The Zika virus presents a major public health concern due to severe fetal neurological disorders associated with infections in pregnant women. In addition to vaccine development, the discovery of ...selective antiviral drugs is essential to combat future epidemic Zika virus outbreaks. The Zika virus NS2B-NS3 protease, which performs replication-critical cleavages of the viral polyprotein, is a promising drug target. We report the first macrocyclic peptide-based inhibitors of the NS2B-NS3 protease, discovered de novo through in vitro display screening of a genetically reprogrammed library including noncanonical residues. Six compounds were selected, resynthesized, and isolated, all of which displayed affinities in the low nanomolar concentration range. Five compounds showed significant protease inhibition. Two of these were validated as hits with submicromolar inhibition constants and selectivity toward Zika over the related proteases from dengue and West Nile viruses. The compounds were characterized as noncompetitive inhibitors, suggesting allosteric inhibition.
Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae family (Lagovirus genus). RHDV is highly contagious and attaches to epithelial cells in the digestive or respiratory tract, ...leading to massive lesions with high mortality rates. A new variant of RHDV (termed RHDVb) recently has emerged, and previously vaccinated rabbits appear to have little protection against this new strain. Similar to human norovirus (Caliciviridae, Norovirus genus), RHDV binds histo-blood group antigens (HBGAs), and this is thought to be important for infection. Here, we report the HBGA binding site on the RHDVb capsid-protruding domain (P domain) using X-ray crystallography. The HBGA binding pocket was located in a negatively charged patch on the side of the P domain and at a dimeric interface. Residues from both monomers contributed to the HBGA binding and involved a network of direct hydrogen bonds and water-mediated interactions. An amino acid sequence alignment of different RHDV strains indicated that the residues directly interacting with the ABH-fucose of the HBGAs (Asp472, Asn474, and Ser479) were highly conserved. This result suggested that different RHDV strains also could bind HBGAs at the equivalent pocket. Moreover, several HBGA binding characteristics between RHDVb and human genogroup II norovirus were similar, which indicated a possible convergent evolution of HBGA binding interactions. Further structural studies with other RHDV strains are needed in order to better understand the HBGA binding mechanisms among the diverse RHDV strains.
We identified, for the first time, the HBGA binding site on an RHDVb P domain using X-ray crystallography. Our results showed that RHDVb and human genogroup II noroviruses had similar HBGA binding interactions. Recently, it was discovered that synthetic HBGAs or HBGA-expressing enteric bacteria could enhance human genogroup II norovirus infection in B cells. Considering that RHDVb and genogroup II norovirus similarly interacted with HBGAs, it may be possible that a comparable cell culture system also could work with RHDVb. Taken together, these new findings will extend our understanding of calicivirus HBGA interactions and may help to elucidate the specific roles of HBGAs in calicivirus infections.
Human norovirus interacts with the polymorphic human histo-blood group antigens (HBGAs), and this interaction is thought to be important for infection. The genogroup II genotype 4 (GII.4) noroviruses ...are the dominant cluster, evolve every other year, and are thought to modify their binding interactions with different HBGA types. Most human noroviruses bind HBGAs, while some strains were found to have minimal or no HBGA interactions. Here, we explain some possible structural constraints for several noroviruses that were found to bind poorly to HBGAs by using X-ray crystallography. We showed that one aspartic acid was flexible or positioned away from the fucose moiety of the HBGAs and this likely hindered binding, although other fucose-interacting residues were perfectly oriented. Interestingly, a neighboring loop also appeared to influence the loop hosting the aspartic acid. These new findings might explain why some human noroviruses bound HBGAs poorly, although further studies are required.
Human noroviruses are the dominant cause of outbreaks of gastroenteritis around the world. Human noroviruses interact with the polymorphic human histo-blood group antigens (HBGAs), and this ...interaction is thought to be important for infection. Indeed, synthetic HBGAs or HBGA-expressing enteric bacteria were shown to enhance norovirus infection in B cells. A number of studies have found a possible relationship between HBGA type and norovirus susceptibility. The genogroup II, genotype 4 (GII.4) noroviruses are the dominant cluster, evolve every other year, and are thought to modify their binding interactions with different HBGA types. Here we show high-resolution X-ray crystal structures of the capsid protruding (P) domains from epidemic GII.4 variants from 2004, 2006, and 2012, cocrystallized with a panel of HBGA types (H type 2, Lewis Y, Lewis B, Lewis A, Lewis X, A type, and B type). Many of the HBGA binding interactions were found to be complex, involving capsid loop movements, alternative HBGA conformations, and HBGA rotations. We showed that a loop (residues 391 to 395) was elegantly repositioned to allow for Lewis Y binding. This loop was also slightly shifted to provide direct hydrogen- and water-mediated bonds with Lewis B. We considered that the flexible loop modulated Lewis HBGA binding. The GII.4 noroviruses have dominated outbreaks over the past decade, which may be explained by their exquisite HBGA binding mechanisms, their fondness for Lewis HBGAs, and their temporal amino acid modifications.
Our data provide a comprehensive picture of GII.4 P domain and HBGA binding interactions. The exceptionally high resolutions of our X-ray crystal structures allowed us to accurately recognize novel GII.4 P domain interactions with numerous HBGA types. We showed that the GII.4 P domain-HBGA interactions involved complex binding mechanisms that were not previously observed in norovirus structural studies. Many of the GII.4 P domain-HBGA interactions we identified were negative in earlier enzyme-linked immunosorbent assay (ELISA)-based studies. Altogether, our data show that the GII.4 norovirus P domains can accommodate numerous HBGA types.
HBV persistence is maintained by both an episomal covalently closed circular (ccc)DNA pool and genomic integration of HBV DNA fragments. While cccDNA transcription is regulated by ...Cullin4A-DDB1-HBx-mediated degradation of the SMC5/6 complex, HBsAg expression from integrants is largely SMC5/6 independent. Inhibiting the conjugation of Nedd8 to Cullin-RING ubiquitin ligases impairs degradation of substrates, restoring SMC5/6 levels. Herein, we show that targeting neddylation pathway components by small-interfering (si)RNAs or the drug MLN4924 (pevonedistat) suppresses expression of HBV proteins from both cccDNA and integrants.
An siRNA screen targeting secretory pathway regulators and neddylation genes was performed. Activity of MLN4924 was assessed in infection and integration models. Trans-complementation assays were used to study HBx function in cccDNA and integrant-driven expression.
siRNA screening uncovered neddylation pathway components (Nedd8, Ube2m) that promote HBsAg production post-transcriptionally. Likewise, MLN4924 inhibited production of HBsAg encoded by integrants and reduced intracellular HBsAg levels, independent of HBx. MLN4924 also profoundly inhibited cccDNA transcription in 3 infection models. Using the HBV inducible cell line HepAD38 as a model we verified the dual action of MLN4924 on both cccDNA and integrants with sustained suppression of HBV markers during 42 days of treatment.
Neddylation is required both for the formation of a cccDNA reservoir and for the genomic integration of viral DNA. Therefore, blocking neddylation might offer an attractive approach towards functional cure of chronic hepatitis B.
While there are effective treatments for chronic hepatitis B, they are rarely able to induce functional cure. This is partly because of the presence of a pool of circular viral DNA in the host nucleus, as well as viral DNA fragments that are integrated into the host genome. Herein, we show that a host biological pathway called neddylation could play a key role in infection and viral DNA integration. Inhibiting this pathway could hold therapeutic promise for patients with chronic hepatitis B.
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•Neddylation plays a dual role in HBV expression from viral integrants and episomal cccDNA.•Impaired neddylation suppresses production of HBsAg expressed from viral integrants.•Neddylation promotes HBsAg generation from viral integrants in an HBx-independent manner.•MLN4924 also inhibits the synthesis of viral transcripts from episomal cccDNA.
The RNA viruses SARS-CoV-2 and dengue pose a major threat to human health worldwide and their proteases (Mpro; NS2B/NS3) are considered as promising targets for drug development. We present the ...synthesis and biological evaluation of novel benzoxaborole inhibitors of these two proteases. The most active compound achieves single-digit micromolar activity against SARS-CoV-2 Mpro in a biochemical assay. The most active substance against dengue NS2B/NS3 protease has submicromolar activity in cells (EC50 0.54 μM) and inhibits DENV-2 replication in cell culture. Most benzoxaboroles had no relevant cytotoxicity or significant off-target inhibition. Furthermore, the class demonstrated passive membrane penetration and stability against the evaluated proteases. This compound class may contribute to the development of antiviral agents with activity against DENV or SARS-CoV-2.
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•Benzoxaboroles are inhibitors of the dengue protease and SARS CoV-2 main protease.•Activity against DENV protease is shown in a cellular reporter-gene assay.•The compounds have low cytotoxicity and do not interfere with off-target proteases.
Abstract Norovirus infects different animals, including humans, mice, dogs, and cats. Here, we show an X-ray crystal structure of a feline GIV.2 norovirus capsid-protruding (P) domain to 2.35 Å ...resolution. The feline GIV.2 P domain was reminiscent of human norovirus P domains, except for a novel P2 subdomain α-helix and an extended P1 subdomain interface loop. These new structural features likely obstructed histo-blood group antigens, which are attachment factors for human norovirus, from binding at the equivalent sites on the feline GIV.2 P domain. Additionally, an ELISA showed that the feline GIV.2 was antigenically distinct from a human GII.10 norovirus.
•Direct capture of proteins from particle-containing solutions.•Process simplification by using tangential flow through membranes.•Lower frequency of purging compared to classic ...MA-IEX-capsules.•Modules with extruded polymer spacers (250μm) show highest binding capacity.•Modules with extruded polymer spacers (480μm) show best long term stability.
Potato fruit juice (PFJ) is a by-product from industrial starch production. It still contains several valuable components such as amino acids, minerals and proteins. An economic technology for the isolation and purification of different native potato proteins is the ion exchange chromatography, which can be performed either by classical bed chromatography or by membrane adsorption chromatography (MA-IEX). An already published MA-IEX process for the downstreaming of PFJ is based on the following steps: prefiltration/microfiltration, fractionation with MA-IEX, ultra-/diafiltration and finally drying. In order to further minimize process complexity and costs, new MA-IEX-modules were designed and tested in this research project to facilitate the processing of crude, particle-containing solutions using a tangential flow through the membranes. Modules with fleece polymer spacers and extruded polymer spacers, as well as different spacer channel sizes were tested for their binding capacities and their long-term stability. An optimized setup was found for the technical scale. Modules with extruded polymer spacers channel size 250μm show the highest binding capacities (anion exchanger approx. 0.34mg/cm2, cation exchanger approx. 0.16mg/cm2), while the modules with extruded polymer spacers channel size 480μm show the best long-term stability with 23 passes without intermediary cleaning.
The β-lactam ring represents a valuable moiety that can induce covalent binding of an inhibitor to its target. In this study, we explored di- and tripeptides with β-lactam electrophilic warheads as ...inhibitors of dengue and West Nile virus NS2B-NS3 protease. Tripeptides with a (3S)-β-lactam moiety displayed the highest activity, with IC50 and EC50 values in the lower micromolar range in biochemical and cellular assays. The activity against dengue protease was in general higher than against West Nile virus protease. The compounds were inactive against the off-targets thrombin and trypsin. Liquid chromatography–mass spectrometry experiments revealed that tripeptide-β-lactam inhibitors bind to the protease in two distinct binding modes. Only one binding mode leads to a covalent, but reversible, interaction of the β-lactam ring with the catalytic serine, followed by release of the inhibitor with opened β-lactam ring. The other binding mode leads to the cleavage of the peptide backbone. This observation provides the first experimental evidence that benzyloxyphenylglycine in flaviviral protease inhibitors is positioned in the prime site of the enzyme.
We have remotely mapped optical scattering and absorption in glacial ice at the South Pole for wavelengths between 313 and 560 nm and depths between 1100 and 2350 m. We used pulsed and continuous ...light sources embedded with the AMANDA neutrino telescope, an array of more than six hundred photomultiplier tubes buried deep in the ice. At depths greater than 1300 m, both the scattering coefficient and absorptivity follow vertical variations in concentration of dust impurities, which are seen in ice cores from other Antarctic sites and which track climatological changes. The scattering coefficient varies by a factor of seven, and absorptivity (for wavelengths less than ∼450 nm) varies by a factor of three in the depth range between 1300 and 2300 m, where four dust peaks due to stadials in the late Pleistocene have been identified. In our absorption data, we also identify a broad peak due to the Last Glacial Maximum around 1300 m. In the scattering data, this peak is partially masked by scattering on residual air bubbles, whose contribution dominates the scattering coefficient in shallower ice but vanishes at ∼1350 m where all bubbles have converted to nonscattering air hydrates. The wavelength dependence of scattering by dust is described by a power law with exponent −0.90 ± 0.03, independent of depth. The wavelength dependence of absorptivity in the studied wavelength range is described by the sum of two components: a power law due to absorption by dust, with exponent −1.08 ± 0.01 and a normalization proportional to dust concentration that varies with depth; and a rising exponential due to intrinsic ice absorption which dominates at wavelengths greater than ∼500 nm.
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