Structural and inhibitor studies of norovirus 3C-like proteases
Introduction
Noroviruses (genus norovirus in the family Caliciviridae) are the most common cause of food- and water-borne acute viral gastroenteritis worldwide (Eckardt and Baumgart, 2011, Green, 2007, Koo et al., 2010, Patel et al., 2009). In the US alone, up to 21 million estimated cases of norovirus-associated gastroenteritis occur each year (2010). Due to its high contagious nature, norovirus outbreaks in cruise ships, army barracks, schools, and hospitals can cause significant morbidity. Furthermore, norovirus infections may lead to fatality in children, the elderly and immuno-compromised patients (Green, 2007). Therefore, norovirus infection constitutes an important public health problem, as well as a potential bioterrorism threat.
Noroviruses are highly diverse with multiple genotypes (GI to GV) with currently genotype II as the most prevalent in the field. Recently, outbreaks of extremely virulent norovirus, GII.4 Syndey strain (first discovered in Australia in March 2012) continues to spread globally, including the US (Desai et al., 2012, MMWR, 2013, van Beek et al., 2013). This emerging norovirus strain is associated with increased rates of hospitalizations and deaths (Desai et al., 2012). Although norovirus is classified as a Category B bioterrorism agent by CDC (2010)), currently there is no norovirus-specific antiviral therapeutics or vaccine partly due to the inability to grow human noroviruses in cell culture (Atmar et al., 2011, Tan and Jiang, 2008). Thus there is an urgent and unmet need for the discovery and development of antiviral therapeutics for the prevention and treatment of norovirus infection. Our group has focused on the discovery and development of small molecules as anti-norovirus therapeutics with enzyme and/or cell based assay systems (Dou et al., 2011a, Dou et al., 2011b, Dou et al., 2011c, Dou et al., 2012, Kim et al., 2011, Mandadapu et al., 2012, Mandadapu et al., 2013a, Mandadapu et al., 2013b, Pokhrel et al., 2012, Tiew et al., 2011).
Noroviruses have a single-stranded, positive sense 7–8 kb RNA genome, which encodes a polyprotein precursor processed by a virus-encoded 3C-like cysteine protease (3CLpro) to generate intermediate or mature functional non-structural proteins. Since processing of the polyprotein is essential for virus replication, viral 3CLpro has been targeted for the discovery of anti-norovirus small molecule therapeutics. Norovirus 3CLpro is characterized with a chymotrypsin-like fold and a cysteine residue acting as a nucleophile in the active site. Norovirus 3CLpro has N-terminal anti-parallel β-sheet domain and C-terminal β-barrel domain and the catalytic site consisting of C139, H30, and E54 is present in the cleft between the domains. The structures of Norwalk virus protease (NV 3CLpro) (Zeitler et al., 2006), Chiba virus protease (CV 3CLpro) (Nakamura et al., 2005), Southampton virus protease (SV 3CLpro) bound with a Michael acceptor peptidyl inhibitor (Hussey et al., 2011), and MNV 3CLpro (Leen et al., 2012) have been determined by X-ray crystallography.
Our group has previously reported the backbone and side-chain resonance assignments of NV 3CLpro by solution NMR spectroscopy (PDB: 2LNC) (Takahashi et al., 2012, Takahashi et al., 2013), and 3D structure of NV 3CLpro without or with a protease inhibitor (GC376) by X-ray crystallography (PDB: 3UR6, 3UR9) (Kim et al., 2012). In addition, the design, synthesis, and evaluation of transition state inhibitors of norovirus 3CLpro, including GC376 (a dipeptidyl compound), was recently reported by our group (Kim et al., 2012, Mandadapu et al., 2012, Tiew et al., 2011). In this study, the proteolytic activities of three 3CLpro from Norwalk virus (NV, genogroup I), MD145 (genogroup II) and murine norovirus-1 (MNV-1, genogroup V) were optimized for a fluorescence resonance energy transfer (FRET) assay, and compared for the inhibitory activities of a synthetic protease inhibitor (GC376). The comparative analysis of 3CLpro from different genogroups of noroviruses may provide further basic knowledge of substrate (inhibitor)-protease interactions, as well as finding broadly active inhibitors against diverse noroviruses. In addition, we describe the comparative analysis of the apo NV 3CLpro and NV 3CLpro-GC376 determined by X-ray crystallography and NMR spectroscopy.
Section snippets
The expression and purification of proteases from MNV-1
The expression of 3CLpro from NV and MD145 was described previously by our lab (Kim et al., 2012). The 3CLpro from MNV-1 was expressed for the comparative studies in this study. The multi-alignment of 3CLpro from NV, MD145 and MNV-1 showed that overall amino acid sequences of three 3CLpro are conserved well (Fig. 1). The full-length cDNA corresponding to the complete amino acid sequence of MNV-1 3CLpro was amplified using primers of MNV 3CLpro-6hisXba-F (5′-tctagaaaggagatataccATG
Proteases from NV, MD145 or MNV-1 were inhibited by GC376 with a similar potency
The optimal concentrations of each protease and the substrate (5-FAM-DFHLQGP-QXL520) were determined for the FRET assay. The optimal concentrations of enzyme and the substrate were 0.03 and 1.6 μM for NV and MD145 3CLpro, respectively, and 0.057 and 1.6 μM for MNV 3CLpro, respectively. These combinations of 3CLpro and substrate gave strong signals with minimal backgrounds. A protease inhibitor, GC376, previously designed and reported from our lab was used to probe the inhibition of 3CLpro from
Discussion
Norovirus 3CLpro is a cysteine protease with chymotrypsin-like folds with two domains, N-terminal twisted anti-parallel β-sheet domain and C-terminal β-barrel domain. The active site with catalytic residues, C139, H30, and E54 are present in the cleft between the domains. The 3D crystal structures of NV 3CLpro (PDB: 3UR6, 3UR9, 2FYQ, 2FYR) (Kim et al., 2012, Zeitler et al., 2006), CV 3CLpro (PDB: 1WQS) (Nakamura et al., 2005), and Southampton virus (SV) 3CLpro bound with a Michael acceptor
Acknowledgments
We are grateful to David George for technical assistance. This work was supported by NIH grant AI081891. Use of the Advanced Photon Source was supported by the U.S. Department of Energy under Contract DE-AC02-06CH11357. Use of the KU COBRE-PSF Protein Structure Laboratory was supported by NIH grant P20 RR-17708. NMR instrumentation at KSU was funded by NIH grant (S10-RR 025441).
References (34)
- et al.
Characterization and inhibition of norovirus proteases of genogroups I and II using a fluorescence resonance energy transfer assay
Virology
(2012) - et al.
Design and synthesis of inhibitors of noroviruses by scaffold hopping
Bioorg. Med. Chem.
(2011) - et al.
Cyclosulfamide-based derivatives as inhibitors of noroviruses
Eur. J. Med. Chem.
(2012) - et al.
Potent inhibition of Norwalk virus by cyclic sulfamide derivatives
Bioorg. Med. Chem.
(2011) - et al.
Biodegradable nanogels for oral delivery of interferon for norovirus infection
Antiviral Res.
(2011) - et al.
Inhibition of norovirus 3CL protease by bisulfite adducts of transition state inhibitors
Bioorg. Med. Chem. Lett.
(2013) - et al.
Potent inhibition of Norwalk virus 3C protease by peptidyl α-ketoamides and α-ketoheterocycles
Bioorg. Med. Chem.
(2012) - et al.
Macrocyclic inhibitors of 3C and 3C-like proteases of picornavirus, norovirus, and coronavirus
Bioorg. Med. Chem. Lett.
(2013) - et al.
Noroviruses: a comprehensive review
J. Clin. Virol.
(2009) - et al.
Synthesis and anti-norovirus activity of pyranobenzopyrone compounds
Bioorg. Med. Chem. Lett.
(2012)