animal model using mouse-adapted EV71 (EV71/MP4) and ICR mice, and PR66 efficiently reduced the neurological disease score and increased the survival rate in EV71/MP4-infected mice. We therefore propose the following aims in order to gain insight into the antiviral mechanisms and in vivo efficacies of PR66 and PR55. First, we propose to thoroughly study the inhibitory mechanism of viral entry by PR66 by performing viral attachment and penetration assays. In addition, we will determine whether PR66 can inhibit virus-receptor binding and viral stability. We will screen for resistant viruses to further elucidate the mechanism through which PR66 inhibits EV71. Because MP4/EV71 is not found in clinical isolates, it thus cannot be generalized to natural EV71 infection. Our second aim is to verify the in vivo efficacy of PR66 in cellular receptor (human SCARB2) transgenic mice recently developed by Dr. Koike. Third, we will establish an EV-D68 infection model in mice and examine the in vivo efficacy of PR55. In conclusion, the results of our study could lead to the preclinical development of PR66 and PR55 as new and broad-spectrum anti-enterovirus agents.

Project ID:PA10507-1178
External Project ID:MOST105-2311-B182-001