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1.
Methods Mol Biol ; 2303: 319-327, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34626390

RESUMO

Glycosaminoglycans (GAGs) are important sulfated carbohydrates prevalently found in the extracellular matrix that serve many biological functions. The synthesis of structurally diverse but defined GAGs is extremely challenging as one has to account for the various sulfation patterns. Described is the automated synthesis of chondroitin sulfate hexasaccharides on a solid support equipped with a photolabile linker. The linker cleavage from the resin is performed in a continuous-flow photoreactor under chemically mild conditions. This approach serves as a general scheme to access oligosaccharides of all GAG families.

2.
Org Biomol Chem ; 2021 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-34734957

RESUMO

We report the automated glycan assembly (AGA) of different oligosaccharide fragments of the bacterial peptidoglycan (PGN) backbone. Iterative addition on a solid support of an acetyl glucosamine and a new muramic acid building block is followed by cleavage from the solid support and final deprotection providing 10 oligosaccharides up to six units.

3.
Front Chem ; 9: 766932, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34778215

RESUMO

Multivalent ligand-protein interactions are a commonly employed approach by nature in many biological processes. Single glycan-protein interactions are often weak, but their affinity and specificity can be drastically enhanced by engaging multiple binding sites. Microarray technology allows for quick, parallel screening of such interactions. Yet, current glycan microarray methodologies usually neglect defined multivalent presentation. Our laser-based array technology allows for a flexible, cost-efficient, and rapid in situ chemical synthesis of peptide scaffolds directly on functionalized glass slides. Using copper(I)-catalyzed azide-alkyne cycloaddition, different monomer sugar azides were attached to the scaffolds, resulting in spatially defined multivalent glycopeptides on the solid support. Studying their interaction with several different lectins showed that not only the spatially defined sugar presentation, but also the surface functionalization and wettability, as well as accessibility and flexibility, play an essential role in such interactions. Therefore, different commercially available functionalized glass slides were equipped with a polyethylene glycol (PEG) linker to demonstrate its effect on glycan-lectin interactions. Moreover, different monomer sugar azides with and without an additional PEG-spacer were attached to the peptide scaffold to increase flexibility and thereby improve binding affinity. A variety of fluorescently labeled lectins were probed, indicating that different lectin-glycan pairs require different surface functionalization and spacers for enhanced binding. This approach allows for rapid screening and evaluation of spacing-, density-, ligand and surface-dependent parameters, to find optimal lectin binders.

4.
J Am Chem Soc ; 143(45): 18977-18988, 2021 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-34748320

RESUMO

Dendritic cells (DC) are antigen-presenting cells coordinating the interplay of the innate and the adaptive immune response. The endocytic C-type lectin receptors DC-SIGN and Langerin display expression profiles restricted to distinct DC subtypes and have emerged as prime targets for next-generation immunotherapies and anti-infectives. Using heteromultivalent liposomes copresenting mannosides bearing aromatic aglycones with natural glycan ligands, we serendipitously discovered striking cooperativity effects for DC-SIGN+ but not for Langerin+ cell lines. Mechanistic investigations combining NMR spectroscopy with molecular docking and molecular dynamics simulations led to the identification of a secondary binding pocket for the glycomimetics. This pocket, located remotely of DC-SIGN's carbohydrate bindings site, can be leveraged by heteromultivalent avidity enhancement. We further present preliminary evidence that the aglycone allosterically activates glycan recognition and thereby contributes to DC-SIGN-specific cell targeting. Our findings have important implications for both translational and basic glycoscience, showcasing heteromultivalent targeting of DCs to improve specificity and supporting potential allosteric regulation of DC-SIGN and CLRs in general.

5.
Chembiochem ; 2021 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-34788491

RESUMO

Pseudomonas aeruginosa is an opportunistic ESKAPE pathogen that produces two lectins, LecA and LecB, as part of its large arsenal of virulence factors. Both carbohydrate-binding proteins are central to the initial and later persistent infection processes, i.e. bacterial adhesion and biofilm formation. The biofilm matrix is a major resistance determinant and protects the bacteria against external threats such as the host immune system or antibiotic treatment. Therefore, the development of drugs against the P. aeruginosa biofilm is of particular interest to restore efficacy of antimicrobials. Carbohydrate-based inhibitors for LecA and LecB were previously shown to efficiently reduce biofilm formations. Here, we report a new approach for inhibiting LecA with synthetic molecules bridging the established carbohydrate-binding site and a central cavity located between two LecA protomers of the lectin tetramer. Inspired by in silico design, we synthesized various galactosidic LecA inhibitors with aromatic moities targeting this central pocket. These compounds reached low micromolar affinities, validated in different biophysical assays. Finally, X-ray diffraction analysis revealed the interactions of this compound class with LecA. This new mode of action paves the way to a novel route towards inhibition of P. aeruginosa biofilms.

6.
Chemistry ; 2021 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-34665908

RESUMO

Acinetobacter baumannii is an opportunistic pathogen that causes serious nosocomial infections. One of the multidrug-resistant strains, AB5075, can result in bacteremia, pneumonia and wound infections associated with high morbidity and mortality. The structurally unique glycans on the surface of these bacteria are attractive targets for the development of glycoconjugate vaccines. Here, we report the first total synthesis of the densely functionalized trisaccharide repeating unit of A. baumannii AB5075 as well as two analogues. The construction of 1,2-cis linkages between the rare sugars relies on a double-serial inversion strategy. The judicious selection of building blocks and reaction conditions allowed for stereoselective glycosylations, the installation of acetamido groups and the (S)-3-hydroxybutanoyl chain.

7.
ACS Chem Biol ; 16(11): 2297-2306, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34618440

RESUMO

The attachment of proteins to the cell membrane using a glycosylphosphatidylinositol (GPI) anchor is a ubiquitous process in eukaryotic cells. Deficiencies in the biosynthesis of GPIs and the concomitant production of GPI-anchored proteins lead to a series of rare and complicated disorders associated with inherited GPI deficiencies (IGDs) in humans. Currently, there is no treatment for patients suffering from IGDs. Here, we report the design, synthesis, and use of GPI fragments to rescue the biosynthesis of GPI-anchored proteins (GPI-APs) caused by mutation in genes involved in the assembly of GPI-glycolipids in cells. We demonstrated that the synthetic fragments GlcNAc-PI (1), Man-GlcN-PI (5), and GlcN-PI with two (3) and three lipid chains (4) rescue the deletion of the GPI biosynthesis in cells devoid of the PIGA, PIGL, and PIGW genes in vitro. The compounds allowed for concentration-dependent recovery of GPI biosynthesis and were highly active on the cytoplasmic face of the endoplasmic reticulum membrane. These synthetic molecules are leads for the development of treatments for IGDs and tools to study GPI-AP biosynthesis.

8.
Artigo em Inglês | MEDLINE | ID: mdl-34713573

RESUMO

Carbohydrate-binding proteins (lectins) are auspicious targets in drug discovery to combat antimicrobial resistance; however, their non-carbohydrate drug-like inhibitors are still unavailable. Here, we present a druggable pocket in a ß-propeller lectin BambL from Burkholderia ambifaria as a potential target for allosteric inhibitors. This site was identified employing 19 F NMR fragment screening and a computational pocket prediction algorithm SiteMap. The structure-activity relationship study revealed the most promising fragment with a dissociation constant of 0.3±0.1 mM and a ligand efficiency of 0.3 kcal mol-1 HA-1 that affected the orthosteric site. This effect was substantiated by site-directed mutagenesis in the orthosteric and secondary pockets. Future drug-discovery campaigns that aim to develop small molecule inhibitors can benefit from allosteric sites in lectins as a new therapeutic approach against antibiotic-resistant pathogens.

9.
Chemistry ; 27(65): 16098-16102, 2021 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-34634174

RESUMO

Fluorescence signals have been widely used in information encryption for a few decades, but still suffer from limited reliability. Here, reversible multichannel fluorescent devices with encrypted information were constructed, based on two fluorescent positional isomers of a diphenylquinoxaline derivative. Possessing the same core fluorescent group and acid-/pH-responsive mechanism, the two isomers showed different fluorescence colors in an acidic environment; this allowed us to realize stepwise encryption of information in orthogonal fluorescence channels. Because the protonation was reversible, the revealed information could be re-encrypted simply by heating. This approach highlights the value of positional isomers to build multichannel encryption devices, improving their reliability on the molecular level.

10.
Virol J ; 18(1): 182, 2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34496903

RESUMO

BACKGROUND: Traditional medicines based on herbal extracts have been proposed as affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Teas and drinks containing extracts of Artemisia annua and Artemisia afra have been widely used in Africa in efforts to prevent SARS-CoV-2 infection and fight COVID-19. METHODS: The plant extracts and Covid-Organics drink produced in Madagascar were tested for plaque reduction using both feline coronavirus and SARS-CoV-2 in vitro. Their cytotoxicities were also investigated. RESULTS: Several extracts as well as Covid-Organics inhibited SARS-CoV-2 and FCoV infection at concentrations that did not affect cell viability. CONCLUSIONS: Some plant extracts show inhibitory activity against FCoV and SARS-CoV-2. However, it remains unclear whether peak plasma concentrations in humans can reach levels needed to inhibit viral infection following consumption of teas or Covid-Organics. Clinical studies are required to evaluate the utility of these drinks for COVID-19 prevention or treatment of patients.


Assuntos
Antivirais/farmacologia , Artemisia/química , Extratos Vegetais/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Coronavirus Felino/efeitos dos fármacos , Coronavirus Felino/crescimento & desenvolvimento , Extratos Vegetais/química , SARS-CoV-2/crescimento & desenvolvimento , Ensaio de Placa Viral
11.
Nanoscale ; 13(35): 15067-15073, 2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34533554

RESUMO

The most efficient approach for cancer identification and monitoring is the detection of cancer-associated protein biomarkers but an accurate diagnosis requires multiple analyses. Glycosylation profiling can provide important biological information since different glycoforms are involved in malignant transformation. Here, a near-infrared (NIR) light activated fluorescence resonance energy transfer (FRET) strategy for the efficient and reliable simultaneous dual imaging of the mucin 1 (MUC1) protein backbone and MUC1-specific sialic acid (Sia) is reported. MUC1, an important tumor biomarker, is overexpressed and under-glycosylated in most tumor cells. Two aptamer-functionalized nanoprobes, Cy5-labeled Sia aptamer-functionalized gold nanostars (Sia-GNSs) and MUC1 aptamer-functionalized quantum dots (MUC1-QDs), were successfully constructed with high specificity and biocompatibility. Upon excitation with NIR light, Sia-GNSs endothermically released the Cy5-labeled Sia aptamer that specifically binds to Sia. The Cy5 fluorescence can be observed due to the FRET effect when the Cy5-labeled Sia aptamer and MUC1-QDs bind to the same MUC1 molecule. Dual imaging and relative quantification of MUC1 and its sialylation were achieved in vitro, in vivo and in clinical tissue samples. This efficient platform allows for the simultaneous detection of protein biomarkers and their glycosylation pattern, with significant potential for clinical cancer diagnostics.


Assuntos
Aptâmeros de Nucleotídeos , Pontos Quânticos , Transferência Ressonante de Energia de Fluorescência , Mucina-1 , Imagem Óptica
12.
Biochem Soc Trans ; 49(5): 2411-2429, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34495299

RESUMO

The importance of vaccine-induced protection was repeatedly demonstrated over the last three decades and emphasized during the recent COVID-19 pandemic as the safest and most effective way of preventing infectious diseases. Vaccines have controlled, and in some cases, eradicated global viral and bacterial infections with high efficiency and at a relatively low cost. Carbohydrates form the capsular sugar coat that surrounds the outer surface of human pathogenic bacteria. Specific surface-exposed bacterial carbohydrates serve as potent vaccine targets that broadened our toolbox against bacterial infections. Since first approved for commercial use, antibacterial carbohydrate-based vaccines mostly rely on inherently complex and heterogenous naturally derived polysaccharides, challenging to obtain in a pure, safe, and cost-effective manner. The introduction of synthetic fragments identical with bacterial capsular polysaccharides provided well-defined and homogenous structures that resolved many challenges of purified polysaccharides. The success of semisynthetic glycoconjugate vaccines against bacterial infections, now in different phases of clinical trials, opened up new possibilities and encouraged further development towards fully synthetic antibacterial vaccine solutions. In this mini-review, we describe the recent achievements in semi- and fully synthetic carbohydrate vaccines against a range of human pathogenic bacteria, focusing on preclinical and clinical studies.


Assuntos
Antibacterianos/imunologia , Bactérias/imunologia , Infecções Bacterianas/imunologia , Carboidratos/imunologia , Glicoconjugados/imunologia , Vacinas Sintéticas/imunologia , Antibacterianos/química , Antibacterianos/uso terapêutico , Bactérias/efeitos dos fármacos , Infecções Bacterianas/microbiologia , Infecções Bacterianas/prevenção & controle , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/uso terapêutico , Sequência de Carboidratos , Carboidratos/química , Glicoconjugados/química , Glicoconjugados/uso terapêutico , Humanos , Vacinas Sintéticas/química , Vacinas Sintéticas/uso terapêutico
13.
J Virol ; 95(23): e0124921, 2021 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-34549984

RESUMO

Recombinant adeno-associated virus (rAAV) vectors are one of the leading tools for the delivery of therapeutic genes in human gene therapy applications. For a successful transfer of their payload, the AAV vectors have to circumvent potential preexisting neutralizing host antibodies and bind to the receptors of the target cells. Both of these aspects have not been structurally analyzed for AAVrh.10. Here, cryo-electron microscopy and three-dimensional image reconstruction were used to map the binding site of sulfated N-acetyllactosamine (LacNAc; previously shown to bind AAVrh.10) and a series of four monoclonal antibodies (MAbs). LacNAc was found to bind to a pocket located on the side of the 3-fold capsid protrusion that is mostly conserved to AAV9 and equivalent to its galactose-binding site. As a result, AAVrh.10 was also shown to be able to bind to cell surface glycans with terminal galactose. For the antigenic characterization, it was observed that several anti-AAV8 MAbs cross-react with AAVrh.10. The binding sites of these antibodies were mapped to the 3-fold capsid protrusions. Based on these observations, the AAVrh.10 capsid surface was engineered to create variant capsids that escape these antibodies while maintaining infectivity. IMPORTANCE Gene therapy vectors based on adeno-associated virus rhesus isolate 10 (AAVrh.10) have been used in several clinical trials to treat monogenetic diseases. However, compared to other AAV serotypes little is known about receptor binding and antigenicity of the AAVrh.10 capsid. Particularly, preexisting neutralizing antibodies against capsids are an important challenge that can hamper treatment efficiency. This study addresses both topics and identifies critical regions of the AAVrh.10 capsid for receptor and antibody binding. The insights gained were utilized to generate AAVrh.10 variants capable of evading known neutralizing antibodies. The findings of this study could further aid the utilization of AAVrh.10 vectors in clinical trials and help the approval of the subsequent biologics.

14.
Int J Mol Sci ; 22(16)2021 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-34445445

RESUMO

Ascariasis is a global health problem for humans and animals. Adult Ascaris nematodes are long-lived in the host intestine where they interact with host cells as well as members of the microbiota resulting in chronic infections. Nematode interactions with host cells and the microbial environment are prominently mediated by parasite-secreted proteins and peptides possessing immunomodulatory and antimicrobial activities. Previously, we discovered the C-type lectin protein AsCTL-42 in the secreted products of adult Ascaris worms. Here we tested recombinant AsCTL-42 for its ability to interact with bacterial and host cells. We found that AsCTL-42 lacks bactericidal activity but neutralized bacterial cells without killing them. Treatment of bacterial cells with AsCTL-42 reduced invasion of intestinal epithelial cells by Salmonella. Furthermore, AsCTL-42 interacted with host myeloid C-type lectin receptors. Thus, AsCTL-42 is a parasite protein involved in the triad relationship between Ascaris, host cells, and the microbiota.


Assuntos
Ascaris suum/metabolismo , Interações Hospedeiro-Parasita , Mucosa Intestinal/metabolismo , Lectinas Tipo C/metabolismo , Lectinas/metabolismo , Salmonella , Animais , Ascaríase/metabolismo , Ascaríase/microbiologia , Ascaris suum/microbiologia , Ascaris suum/fisiologia , Linhagem Celular , Lectinas/fisiologia , Proteínas Recombinantes , Sus scrofa/microbiologia , Sus scrofa/parasitologia
15.
Cell Chem Biol ; 2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34358442

RESUMO

Protein S-palmitoylation is a post-translational modification that plays a crucial role in cancer cells by regulating the function and localization of oncoproteins and tumor suppressor proteins. Here, we identify artemisinin (ART), a clinically approved antimalarial endoperoxide natural product with promising anticancer activities, as an inhibitor of the ER-residing palmitoyl transferase ZDHHC6 in cancer cells using a chemoproteomic approach. We show that ART covalently binds and inhibits ZDHHC6 to reduce palmitoylation of the oncogenic protein NRas, disrupt NRas subcellular localization, and attenuate the downstream pro-proliferative signaling cascades. Our study identifies artemisinin as a non-lipid-based palmitoylation inhibitor targeting a specific palmitoyl acyltransferase and provides valuable mechanistic insights into the anticancer activity of artemisinins that are currently being studied in human clinical trials for different cancers.

16.
J Med Chem ; 64(17): 12774-12789, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34432457

RESUMO

The development of multivalent sialic acid-based inhibitors active against a variety of influenza A virus (IAV) strains has been hampered by high genetic and structural variability of the targeted viral hemagglutinin (HA). Here, we addressed this challenge by employing sialylated polyglycerols (PGs). Efficacy of prototypic PGs was restricted to a narrow spectrum of IAV strains. To understand this restriction, we selected IAV mutants resistant to a prototypic multivalent sialylated PG by serial passaging. Resistance mutations mapped to the receptor binding site of HA, which was accompanied by altered receptor binding profiles of mutant viruses as detected by glycan array analysis. Specifying the inhibitor functionalization to 2,6-α-sialyllactose (SL) and adjusting the linker yielded a rationally designed inhibitor covering an extended spectrum of inhibited IAV strains. These results highlight the importance of integrating virological data with chemical synthesis and structural data for the development of sialylated PGs toward broad anti-influenza compounds.

17.
Front Microbiol ; 12: 712538, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335547

RESUMO

Plasmodium parasites cause malaria disease, one of the leading global health burdens for humanity, infecting hundreds of millions of people each year. Different glycans on the parasite and the host cell surface play significant roles in both malaria pathogenesis and host defense mechanisms. So far, only small, truncated N- and O-glycans have been identified in Plasmodium species. In contrast, complex glycosylphosphatidylinositol (GPI) glycolipids are highly abundant on the parasite's cell membrane and are essential for its survival. Moreover, the parasites express lectins that bind and exploit the host cell surface glycans for different aspects of the parasite life cycle, such as adherence, invasion, and evasion of the host immune system. In parallel, the host cell glycocalyx and lectin expression serve as the first line of defense against Plasmodium parasites and directly dictate susceptibility to Plasmodium infection. This review provides an overview of the glycobiology involved in Plasmodium-host interactions and its contribution to malaria pathogenesis. Recent findings are presented and evaluated in the context of potential therapeutic exploitation.

18.
Chemistry ; 27(52): 13139-13143, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34251709

RESUMO

The molecular level description of carbohydrate assemblies is hampered by their structural complexity and the lack of suitable analytical methods. Here, we employed systematic chemical modifications to identify key non-covalent interactions that triggered the supramolecular assembly of a disaccharide model. While some modifications disrupted the supramolecular organization, others were tolerated, delivering important information on the aggregation process. The screening identified new geometries, including nanotubes, and twisted ribbons that were characterized with electron tomography and electron diffraction (ED) methods. This work demonstrates that the combination of synthetic chemistry and ED methods is a powerful tool to draw correlations between the molecular structure and the nanoscale architecture of carbohydrate assemblies.


Assuntos
Carboidratos , Nanotubos , Estrutura Molecular
19.
Org Process Res Dev ; 25(7): 1573-1578, 2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34305386

RESUMO

Palladium-catalyzed hydrogenolysis is often the final step in challenging natural product total syntheses and a key step in industrial processes producing fine chemicals. Here, we demonstrate that there is wide variability in the efficiency of commercial sources of palladium on carbon (Pd/C) resulting in significant differences in selectivity, reaction times, and yields. We identified the physicochemical properties of efficient catalysts for hydrogenolysis: (1) small Pd/PdO particle size (2) homogeneous distribution of Pd/PdO on the carbon support, and (3) palladium oxidation state are good predictors of catalytic efficiency. Now chemists can identify and predict a catalyst's efficiency prior to the use of valuable synthetic material and time.

20.
Sci Rep ; 11(1): 14571, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272426

RESUMO

Effective and affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are needed. We report in vitro efficacy of Artemisia annua extracts as well as artemisinin, artesunate, and artemether against SARS-CoV-2. The latter two are approved active pharmaceutical ingredients of anti-malarial drugs. Concentration-response antiviral treatment assays, based on immunostaining of SARS-CoV-2 spike glycoprotein, revealed that treatment with all studied extracts and compounds inhibited SARS-CoV-2 infection of VeroE6 cells, human hepatoma Huh7.5 cells and human lung cancer A549-hACE2 cells, without obvious influence of the cell type on antiviral efficacy. In treatment assays, artesunate proved most potent (range of 50% effective concentrations (EC50) in different cell types: 7-12 µg/mL), followed by artemether (53-98 µg/mL), A. annua extracts (83-260 µg/mL) and artemisinin (151 to at least 208 µg/mL). The selectivity indices (SI), calculated based on treatment and cell viability assays, were mostly below 10 (range 2 to 54), suggesting a small therapeutic window. Time-of-addition experiments in A549-hACE2 cells revealed that artesunate targeted SARS-CoV-2 at the post-entry level. Peak plasma concentrations of artesunate exceeding EC50 values can be achieved. Clinical studies are required to further evaluate the utility of these compounds as COVID-19 treatment.


Assuntos
Artemisininas/farmacologia , Extratos Vegetais/farmacologia , SARS-CoV-2/efeitos dos fármacos , Células A549 , Animais , Artemisia annua/química , COVID-19/tratamento farmacológico , Chlorocebus aethiops , Humanos , Células Vero
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