CX-6258 hydrochloride hydrate: A potential non-nucleoside inhibitor targeting the RNA-dependent RNA polymerase of norovirus.

Human norovirus (HuNoV), a primary cause of non-bacterial gastroenteritis, currently lacks approved treatment. RdRp is vital for virus replication, making it an attractive target for therapeutic intervention. By application of structure-based virtual screening procedure, we present CX-6258 hydrochloride hydrate as a potent RdRp non-nucleoside inhibitor, effectively inhibiting HuNoV RdRp activity with an IC50 of 3.61 µM. Importantly, this compound inhibits viral replication in cell culture, with an EC50 of 0.88 µM. In vitro binding assay validate that CX-6258 hydrochloride hydrate binds to RdRp through interaction with the "B-site" binding pocket. Interestingly, CX-6258-contacting residues such as R392, Q439, and Q414 are highly conserved among major norovirus GI and GII variants, suggesting that it may be a general inhibitor of norovirus RdRp. Given that CX-6258 hydrochloride hydrate is already utilized as an orally efficacious pan-Pim kinase inhibitor, it may serve as a potential lead compound in the effort to control HuNoV infections.

Norovirus MLKL-like protein initiates cell death to induce viral egress.

Non-enveloped viruses require cell lysis to release new virions from infected cells, suggesting that these viruses require mechanisms to induce cell death. Noroviruses are one such group of viruses, but there is no known mechanism that causes norovirus infection-triggered cell death and lysis1-3. Here we identify a molecular mechanism of norovirus-induced cell death. We found that the norovirus-encoded NTPase NS3 contains an N-terminal four-helix bundle domain homologous to the membrane-disruption domain of the pseudokinase mixed lineage kinase domain-like (MLKL). NS3 has a mitochondrial localization signal and thus induces cell death by targeting mitochondria. Full-length NS3 and an N-terminal fragment of the protein bound the mitochondrial membrane lipid cardiolipin, permeabilized the mitochondrial membrane and induced mitochondrial dysfunction. Both the N-terminal region and the mitochondrial localization motif of NS3 were essential for cell death, viral egress from cells and viral replication in mice. These findings suggest that noroviruses have acquired a host MLKL-like pore-forming domain to facilitate viral egress by inducing mitochondrial dysfunction.

Structural Investigations on Novel Non-Nucleoside Inhibitors of Human Norovirus Polymerase.

Human norovirus is the first cause of foodborne disease worldwide, leading to extensive outbreaks of acute gastroenteritis, and causing around 200,000 children to die annually in developing countries. No specific vaccines or antiviral agents are currently available, with therapeutic options limited to supportive care to prevent dehydration. The infection can become severe and lead to life-threatening complications in young children, the elderly and immunocompromised individuals, leading to a clear need for antiviral agents, to be used as treatments and as prophylactic measures in case of outbreaks. Due to the key role played by the viral RNA-dependent RNA polymerase (RdRp) in the virus life cycle, this enzyme is a promising target for antiviral drug discovery. In previous studies, following in silico investigations, we identified different small-molecule inhibitors of this enzyme. In this study, we rationally modified five identified scaffolds, to further explore structure-activity relationships, and to enhance binding to the RdRp. The newly designed compounds were synthesized according to multiple-step synthetic routes and evaluated for their inhibition of the enzyme in vitro. New inhibitors with low micromolar inhibitory activity of the RdRp were identified, which provide a promising basis for further hit-to-lead optimization.

Norovirus Protease Structure and Antivirals Development.

Human norovirus (HuNoV) infection is a global health and economic burden. Currently, there are no licensed HuNoV vaccines or antiviral drugs available. The protease encoded by the HuNoV genome plays a critical role in virus replication by cleaving the polyprotein and is an excellent target for developing small-molecule inhibitors. The current strategy for developing HuNoV protease inhibitors is by targeting the enzyme's active site and designing inhibitors that bind to the substrate-binding pockets located near the active site. However, subtle differential conformational flexibility in response to the different substrates in the polyprotein and structural differences in the active site and substrate-binding pockets across different genogroups, hamper the development of effective broad-spectrum inhibitors. A comparative analysis of the available HuNoV protease structures may provide valuable insight for identifying novel strategies for the design and development of such inhibitors. The goal of this review is to provide such analysis together with an overview of the current status of the design and development of HuNoV protease inhibitors.

A Novel Class of Norovirus Inhibitors Targeting the Viral Protease with Potent Antiviral Activity In Vitro and In Vivo.

Human noroviruses (HuNoVs) are the most common cause of viral gastroenteritis resulting annually in ~219,000 deaths and a societal cost of ~USD 60 billion, and no antivirals or vaccines are available. Here, we assess the anti-norovirus activity of new peptidomimetic aldehydes related to the protease inhibitor rupintrivir. The early hit compound 4 inhibited the replication of murine norovirus (MNV) and the HuNoV GI.1 replicon in vitro (EC50 ~1 µM) and swiftly cleared the HuNoV GI.1 replicon from the cells. Compound 4 still inhibits the proteolytic activity. We selected a resistant GI.1 replicon, with a mutation (I109V) in a highly conserved region of the viral protease, conferring a low yield of resistance against compound 4 and rupintrivir. After testing new derivatives, compound 10d was the most potent (EC50 nanomolar range). Molecular docking indicated that the aldehyde group of compounds 4 and 10d bind with Cys139 in the HuNoV 3CL protease by a covalent linkage. Finally, compound 10d inhibited the replication of HuNoV GII.4 in infected zebrafish larvae, and PK studies in mice showed an adequate profile.

Identification and Characterization of Human Norovirus NTPase Regions Required for Lipid Droplet Localization, Cellular Apoptosis, and Interaction with the Viral P22 Protein.

The human norovirus (HuNV)-encoded nucleoside-triphosphatase (NTPase) is a multifunctional protein critically involved in viral replication and pathogenesis. Previously, we have shown that the viral NTPase is capable of forming vesicle clusters in cells, interacting with other viral proteins such as P22, and promoting cellular apoptosis. Herein, we demonstrate that NTPase-associated vesicle clusters correspond to lipid droplets (LDs) wrapped by the viral protein and show that NTPase-induced apoptosis is mediated through both caspase-8- and caspase-9-dependent pathways. Deletion analysis revealed that the N-terminal 179-amino-acid (aa) region of NTPase encompasses two LD-targeting motifs (designated LTM-1 and LTM-2), two apoptosis-inducing motifs, and multiple regulatory regions. Interestingly, the identified LTM-1 and LTM-2, which are located from aa 1 to 50 and from aa 51 to 90, respectively, overlap with the two apoptosis-inducing motifs. Although there was no positive correlation between the extent of LD localization and the degree of cellular apoptosis for NTPase mutants, we noticed that mutant proteins defective in LD-targeting ability could not induce cellular apoptosis. In addition to LD targeting, the amphipathic LTM-1 and LTM-2 motifs could have the potential to direct fusion proteins to the endoplasmic reticulum (ER). Furthermore, we found that the LTM-1 motif is a P22-interacting motif. However, P22 functionally augmented the proapoptotic activity of the LTM-2 fusion protein but not the LTM-1 fusion protein. Overall, our findings propose that NTPase may participate in multiple cellular processes through binding to LDs or to the ER via its N-terminal amphipathic helix motifs. IMPORTANCE Human noroviruses (HuNVs) are the major agent of global gastroenteritis outbreaks. However, due to the lack of an efficient cell culture system for HuNV propagation, functions of the viral-encoded proteins in host cells are still poorly understood. In the current study, we present that the viral NTPase is a lipid droplet (LD)-associated protein, and we identify two LD-targeting motifs, LTM-1 and LTM-2, in its N-terminal domain. In particular, the identified LTM-1 and LTM-2 motifs, which contain a hydrophobic region and an amphipathic helix, are also capable of delivering the fusion protein to the endoplasmic reticulum (ER), promoting cellular apoptosis, and physically or functionally associating with another viral protein P22. Since LDs and the ER have been linked to several biological functions in cells, our study therefore proposes that the norovirus NTPase may utilize LDs or the ER as replication platforms to benefit viral replication and pathogenesis.

Structure-Guided Optimization of Dipeptidyl Inhibitors of Norovirus 3CL Protease.

Acute gastroenteritis caused by noroviruses has a major impact on public health worldwide in terms of morbidity, mortality, and economic burden. The disease impacts most severely immunocompromised patients, the elderly, and children. The current lack of approved vaccines and small-molecule therapeutics for the treatment and prophylaxis of norovirus infections underscores the need for the development of norovirus-specific drugs. The studies described herein entail the use of the gem-dimethyl moiety as a means of improving the pharmacological activity and physicochemical properties of a dipeptidyl series of transition state inhibitors of norovirus 3CL protease, an enzyme essential for viral replication. Several compounds were found to be potent inhibitors of the enzyme in biochemical and cell-based assays. The pharmacological activity and cellular permeability of the inhibitors were found to be sensitive to the location of the gem-dimethyl group.

Murine norovirus replicase augments RIG-I-like receptors-mediated antiviral interferon response.

Noroviruses are the main causative agents for acute viral gastroenteritis worldwide. RIG-I-like receptors (RLRs) triggered interferon (IFN) activation is essential for host defense against viral infections. In turn, viruses have developed sophisticated strategies to counteract host antiviral response. This study aims to investigate how murine norovirus (MNV) replicase interacts with RLRs-mediated antiviral IFN response. Counterintuitively, we found that the MNV replicase NS7 enhances the activation of poly (I:C)-induced IFN response and the transcription of downstream interferon-stimulated genes (ISGs). Interestingly, NS7 protein augments RIG-I and MDA5-triggered antiviral IFN response, which conceivably involves direct interactions with the caspase activation and recruitment domains (CARDs) of RIG-I and MDA5. Consistently, RIG-I and MDA5 exert anti-MNV activity in human HEK293T cells with ectopic expression of viral receptor CD300lf. This effect requires the activation of JAK/STAT pathway, and is further enhanced by NS7 overexpression. These findings revealed an unconventional role of MNV NS7 as augmenting RLRs-mediated IFN response to inhibit viral replication.

A new antiviral scaffold for human norovirus identified with computer-aided approaches on the viral polymerase.

Human norovirus is the leading cause of acute gastroenteritis worldwide, affecting every year 685 million people. In about one third of cases, this virus affects children under five years of age, causing each year up to 200,000 child deaths, mainly in the developing countries. Norovirus outbreaks are associated with very significant economic losses, with an estimated societal cost of 60 billion dollars per year. Despite the marked socio-economic consequences associated, no therapeutic options or vaccines are currently available to treat or prevent this infection. One promising target to identify new antiviral agents for norovirus is the viral polymerase, which has a pivotal role for the viral replication and lacks closely homologous structures in the host. Starting from the scaffold of a novel class of norovirus polymerase inhibitors recently discovered in our research group with a computer-aided method, different new chemical modifications were designed and carried out, with the aim to identify improved agents effective against norovirus replication in cell-based assays. While different new inhibitors of the viral polymerase were found, a further computer-aided ligand optimisation approach led to the identification of a new antiviral scaffold for norovirus, which inhibits human norovirus replication at low-micromolar concentrations.

Putative structural rearrangements associated with the interaction of macrocyclic inhibitors with norovirus 3CL protease.

Human noroviruses are the primary cause of outbreaks of acute gastroenteritis worldwide. The problem is further compounded by the current lack of norovirus-specific antivirals or vaccines. Noroviruses have a single-stranded, positive sense 7 to 8 kb RNA genome which encodes a polyprotein precursor that is processed by a virus-encoded 3C-like cysteine protease (NV 3CLpro) to generate at least six mature nonstructural proteins. Processing of the polyprotein is essential for virus replication, consequently, NV 3CLpro has emerged as an attractive target for the discovery of norovirus therapeutics and prophylactics. We have recently described the structure-based design of macrocyclic transition state inhibitors of NV 3CLpro. In order to gain insight and understanding into the interaction of macrocyclic inhibitors with the enzyme, as well as probe the effect of ring size on pharmacological activity and cellular permeability, additional macrocyclic inhibitors were synthesized and high resolution cocrystal structures determined. The results of our studies tentatively suggest that the macrocyclic scaffold may hamper optimal binding to the active site by impeding concerted cross-talk between the S2 and S4 subsites.

Antiviral Drug Discovery: Norovirus Proteases and Development of Inhibitors.

Proteases are a major enzyme group playing important roles in a wide variety of biological processes in life forms ranging from viruses to mammalians. The aberrant activity of proteases can lead to various diseases; consequently, host proteases have been the focus of intense investigation as potential therapeutic targets. A wide range of viruses encode proteases which play an essential role in viral replication and, therefore, constitute attractive targets for the development of antiviral therapeutics. There are numerous examples of successful drug development targeting cellular and viral proteases, including antivirals against human immunodeficiency virus and hepatitis C virus. Most FDA-approved antiviral agents are peptidomimetics and macrocyclic compounds that interact with the active site of a targeted protease. Norovirus proteases are cysteine proteases that contain a chymotrypsin-like fold in their 3D structures. This review focuses on our group's efforts related to the development of norovirus protease inhibitors as potential anti-norovirus therapeutics. These protease inhibitors are rationally designed transition-state inhibitors encompassing dipeptidyl, tripeptidyl and macrocyclic compounds. Highly effective inhibitors validated in X-ray co-crystallization, enzyme and cell-based assays, as well as an animal model, were generated by launching an optimization campaign utilizing the initial hit compounds. A prodrug approach was also explored to improve the pharmacokinetics (PK) of the identified inhibitors.

Targeting the Viral Polymerase of Diarrhea-Causing Viruses as a Strategy to Develop a Single Broad-Spectrum Antiviral Therapy.

Viral gastroenteritis is an important cause of morbidity and mortality worldwide, being particularly severe for children under the age of five. The most common viral agents of gastroenteritis are noroviruses, rotaviruses, sapoviruses, astroviruses and adenoviruses, however, no specific antiviral treatment exists today against any of these pathogens. We here discuss the feasibility of developing a broad-spectrum antiviral treatment against these diarrhea-causing viruses. This review focuses on the viral polymerase as an antiviral target, as this is the most conserved viral protein among the diverse viral families to which these viruses belong to. We describe the functional and structural similarities of the different viral polymerases, the antiviral effect of reported polymerase inhibitors and highlight common features that might be exploited in an attempt of designing such pan-polymerase inhibitor.

Polyprotein processing and intermolecular interactions within the viral replication complex spatially and temporally control norovirus protease activity.

Norovirus infections are a major cause of acute viral gastroenteritis and a significant burden on global human health. A vital process for norovirus replication is the processing of the nonstructural polyprotein by a viral protease into the viral components required to form the viral replication complex. This cleavage occurs at different rates, resulting in the accumulation of stable precursor forms. Here, we characterized how precursor forms of the norovirus protease accumulate during infection. Using stable forms of the protease precursors, we demonstrated that all of them are proteolytically active in vitro, but that when expressed in cells, their activities are determined by both substrate and protease localization. Although all precursors could cleave a replication complex-associated substrate, only a subset of precursors lacking the NS4 protein were capable of efficiently cleaving a cytoplasmic substrate. By mapping the full range of protein-protein interactions among murine and human norovirus proteins with the LUMIER assay, we uncovered conserved interactions between replication complex members that modify the localization of a protease precursor subset. Finally, we demonstrate that fusion to the membrane-bound replication complex components permits efficient cleavage of a fused substrate when active polyprotein-derived protease is provided in trans These findings offer a model for how norovirus can regulate the timing of substrate cleavage throughout the replication cycle. Because the norovirus protease represents a key target in antiviral therapies, an improved understanding of its function and regulation, as well as identification of interactions among the other nonstructural proteins, offers new avenues for antiviral drug design.

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

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.

Nucleotide triphosphatase and RNA chaperone activities of murine norovirus NS3.

Modulation of RNA structure is essential in the life cycle of RNA viruses. Immediate replication upon infection requires RNA unwinding to ensure that RNA templates are not in intra- or intermolecular duplex forms. The calicivirus NS3, one of the highly conserved nonstructural (NS) proteins, has conserved motifs common to helicase superfamily 3 among six genogroups. However, its biological functions are not fully understood. In this study we report the oligomeric state and the nucleotide triphosphatase (NTPase) and RNA chaperone activities of the recombinant full-length NS3 derived from murine norovirus (MNV). The MNV NS3 has an Mg2+-dependent NTPase activity, and site-directed mutagenesis of the conserved NTPase motifs blocked enzyme activity and viral replication in cells. Further, the NS3 was found via fluorescence resonance energy transfer (FRET)-based assays to destabilize double-stranded RNA in the presence of Mg2+ or Mn2+ in an NTP-independent manner. However, the RNA destabilization activity was not affected by mutagenesis of the conserved motifs of NTPase. These results reveal that the MNV NS3 has an NTPase-independent RNA chaperone-like activity, and that a FRET-based RNA destabilization assay has the potential to identify new antiviral drugs targeting NS3.

Expression, Purification and Characterization of a GII.4 Norovirus Protease from Minerva Virus.

BACKGROUND: Noroviruses are the leading cause of acute gastroenteritis worldwide. Norovirus proteases, which are responsible for cleavage of the viral polyprotein, have become an attractive drug target to treat norovirus infections. Genogroup II (GII) noroviruses are responsible for a majority of outbreaks; however, limited data exists regarding GII norovirus proteases. METHODS: We report here successful expression, purification, characterization, and inhibition of the Minerva virus protease (MVpro), a genogroup II genotype 4 (GII.4) norovirus protease. We observed MVpro as both a monomer and dimer in solution through sizeexclusion chromatography. In addition, MVpro cleaves the synthetic substrate mimicking the MVpro NS2/NS3 cleavage site more efficiently than other norovirus proteases such as the Norwalk virus protease (GI.1) and the MD145 protease (GII.4). RESULTS AND CONCLUSION: Compound A, a potent inhibitor of MVpro, is a good starting point for the design of inhibitors to target GII.4 noroviruses. Furthermore, the results presented here will allow for future characterization of MVpro inhibitors as they are synthesized.

Synthesis and antiviral evaluation of novel peptidomimetics as norovirus protease inhibitors.

A series of tripeptidyl transition state inhibitors with new P1 and warhead moieties were synthesized and evaluated in a GI-1 norovirus replicon system and against GII-4 and GI-1 norovirus proteases. Compound 19, containing a 6-membered ring at the P1 position and a reactive aldehyde warhead exhibited sub-micromolar replicon inhibition. Retaining the same peptidyl scaffold, several reactive warheads were tested for protease inhibition and norovirus replicon inhibition. Of the six that were synthesized and tested, compounds 42, 43, and 45 potently inhibited the protease in biochemical assay and GI-1 norovirus replicon in the nanomolar range.

In silico screening for human norovirus antivirals reveals a novel non-nucleoside inhibitor of the viral polymerase.

Human norovirus causes approximately 219,000 deaths annually, yet there are currently no antivirals available. A virtual screening of commercially available drug-like compounds (~300,000) was performed on the suramin and PPNDS binding-sites of the norovirus RNA-dependent RNA polymerase (RdRp). Selected compounds (n = 62) were examined for inhibition of norovirus RdRp activity using an in vitro transcription assay. Eight candidates demonstrated RdRp inhibition (>25% inhibition at 10 µM), which was confirmed using a gel-shift RdRp assay for two of them. The two molecules were identified as initial hits and selected for structure-activity relationship studies, which resulted in the synthesis of novel compounds that were examined for inhibitory activity. Five compounds inhibited human norovirus RdRp activity (>50% at 10 µM), with the best candidate, 54, demonstrating an IC50 of 5.6 µM against the RdRp and a CC50 of 62.8 µM. Combinational treatment of 54 and the known RdRp site-B inhibitor PPNDS revealed antagonism, indicating that 54 binds in the same binding pocket. Two RdRps with mutations (Q414A and R419A) previously shown to be critical for the binding of site-B compounds had no effect on inhibition, suggesting 54 interacts with distinct site-B residues. This study revealed the novel scaffold 54 for further development as a norovirus antiviral.

Human Norovirus NS3 Has RNA Helicase and Chaperoning Activities.

RNA-remodeling proteins, including RNA helicases and chaperones, act to remodel RNA structures and/or protein-RNA interactions and are required for all processes involving RNAs. Although many viruses encode RNA helicases and chaperones, their in vitro activities and their roles in infected cells largely remain elusive. Noroviruses are a diverse group of positive-strand RNA viruses in the family Caliciviridae and constitute a significant and potentially fatal threat to human health. Here, we report that the protein NS3 encoded by human norovirus has both ATP-dependent RNA helicase activity that unwinds RNA helices and ATP-independent RNA-chaperoning activity that can remodel structured RNAs and facilitate strand annealing. Moreover, NS3 can facilitate viral RNA synthesis in vitro by norovirus polymerase. NS3 may therefore play an important role in norovirus RNA replication. Lastly, we demonstrate that the RNA-remodeling activity of NS3 is inhibited by guanidine hydrochloride, an FDA-approved compound, and, more importantly, that it reduces the replication of the norovirus replicon in cultured human cells. Altogether, these findings are the first to demonstrate the presence of RNA-remodeling activities encoded by Caliciviridae and highlight the functional significance of NS3 in the noroviral life cycle.IMPORTANCE Noroviruses are a diverse group of positive-strand RNA viruses, which annually cause hundreds of millions of human infections and over 200,000 deaths worldwide. For RNA viruses, cellular or virus-encoded RNA helicases and/or chaperones have long been considered to play pivotal roles in viral life cycles. However, neither RNA helicase nor chaperoning activity has been demonstrated to be associated with any norovirus-encoded proteins, and it is also unknown whether norovirus replication requires the participation of any viral or cellular RNA helicases/chaperones. We found that a norovirus protein, NS3, not only has ATP-dependent helicase activity, but also acts as an ATP-independent RNA chaperone. Also, NS3 can facilitate in vitro viral RNA synthesis, suggesting the important role of NS3 in norovirus replication. Moreover, NS3 activities can be inhibited by an FDA-approved compound, which also suppresses norovirus replicon replication in human cells, raising the possibility that NS3 could be a target for antinoroviral drug development.

Structure-guided design, synthesis and evaluation of oxazolidinone-based inhibitors of norovirus 3CL protease.

Acute nonbacterial gastroenteritis caused by noroviruses constitutes a global public health concern and a significant economic burden. There are currently no small molecule therapeutics or vaccines for the treatment of norovirus infections. A structure-guided approach was utilized in the design of a series of inhibitors of norovirus 3CL protease that embody an oxazolidinone ring as a novel design element for attaining optimal binding interactions. Low micromolar cell-permeable inhibitors that display anti-norovirus activity have been identified. The mechanism of action, mode of binding, and structural rearrangements associated with the interaction of the inhibitors and the enzyme were elucidated using X-ray crystallography.