Display Selection of a Hybrid Foldamer-Peptide Macrocycle.

Expanding the chemical diversity of peptide macrocycle libraries for display selection is desirable to improve their potential to bind biomolecular targets. We now have implemented a considerable expansion through a large aromatic helical foldamer inclusion. A foldamer was first identified that undergoes flexizyme-mediated tRNA acylation and that is capable of initiating ribosomal translation with yields sufficiently high to perform an mRNA display selection of macrocyclic foldamer-peptide hybrids. A hybrid macrocyclic nanomolar binder to the C-lobe of the E6AP HECT domain was selected that showed a highly converged peptide sequence. A crystal structure and molecular dynamics simulations revealed that both the peptide and foldamer are helical in an intriguing reciprocal stapling fashion. The strong residue convergence could be rationalized based on their involvement in specific interactions with the target protein. The foldamer stabilizes the peptide helix through stapling and through contacts with key residues. These results altogether represent a significant extension of the chemical space amenable to display selection and highlight possible benefits of inserting an aromatic foldamer into a peptide macrocycle for the purpose of protein recognition.

Biotechnologically produced fucosylated oligosaccharides inhibit the binding of human noroviruses to their natural receptors.

Norovirus infections cause severe gastroenteritis in millions of people every year. Infection requires the recognition of histo-blood group antigens (HBGAs), but such interactions can be inhibited by human milk oligosaccharides (HMOs), which act as structurally-similar decoys. HMO supplements could help to prevent norovirus infections, but the industrial production of complex HMOs is challenging. Here we describe a large-scale fermentation process that yields several kilograms of lacto-N-fucopentaose I (LNFP I). The product was synthesized in Escherichia coli BL21(DE3) cells expressing a recombinant N-acetylglucosaminyltransferase, ß(1,3)galactosyltransferase and α(1,2)fucosyltransferase. Subsequent in vitro enzymatic conversion produced HBGA types A1 and B1 for norovirus inhibition assays. These carbohydrates inhibited the binding of GII.17 virus-like particles (VLPs) to type A1 and B1 trisaccharides more efficiently than simpler fucosylated HMOs, which were in turn more effective than any non-fucosylated structures. However, we found that the simpler fucosylated HMOs were more effective than complex molecules such as LNFP I when inhibiting the binding of GII.17 and GII.4 VLPs to human gastric mucins and mucins from human amniotic fluid. Our results show that complex fucosylated HMOs can be produced by large-scale fermentation and that a combination of simple and complex fucosylated structures is more likely to prevent norovirus infections.

Human Norovirus Neutralized by a Monoclonal Antibody Targeting the Histo-Blood Group Antigen Pocket.

Temporal changes in the GII.4 human norovirus capsid sequences occasionally result in the emergence of genetic variants capable of causing new epidemics. The persistence of GII.4 is believed to be associated with the recognition of numerous histo-blood group antigen (HBGA) types and antigenic drift. We found that one of the earliest known GII.4 isolates (in 1974) and a more recent epidemic GII.4 variant (in 2012) had varied norovirus-specific monoclonal antibody (MAb) reactivities but similar HBGA binding profiles. To better understand the binding interaction of one MAb (10E9) that had varied reactivity with these GII.4 variants, we determined the X-ray crystal structure of the NSW-2012 GII.4 P domain 10E9 Fab complex. We showed that the 10E9 Fab interacted with conserved and variable residues, which could be associated with antigenic drift. Interestingly, the 10E9 Fab binding pocket partially overlapped the HBGA pocket and had direct competition for conserved HBGA binding residues (i.e., Arg345 and Tyr444). Indeed, the 10E9 MAb blocked norovirus virus-like particles (VLPs) from binding to several sources of HBGAs. Moreover, the 10E9 antibody completely abolished virus replication in the human norovirus intestinal enteroid cell culture system. Our new findings provide the first direct evidence that competition for GII.4 HBGA binding residues and steric obstruction could lead to norovirus neutralization. On the other hand, the 10E9 MAb recognized residues flanking the HBGA pocket, which are often substituted as the virus evolves. This mechanism of antigenic drift likely influences herd immunity and impedes the possibility of acquiring broadly reactive HBGA-blocking antibodies.IMPORTANCE The emergence of new epidemic GII.4 norovirus variants is thought to be associated with changes in antigenicity and HBGA binding capacity. Here, we show that HBGA binding profiles remain unchanged between the 1974 and 2012 GII.4 variants, whereas these variants showed various levels of reactivity against a panel of GII.4 MAbs. We identified a MAb that bound at the HBGA pocket, blocked norovirus VLPs from binding to HBGAs, and neutralized norovirus virions in the cell culture system. Raised against a GII.4 2006 strain, this MAb was unreactive to a GII.4 1974 isolate but was able to neutralize the newer 2012 strain, which has important implications for vaccine design. Altogether, these new findings suggest that the amino acid variations surrounding the HBGA pocket lead to temporal changes in antigenicity without affecting the ability of GII.4 variants to bind HBGAs, which are known cofactors for infection.

Pandemic GII.4 Sydney and Epidemic GII.17 Kawasaki308 Noroviruses Display Distinct Specificities for Histo-Blood Group Antigens Leading to Different Transmission Vector Dynamics in Pacific Oysters.

Noroviruses are the major cause of foodborne outbreaks of acute gastroenteritis, which are often linked to raw oyster consumption. Previous studies have suggested histo-blood group antigens (HBGA)-like structures in the oyster tissues as ligands for norovirus binding and persistence. To better understand how oysters function as vectors for the most common human noroviruses, we first tested the ability of the norovirus strains GI.1 West Chester, the pandemic GII.4 Sydney, and the epidemic GII.17 Kawasaki308 strains to interact with oyster tissues. Secondly, we explored how the HBGA preferences of these strains can affect their persistence in oyster tissues. We found limited HBGA expression in oyster tissues. HBGAs of A and H type 1 were present in the digestive tissues and palps of the Pacific oyster Crassostrea gigas, while the gills and mantle lacked any HBGA structures. By using Virus-like particles (VLPs), which are antigenically and morphologically similar to native virions, we were able to demonstrate that VLPs of GI.1 West Chester norovirus reacted with the digestive tissues and palps. Despite of the lack of HBGA expression in mantle, dominant GII.4 Sydney strain readily bound to all the oyster tissues, including the digestive tissues, gills, palps, and mantle. In contrast, no binding of the epidemic GII.17 Kawasaki308 VLPs to any of the investigated oyster tissues was observed. In synthetic HBGA and saliva-binding assays, GI.1 reacted with A type, H type, and Leb (Lewis b) HBGAs. GII.4 Sydney VLPs showed a broad binding pattern and interacted with various HBGA types. Compared to GI.1 and GII.4 VLPs, the GII.17 Kawasaki308 VLPs only weakly associated with long-chain saccharides containing A type, B type, H type, and Leb blood group epitopes. Our findings indicate that GI.1 and GII.4 noroviruses are likely to be concentrated in oysters, by binding to HBGA-like glycans, and therefore potentially leading to increased long term transmission. In regards to the GII.17 Kawasaki308 strain, we suggest that oysters can only function as short term transmission vector in periods of high environmental virus concentrations.

Avidity of α-fucose on human milk oligosaccharides and blood group-unrelated oligo/polyfucoses is essential for potent norovirus-binding targets.

There is agreement with respect to norovirus infection routes in humans regarding binding of the pathogen to gastrointestinal epithelia via recognition of blood group-active mucin-typeO-glycans as the initiating and essential event. Among food additives playing a potential role in applications to protect newborns, human milk oligosaccharides (HMOs) as competitors are of major importance. By focusing on fractions of high-molecular mass HMOs with high fucose contents, we attempted to identify the structural elements required for norovirus GII.4 (Sydney 2012, JX459908) capsid binding in neoglycolipid-based arrays. We provide evidence that HMO fractions with the strongest binding capacities contained hepta- to decasaccharides expressing branches with terminal blood group H1 or Lewis-b antigen. H2 antigen, as recognized by UEA-I lectin, is apparently not expressed in high-mass HMOs. Beyond affinity, sterical and valency effects contribute more to virus-like particle binding, as revealed for oligovalent fucose conjugates of α-cyclodextrin and oligofucoses from fucoidan. Accordingly, high-mass HMOs with oligovalent fucose can exhibit stronger binding capacities compared with monovalent fucose HMOs. The above features were revealed for the most clinically relevant and prevalent GII.4 strain and are distinct from other strains, like GII.10 (Vietnam 026, AF504671), which showed a preference for blood group Lewis-a positive glycans.

The Double Face of Mucin-Type O-Glycans in Lectin-Mediated Infection and Immunity.

Epithelial human blood group antigens (HBGAs) on O-glycans play roles in pathogen binding and the initiation of infection, while similar structures on secretory mucins exert protective functions. These double-faced features of O-glycans in infection and innate immunity are reviewed based on two instructive examples of bacterial and viral pathogens. Helicobacter pylori represents a class 1 carcinogen in the human stomach. By expressing blood group antigen-binding adhesin (BabA) and LabA adhesins that bind to Lewis-b and LacdiNAc, respectively, H. pylori colocalizes with the mucin MUC5AC in gastric surface epithelia, but not with MUC6, which is cosecreted with trefoil factor family 2 (TFF2) by deep gastric glands. Both components of the glandular secretome are concertedly up-regulated upon infection. While MUC6 expresses GlcNAc-capped glycans as natural antibiotics for H. pylori growth control, TFF2 may function as a probiotic lectin. In viral infection human noroviruses of the GII genogroup interact with HBGAs via their major capsid protein, VP1. HBGAs on human milk oligosaccharides (HMOs) may exert protective functions by binding to the P2 domain pocket on the capsid. We discuss structural details of the P2 carbohydrate-binding pocket in interaction with blood group H/Lewis-b HMOs and fucoidan-derived oligofucoses as effective interactors for the most prevalent norovirus strains, GII.4 and GII.17.

Strain-dependent effects of clinical echovirus 30 outbreak isolates at the blood-CSF barrier.

BACKGROUND: Echovirus (E) 30 (E-30) meningitis is characterized by neuroinflammation involving immune cell pleocytosis at the protective barriers of the central nervous system (CNS). In this context, infection of the blood-cerebrospinal fluid barrier (BCSFB), which has been demonstrated to be involved in enteroviral CNS pathogenesis, may affect the tight junction (TJ) and adherens junction (AJ) function and morphology. METHODS: We used an in vitro human choroid plexus epithelial (HIBCPP) cell model to investigate the effect of three clinical outbreak strains (13-311, 13-759, and 14-397) isolated in Germany in 2013, and compared them to E-30 Bastianni. Conducting transepithelial electrical resistance (TEER), paracellular dextran flux measurement, quantitative real-time polymerase chain reaction (qPCR), western blot, and immunofluorescence analysis, we investigated TJ and AJ function and morphology as well as strain-specific E-30 infection patterns. Additionally, transmission electron and focused ion beam microscopy electron microscopy (FIB-SEM) was used to evaluate the mode of leukocyte transmigration. Genome sequencing and phylogenetic analyses were performed to discriminate potential genetic differences among the outbreak strains. RESULTS: We observed a significant strain-dependent decrease in TEER with strains E-30 Bastianni and 13-311, whereas paracellular dextran flux was only affected by E-30 Bastianni. Despite strong similarities among the outbreak strains in replication characteristics and particle distribution, strain 13-311 was the only outbreak isolate revealing comparable disruptive effects on TJ (Zonula Occludens (ZO) 1 and occludin) and AJ (E-cadherin) morphology to E-30 Bastianni. Notwithstanding significant junctional alterations upon E-30 infection, we observed both para- and transcellular leukocyte migration across HIBCPP cells. Complete genome sequencing revealed differences between the strains analyzed, but no explicit correlation with the observed strain-dependent effects on HIBCPP cells was possible. CONCLUSION: The findings revealed distinct E-30 strain-specific effects on barrier integrity and junctional morphology. Despite E-30-induced barrier alterations leukocyte trafficking did not exclusively occur via the paracellular route.

Human Milk Oligosaccharides as Promising Antivirals.

Human milk oligosaccharides (HMOs) are diverse unconjugated carbohydrates that are highly abundant in human breast milk. These glycans are investigated in the context of exhibiting multiple functions in infant growth and development. They seem to provide protection against infectious diseases, including a number of poorly manageable viral infections. Although the potential mechanism of the HMO antiviral protection is rather broad, much of the current experimental work has focused on studying of HMO antiadhesive properties. HMOs may mimic structures of viral receptors and block adherence to target cells, thus preventing infection. Still, the potential of HMOs as a source for new antiviral drugs is relatively unexploited. This can be partly attributed to the extreme complexity of the virus-carbohydrate interactions and technical difficulties in HMO isolation, characterization, and manufacturing procedures. Fortunately, we are currently entering a period of major technological advances that have enabled deeper insights into carbohydrate mediated viral entry, rational selection of HMOs as anti-entry inhibitors, and even evaluation of individual synthetic HMO structures. Here, we provide an up-to-date review on glycan binding studies for rotaviruses, noroviruses, influenza viruses, and human immunodeficiency viruses. We also discuss the preventive and therapeutic potential of HMOs as anti-entry inhibitors and address challenges on the route from fundamental studies to clinical trials.

Human norovirus inhibition by a human milk oligosaccharide.

Human noroviruses are the leading cause of outbreaks of acute gastroenteritis. Norovirus interactions with histo-blood group antigens (HBGAs) are known to be important for an infection. In this study, we identified the HBGA binding pocket for an emerging GII genotype 17 (GII.17) variant using X-ray crystallography. The GII.17 variant bound the HBGA with an equivalent set of residues as the leading pandemic GII.4 variants. These structural data highlights the conserved nature of HBGA binding site between prevalent GII noroviruses. Noroviruses also interact with human milk oligosaccharides (HMOs), which mimic HBGAs and may function as receptor decoys. We previously showed that HMOs inhibited the binding of rarely detected GII.10 norovirus to HBGAs. We now found that an HMO, 2'-fucosyllactose (2'FL), additionally blocked both the GI.1 and GII.17 noroviruses from binding to HBGAs. Together, these findings provide evidence that 2'FL might function as a broadly reactive antiviral against multiple norovirus genogroups.

Neuroprotective activity of creatylglycine ethyl ester fumarate.

BACKGROUND: We have recently shown neuroprotective activity of the creatine amides in the focal cerebral ischemia in rats on the 280 mg/kg administration. In the present study, neuroprotective properties of creatylglycine ethyl ester fumarate (CrGEt) in rats with focal cerebral ischemia were explored in a wide dosage range (30-280 mg/kg, intravenous and intragastric). METHODS: Focal cerebral ischemia was induced by the middle cerebral artery occlusion (MCAO). RESULTS: The CrGEt administration 30 minutes before and at the last 5 minutes of MCAO dose dependently attenuated cerebral ischemic damage on 35%-65%, reduced neurobehavioral deficits, led to high neuronal survival in ischemic rat brains. The neuroprotective activity of CrGEt was mediated by its following abilities: (1) normalize the energy metabolism in the ischemic brains, maintaining adenosine triphosphate levels, and reducing lactate concentration; (2) inhibit the ischemia-reperfusion-related oxidative stress as evidenced by the increased activity of superoxide dismutase and the reduced levels of malondialdehyde. CrGEt served as a substrate for creatine kinase and a partial agonist of N-methyl-D-aspartate receptors; this partly explains mechanism of its neuroprotective action. CONCLUSIONS: In view of the previously mentioned results, CrGEt holds a promise as a compound for treatment of ischemic brain disorders.

Controlled formation of emissive silver nanoclusters using rationally designed metal-binding proteins.

The metal-binding properties of rationally designed, synthetic proteins were used to prepare a series of emissive silver nanoclusters having predictable sizes and emission energies. Metal-binding α-helical coiled coils were designed to exist as peptide trimers, tetramers, and hexamers and found to uniquely bind 6, 8, and 12 Ag(+) ions, respectively. Subsequent treatment with a chemical reducing agent produced a series of peptide-bound Ag(0) nanoclusters that display a strong visible fluorescence whose emission energies depend on the number of bound metal ions in excellent agreement with theory.

Metal-binding properties and structural characterization of a self-assembled coiled coil: formation of a polynuclear Cd-thiolate cluster.

This paper describes the design, characterization, and metal-binding properties of a 32-residue polypeptide called AQ-C16C19. The sequence of this peptide is composed of four repeats of the seven residue sequence Ile-Ala-Ala-Leu-Glu-Gln-Lys but with a Cys-X-X-Cys metal-binding motif substituted at positions 16-19. Size exclusion chromatography with multiangle light scattering detection (SEC-MALS) and circular dichroism (CD) spectroscopy studies showed that the apo peptide exhibits a pH-dependent oligomerization state in which a three-stranded α-helical coiled coil is dominant between pH5.4 and 8.5. The Cd(2+)-binding properties of the AQ-C16C19 peptide were studied by ultraviolet-visible spectroscopy (UV-vis), electrospray ionization mass spectrometry (ESI MS), and (113)Cd NMR techniques. The holoprotein was found to contain a polynuclear cadmium-thiolate center formed within the hydrophobic core of the triple-stranded α-helical coiled-coil structure. The X-ray crystal structure of the Cd-loaded peptide, resolved at 1.85Å resolution, revealed an adamantane-like configuration of the polynuclear metal center consisting of four cadmium ions, six thiolate sulfur ligands from cysteine residues and four oxygen-donor ligands. Three of these are from glutamic acid residues and one is from an exogenous water molecule. Thus, each cadmium ion is coordinated in a distorted tetrahedral S(3)O geometry. The metal cluster was found to form cooperatively at pH5.4 but in a stepwise fashion at pH>7. The results demonstrate that synthetic coiled-coils can be designed to incorporate multinuclear metal clusters, a proof-of-concept for their potential use in developing synthetic metalloenzymes and multi-electron redox agents.