Macrocyclic inhibitors of Norovirus 3CL protease

Abstract

Noroviruses are the leading cause of foodborne illness in the U.S., accounting for >21 million infections per year and resulting in >70000 hospitalizations and nearly 800 deaths. Noroviruses are the primary cause of sporadic and epidemic acute gastroenteritis worldwide. Norovirus is an RNA virus that belongs to the Norovirus genus of the Caliciviridae family and carries a positive sense single stranded RNA genome (7.7 Kb). There are currently no vaccines or specific antiviral agents for combating infections caused by norovirus. The virus possesses a 3C-like cysteine protease (3CLpro) that is responsible for the majority of cleavages in the corresponding viral polyprotein into mature and functional proteins and is essential for virus replication. Thus, inhibitors of this enzyme are of potential therapeutic value. Inspection of crystal structures of norovirus 3CLpro with peptidyl inhibitors reveals that peptidyl inhibitors of norovirus 3CLpro bind to the enzyme via a network of backbone hydrogen bonds that mimic an antiparallel ?-sheet. The hydrogen bonding associated with an antiparallel ?-sheet is more feasible when an inhibitor (or substrate) binds to the active site of a protease in a ?-strand conformation. Macrocyclization of linear peptidyl inhibitors helps pre-organizing the macrocycle in a ? -strand conformation prior to binding to the enzyme active site. This approach results in the formation of a pre-organized and semi-rigid entity that displays the amino acid side chains in a well-defined vector relationship for optimal binding and enhances the binding affinity of the inhibitor to the target enzyme by minimizing the entropy loss. Additional potential advantages of macrocyclic inhibitors include high stability to metabolizing enzymes, high selectivity and, frequently, increased cellular permeability. This dissertation describes herein the structure-based design of cell-permeable macrocyclic transition state inhibitors of norovirus 3CLpro, as well as relevant structural, biochemical, spectroscopic, and cell-based studies.