Structural and Antiviral Studies of the Human Norovirus GII.4 Protease

Abstract

Norovirus is the leading cause of acute gastroenteritis worldwide with a yearly reported 700 million cases driving a $60 billion global socioeconomic burden. With no United States Food and Drug Administration approved therapeutics and the chance for severe chronic infection and life-threatening complications, researchers have identified the protease as a potential target. However, drug development has focused on the norovirus GI.1 strain despite its accounting for less than 5% of all outbreaks. Our lab aims to change focus for norovirus drug design from GI.1 to the highly infective GII.4, responsible for more than 50% of all outbreaks worldwide. With the first published crystal structure of the norovirus GII.4 protease, we have identified several significant differences in the structure and active site that have hindered development of a potent inhibitor targeting the norovirus GII.4 protease. With these new insights, we have begun designing compounds that demonstrate increased inhibition of the clinically most relevant norovirus GII.4 strain.

In crystallo-screening for discovery of human norovirus 3C-like protease inhibitors

“…However, a neighbouring dimer in the crystal structure forms an interface of comparable buried surface area (692.3 Å 2 ) between chains labelled A and D chains and likewise for chains labelled the B and C. This result indicates that higher order oligomers may possibly be formed by SV3CP dimers, such as the putative tetramer shown in Fig. 1 b. Intriguingly, a number of other human GI and GII noroviral protease structures ( Nakamura et al, 2005 , RCSB ID: 1wqs; Muzzarelli et al, 2019 , RCSB ID: 6b6i; Viskovska et al, 2019 , RCSB ID: 6nir) form essentially the same tetrameric assembly in the crystals, as shown in Supplementary Fig. 1 .…”

“…These findings, along with the ability of the tetramer cavity to bind small molecule fragments (see later), suggest that this tetrameric form may have functional significance for 3CL pro . Indeed, in the structure of the Minerva virus enzyme (RCSB ID: 6b6i) the tetrameric assembly allows the C-terminal ends of two monomers (equivalent to B and D) to extend into the active sites of adjacent monomers (C and A, respectively) across the dimer-dimer interface ( Muzzarelli et al, 2019 ). Similar tail-interdigitating effects are observed in the structures of the protease from Houston virus (RCSB ID: 6nir; Viskovska et al, 2019 ) and mouse norovirus (RCSB ID: 4x2v; Fernandes et al, 2015 ).…”

“…Other fragments were found to bind at an additional site which is buried deeply in the centre of the crystallographic tetramer. The fact that a C193A mutant of the Minerva virus protease forms the same tetramer in the crystal with the C-terminus of one subunit occupying the active site cleft of another monomer ( Muzzarelli et al, 2019 ), suggests that this assembly may also be involved in proteolytic maturation of noroviruses. Hence, compounds that have the potential to interfere with formation of the tetramer or affect its stability may impact on noroviral replication and therefore deserve to be screened for in vivo activity, e.g.…”

Discovery of Immunoproteasome Inhibitors Using Large-Scale Covalent Virtual Screening

“…It allows users to choose between a “pose prediction” (also named “Lead optimization”, from now on referred to as CovDock-PP) [26]) and a “virtual screening” mode (CovDock-VS) [27]. While the former is designed to predict accurate binding modes via more demanding simulations, the latter allows screening of larger libraries [28,29,30,31] by efficiently decreasing the number of steps in the protocol. However, even CovDock-VS has a throughput (≈15 CPU minutes/ligand) not compatible with the size of our virtual compound collection.…”

Molecular Evolution of the Protease Region in Norovirus Genogroup II

“…Pro could be a major target for antiviral drugs, in addition to RdRp. Many studies have been conducted into the development of drug candidates (Hussey et al, 2011;Muhaxhiri et al, 2013;Muzzarelli et al, 2019;Viskovska et al, 2019). A recent report demonstrated different effectiveness of antivirals against GI and GII NoVs, suggesting that the amino acid diversity in Pro may result in different effectiveness of drugs (Viskovska et al, 2019).…”