Structure-based design and optimization of dipeptidyl inhibitors of norovirus 3CL protease

Abstract

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.