Human noroviruses are the primary cause of sporadic and epidemic acute gastroenteritis in the US and worldwide. Noroviruses constitute an important public health problem, as well as a potential bioterrorism threat. The problem is further compounded by the current dearth of effective vaccines and norovirus-specific antiviral therapeutics and/or prophylactics.

Human noroviruses are single-stranded, positive sense RNA viruses in the Caliciviridae family. Their ~7.5 kb genome encodes a polyprotein precursor that is processed by a virus-encoded 3CL protease (3CLpro) to generate mature non-structural proteins. Processing of the polyprotein is essential for virus replication, consequently NV 3CLpro has emerged as a potential druggable target for the discovery of anti-norovirus small molecule therapeutics and prophylactics. NV 3CLpro is a chymotrypsin-like cysteine protease with a Cys-His-Glu catalytic triad and an extended binding site. The primary substrate specificity of the protease is for P1 glutamine residue and a strong preference for a –D/E-F-X-L-Q-G-P- sequence, where X is H, Q or V, corresponding to the subsites S5-S4-S3-S2-S1-S1'-S2'-. Cleavage is at the P1-P1' (Q-G) scissile bond.

We have recently reported the first high throughput FRET assay of 3CLpro from GI and GII noroviruses as a screening tool for identifying potential protease inhibitors and have determined the first high resolution X-ray crystal structures of NV 3CLpro in complex with peptidyl transition state inhibitors of the protease, as well as the first solution structure of the protease using high-field NMR. We report herein the structure-based optimization of a series of dipeptidyl inhibitors of NV 3CLpro using X-ray crystallography and an array of structure-activity relationship, biochemical, and cell-based studies.