Novel Inhibitors of Neurotropic Alphavirus Replication That Improve Host Survival in a Mouse Model of Acute Viral Encephalitis

Abstract

Arboviral encephalitis is a potentially devastating human disease with no approved therapies that target virus replication. We previously discovered a novel class of thieno[3,2-b]pyrrole-based inhibitors active against neurotropic alphaviruses such as western equine encephalitis virus (WEEV) in cultured cells. In this report we describe initial development of these novel antiviral compounds, including bioisosteric replacement of the 4H-thieno[3,2-b]pyrrole core with indole to improve metabolic stability and the introduction of chirality to assess target enantioselectivity. Selected modifications enhanced antiviral activity while maintaining low cytotoxicity, increased stability to microsomal metabolism, and also revealed striking enantiospecific activity in cultured cells. Furthermore, we demonstrate improved outcomes (both symptoms and survival) following treatment with indole analog 9h (CCG-203926) in an in vivo mouse model of alphaviral encephalitis that closely correlate with the enantiospecific in vitro antiviral activity. These results represent a substantial advancement in the early preclinical development of a promising class of novel antiviral drugs against virulent neurotropic alphaviruses.

Novel Indole-2-Carboxamide Compounds Are Potent Broad-Spectrum Antivirals Active against Western Equine Encephalitis Virus In Vivo

“…Mice were monitored for an additional 7 days after treatment was completed, and all surviving animals were euthanized at day 14. All mice were weighed daily and assigned a clinical score using a 1-to-5 scale as previously described (13).…”

“…Synthesis of compounds CCG205432, CCG206381, and CCG209023 has been previously described (13,20). Ribavirin, mycophenolic acid (MPA), antimycin A, phosphonoacetic acid (PAA), and cycloheximide were all purchased from Sigma-Aldrich (St. Louis, MO).…”

“…Differentiated BE(2)-C/m cells were generated as previously described (23 . BSR-T7 cells were cultured as previously described (13), and all other cells were cultured in complete Dulbecco's modified Eagle medium (cDMEM) containing 5% bovine growth serum, 1% sodium pyruvate, 0.1 mM nonessential amino acids, 10 U/ml penicillin, and 10 g/ml streptomycin.…”

“…Furthermore, the combination of active vaccination plus antiviral therapy may be a more effective response in the setting of an outbreak caused either by natural transmission or by intentional exposure to one of these viral pathogens (12). Although numerous compounds have been reported to inhibit alphavirus replication in cultured cells, only a select few have shown any activity in animal models (13)(14)(15)(16)(17). Thus, there is a pressing need to identify new antiviral compounds and drug targets as part of a comprehensive medical-countermeasure strategy to prevent or mitigate illness, suffering, and death resulting from infections caused by these virulent pathogens (18).…”

“…Through extensive structure-activity relationship (SAR) analyses and targeted medicinal-chemistry efforts, we produced second-generation indole-2-carboxamide derivatives that display increased potency and striking enantiospecific activity against WEEV and related alphaviruses in cultured cells and that also improve survival in mice infected with a neuroadapted strain of Sindbis virus (SINV) (13). We have now produced third-generation analogues through continued medicinal-chemistry-directed efforts that have increased antiviral potency, where we have achieved submicromolar half-maximal inhibitory concentrations (IC 50 ) against WEEV replicons in cultured cells (20).…”