Norovirus 3C-like Protease Research Articles

Valence-driven colorimetric detection of norovirus protease via peptide-AuNP interactions.

We report here a colorimetric method for rapid detection of norovirus based on the valence-driven peptide-AuNP interactions. We engineered a peptide sequence named K1 with a cleavage sequence in between two lysine residues. The positively charged lysine groups aggregated the negatively charged nanoparticles leading to a purple color change. There was a red color when the cleavage sequence was digested by the Southampton norovirus 3C-like protease (SV3CP)-a protease involved in the life cycle of Human norovirus (HNV). The limit of detection was determined to be 320 nM in Tris buffer. We further show that the sensor has good performance in exhaled breath condensate, urine, and faecal matter. This research provides a potential easy and quick way to selectively detect HNV.

An approach to zwitterionic peptide design for colorimetric detection of the Southampton norovirus SV3CP protease.

Noroviruses are highly contagious and are one of the leading causes of acute gastroenteritis worldwide. Due to a lack of effective antiviral therapies, there is a need to diagnose and surveil norovirus infections to implement quarantine protocols and prevent large outbreaks. Currently, the gold standard of diagnosis uses reverse transcription polymerase chain reaction (RT-PCR), but PCR can have limited availability. Here, we propose a combination of a tunable peptide substrate and gold nanoparticles (AuNPs) to colorimetrically detect the Southampton norovirus 3C-like protease (SV3CP), a key protease in viral replication. Careful design of the substrate employs a zwitterionic peptide with opposite charged moieties on the C- and N- termini to induce a rapid color change visible to the naked eye; thus, this color change is indicative of SV3CP activity. This work expands on existing zwitterionic peptide strategies for protease detection by systematically evaluating the effects of lysine and arginine on nanoparticle charge screening. We also determine a limit of detection for SV3CP of 28.0 nM with comparable results in external breath condensate, urine, and fecal matter for 100 nM of SV3CP. The key advantage of this system is its simplicity and accessibility, thus making it an attractive tool for qualitative point-of-care diagnostics.

Norovirus 3C-Like protease antagonizes interferon-β production by cleaving NEMO

An epidemic owing to Norovirus (NoV) has recently been occurring worldwide. Severe cases of NoV can lead to patient death, resulting in significant public health problems. In the early stages of infection, antagonizing the production of host interferon (IFN) is an important strategy for viruses to establish infection. However, the relationship between NoV and interferon and its mechanism remains unclear. In this study, the 3C-like protease encoded by NoV was found to effectively suppress Sendai virus (SEV)-mediated IFN-β production by cleaving the NF-κB essential modulator (NEMO). Glutamine 205 is the site of NoV3CLpro-mediated cleavage of NEMO and this cleavage suppresses the ability of NEMO to activate downstream IFN production. These findings demonstrate that NoV3CLpro-induced cleavage limits NEMO to the activation of type I IFN signaling. In summary, our findings indicate that NoV3CLpro is a new interferon antagonist, and enhances our understanding of the escape of innate immunity mediated by NoV3CLpro.

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

Outbreaks of human epidemic nonbacterial gastroenteritis are mainly caused by noroviruses. Viral replication requires a 3C-like cysteine protease (3CLpro) which processes the 200 kDa viral polyprotein into six functional proteins. The 3CLpro has attracted much interest due to its potential as a target for antiviral drugs. A system for growing high-quality crystals of native Southampton norovirus 3CLpro (SV3CP) has been established, allowing the ligand-free crystal structure to be determined to 1.3 Å in a tetrameric state. This also allowed crystal-based fragment screening to be performed with various compound libraries, ultimately to guide drug discovery for SV3CP. A total of 19 fragments were found to bind to the protease out of the 844 which were screened. Two of the hits were located at the active site of SV3CP and showed good inhibitory activity in kinetic assays. Another 5 were found at the enzyme’s putative RNA-binding site and a further 11 were located in the symmetric central cavity of the tetramer.

Design, Synthesis, and Evaluation of Novel Prodrugs of Transition State Inhibitors of Norovirus 3CL Protease.

Ester and carbamate prodrugs of aldehyde bisulfite adduct inhibitors were synthesized in order to improve their pharmacokinetic and pharmacodynamic properties. The inhibitory activity of the compounds against norovirus 3C-like protease in enzyme and cell-based assays was determined. The ester and carbamate prodrugs displayed equivalent potency to those of the precursor aldehyde bisulfite adducts and precursor aldehydes. Furthermore, the rate of ester cleavage was found to be dependent on alkyl chain length. The generated prodrugs exhibited low cytotoxicity and satisfactory liver microsomes stability and plasma protein binding. The methodology described herein has wide applicability and can be extended to the bisulfite adducts of common warheads employed in the design of transition state inhibitors of serine and cysteine proteases of medical relevance.

Design, synthesis, and evaluation of a novel series of macrocyclic inhibitors of norovirus 3CL protease

Norovirus infections have a major impact on public health worldwide, yet there is a current dearth of norovirus-specific therapeutics and prophylactics. This report describes the discovery of a novel class of macrocyclic inhibitors of norovirus 3C-like protease, a cysteine protease that is essential for virus replication. SAR, structural, and biochemical studies were carried out to ascertain the effect of structure on pharmacological activity and permeability. Insights gained from these studies have laid a solid foundation for capitalizing on the therapeutic potential of the series of inhibitors described herein.

Structure-based design and synthesis of triazole-based macrocyclic inhibitors of norovirus protease: Structural, biochemical, spectroscopic, and antiviral studies

Outbreaks of acute gastroenteritis caused by noroviruses constitute a public health concern worldwide. To date, there are no approved drugs or vaccines for the management and prophylaxis of norovirus infections. A potentially effective strategy for the development of norovirus therapeutics entails the discovery of inhibitors of norovirus 3CL protease, an enzyme essential for noroviral replication. We describe herein the structure-based design of the first class of permeable, triazole-based macrocyclic inhibitors of norovirus 3C-like protease, as well as pertinent X-ray crystallographic, biochemical, spectroscopic, and antiviral studies.

Recent Advances in the Discovery of Norovirus Therapeutics.

Noroviruses are members of the family Caliciviridae. Norovirus infections are a global health burden that impacts >20 million individuals annually in the U.S. alone. Noroviruses are associated with high morbidity among vulnerable populations, particularly immunocompromised patients. This perspective highlights recent developments related to the discovery and development of norovirus-specific small-molecule therapeutics as well as recent advances in our understanding of norovirus biology and pathogenesis. Most of the work in this area is at the early discovery stage and has been primarily focused on inhibitors of norovirus 3C-like protease and RNA dependent RNA polymerase. However, recent discoveries emanating from basic studies in norovirus research have resulted in the identification of new host-related drug targets that can be exploited. A repurposed compound has been advanced to human clinical studies.

Structure-guided design and optimization of dipeptidyl inhibitors of norovirus 3CL protease. Structure-activity relationships and biochemical, X-ray crystallographic, cell-based, and in vivo studies.

Norovirus infection constitutes the primary cause of acute viral gastroenteritis. There are currently no vaccines or norovirus-specific antiviral therapeutics available for the management of norovirus infection. Norovirus 3C-like protease is essential for viral replication, consequently, inhibition of this enzyme is a fruitful avenue of investigation that may lead to the emergence of antinorovirus therapeutics. We describe herein the optimization of dipeptidyl inhibitors of norovirus 3C-like protease using iterative SAR, X-ray crystallographic, and enzyme and cell-based studies. We also demonstrate herein in vivo efficacy of an inhibitor using the murine model of norovirus infection.

Inhibition of norovirus 3CL protease by bisulfite adducts of transition state inhibitors

Noroviruses are the most common cause of acute viral gastroenteritis, accounting for >21 million cases annually in the US alone. Norovirus infections constitute an important health problem for which there are no specific antiviral therapeutics or vaccines. In this study, a series of bisulfite adducts derived from representative transition state inhibitors (dipeptidyl aldehydes and α-ketoamides) was synthesized and shown to exhibit anti-norovirus activity in a cell-based replicon system. The ED50 of the most effective inhibitor was 60nM. This study demonstrates for the first time the utilization of bisulfite adducts of transition state inhibitors in the inhibition of norovirus 3C-like protease in vitro and in a cell-based replicon system. The approach described herein can be extended to the synthesis of the bisulfite adducts of other classes of transition state inhibitors of serine and cysteine proteases, such as α-ketoheterocycles and α-ketoesters.

From head to toe of the norovirus 3C-like protease.

Abstract Noroviruses are major causative agents of viral gastroenteritis in humans. Currently, there are no therapeutic medications to treat noroviral infections, nor are there effective vaccines against these pathogens. The viral 3C-like protease is solely responsible for the maturation of viral protein components. The crystal structures of the proteases were resolved at high atomic resolution. The protease was also explored by means of mutagenesis. These studies revealed the active-site amino acid residues and factors determining and affecting substrate specificity as well as the principle of architecting the protease molecule. The possible mechanism of proteolysis was also suggested. Consideration of the data accumulated thus far will be useful for development of therapeutic drugs targeting the viral protease.

Insights into the Enzyme-Substrate Interaction in the Norovirus 3C-like Protease

The Glu54 residue of the norovirus 3C-like protease was implicated in proteolysis as a third-member carboxylate of the catalytic triad. The E54L mutant protease cleaved the sequence (133)LSFE/AP between the 3B and 3C regions of norovirus polyprotein, but did not cleave the sequence (198)ATSE/GK between the 3A and 3B. The 3BC junction mutation (3B-L133A or 3B-F135S) hampered the cleavage by the E54L protease, whereas the 3AB junction mutation (3A-A198L, S200F) allowed the E54L protease to digest. These results indicate that the E54L mutant protease is a substrate-specificity mutant and requires large hydrophobic amino acid residues at both P4 and P2 positions of the substrate. It was notable that the 3A-S200F P2 position mutation caused tight interaction between the wild-type protease and the C-terminus of the 3A protein, hence a decreased release rate of the product from the enzyme. This tight binding was dependent on the hydrophobicity of amino acid residues introduced at position 200 of the 3A region and was affected by the mutation in the bII-cII loop of the protease or the mutation of position 198 of 3A corresponding to the P4 position of the substrate. These results suggest that the protease and the substrate sense each other in the process of the proteolysis, being supported by crystal structures.

Saturation mutagenesis reveals that GLU54 of norovirus 3C-like protease is not essential for the proteolytic activity.

The norovirus 3C-like protease is a member of the chymotrypsin-like serine protease superfamily. Previous characterization of its crystal structure has implicated the Glu54–His30–Cys139 triad in the catalysis. In the present study, the Glu54 residue of the protease was subjected to site-saturation mutagenesis, with the result that nearly half of the mutants retained the significant proteolytic activity. It was suggested that a carboxylate at position 54 was not essential for the activity. The in vitro assays of the proteolysis revealed that most of Glu54 mutants retained relatively high proteolytic activity. When the Glu54 mutation was combined with the Ser mutation of the Cys139 residue, a nucleophile, only the Asp54 and Gln54 mutations showed proteolytic activity comparable to that of the Ser139 single mutant, suggesting that a hydrogen bond between Glu54 and His30 was critical in the Ser139 background. These results suggested that the mechanism of the proteolysis by the wild-type norovirus 3C-like protease was different from that of typical chymotrypsin-like serine proteases.

Characterization of the norovirus 3C-like protease

The recombinant 3C-like protease of Chiba virus, a Norovirus, expressed in Escherichia coli cells was purified and characterized as to effects of pH, temperature, salt contents, and SH reagents on its proteolytic activity. The optimal pH and temperature of the 3C-like protease for the proteolytic activity were 8.6 and 37 °C, respectively. Increased concentration (∼100 mM) of monovalent cations such as Na + and K + was inhibitory to the activity. Hg 2+ and Zn 2+ remarkably inhibited the protease activity, while Mg 2+ and Ca 2+ had no virtual effect. Several sulfhydryl reagents such as p-chloromercuribenzoic acid, methyl methanethiosulfonate, N-ethylmaleimide and N-phenylmaleimide also blocked the activity, confirming the previous result that cysteine residue(s) were responsible for the proteolysis.