RESUMO
Blockade of vascular endothelial growth factor (VEGF) signaling with bevacizumab, a humanized anti-VEGF monoclonal antibody (mAb), or with receptor tyrosine kinase inhibitors, has improved progression-free survival and, in some indications, overall survival across several types of cancers by interrupting tumor angiogenesis. However, the clinical benefit conferred by these therapies is variable, and tumors from treated patients eventually reinitiate growth. Previously we demonstrated, in mouse tumor models, that galectin-1 (Gal1), an endogenous glycan-binding protein, preserves angiogenesis in anti-VEGF-resistant tumors by co-opting the VEGF receptor (VEGFR)2 signaling pathway in the absence of VEGF. However, the relevance of these findings in clinical settings is uncertain. Here, we explored, in a cohort of melanoma patients from AVAST-M, a multicenter, open-label, randomized controlled phase 3 trial of adjuvant bevacizumab versus standard surveillance, the role of circulating plasma Gal1 as part of a compensatory mechanism that orchestrates endothelial cell programs in bevacizumab-treated melanoma patients. We found that increasing Gal1 levels over time in patients in the bevacizumab arm, but not in the observation arm, significantly increased their risks of recurrence and death. Remarkably, plasma Gal1 was functionally active as it was able to reprogram endothelial cell biology, promoting migration, tubulogenesis, and VEGFR2 phosphorylation. These effects were prevented by blockade of Gal1 using a newly developed fully human anti-Gal1 neutralizing mAb. Thus, using samples from a large-scale clinical trial from stage II and III melanoma patients, we validated the clinical relevance of Gal1 as a potential mechanism of resistance to bevacizumab treatment.
Assuntos
Melanoma , Fator A de Crescimento do Endotélio Vascular , Animais , Camundongos , Humanos , Bevacizumab/farmacologia , Bevacizumab/uso terapêutico , Galectina 1 , Melanoma/tratamento farmacológico , Melanoma/patologia , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Células Endoteliais/patologia , Fatores de Crescimento do Endotélio Vascular , Biologia , Inibidores da Angiogênese/farmacologiaRESUMO
The dimeric architecture of tandem-repeat type galectins, such as galectin-4 (Gal-4), modulates their biological activities, although the underlying molecular mechanisms have remained elusive. Emerging evidence show that tandem-repeat galectins play an important role in innate immunity by recognizing carbohydrate antigens present on the surface of certain pathogens, which very often mimic the structures of the human self-glycan antigens. Herein, we have analyzed the binding preferences of the C-domain of Gal-4 (Gal-4C) toward the ABH-carbohydrate histo-blood antigens with different core presentations and their recognition features have been rationalized by using a combined experimental approach including NMR, solid-phase and hemagglutination assays, and molecular modeling. The data show that Gal-4C prefers A over B antigens (two-fold in affinity), contrary to the N-domain (Gal-4N), although both domains share the same preference for the type-6 presentations. The behavior of the full-length Gal-4 (Gal-4FL) tandem-repeat form has been additionally scrutinized. Isothermal titration calorimetry and NMR data demonstrate that both domains within full-length Gal-4 bind to the histo-blood antigens independently of each other, with no communication between them. In this context, the heterodimeric architecture does not play any major role, apart from the complementary A and B antigen binding preferences. However, upon binding to a bacterial lipopolysaccharide containing a multivalent version of an H-antigen mimetic as O-antigen, the significance of the galectin architecture was revealed. Indeed, our data point to the linker peptide domain and the F-face of the C-domain as key elements that provide Gal-4 with the ability to cross-link multivalent ligands, beyond the glycan binding capacity of the dimer.
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Galectins (Gals), a family of multifunctional glycan-binding proteins, have been traditionally defined as ß-galactoside binding lectins. However, certain members of this family have shown selective affinity toward specific glycan structures including human milk oligosaccharides (HMOs) and blood group antigens. In this work, we explored the affinity of human galectins (particularly Gal-1, -3, -4, -7, and -12) toward a panel of oligosaccharides including HMOs and blood group antigens using a complementary approach based on both experimental and computational techniques. While prototype Gal-1 and Gal-7 exhibited differential affinity for type I versus type II Lac/LacNAc residues and recognized fucosylated neutral glycans, chimera-type Gal-3 showed high binding affinity toward poly-LacNAc structures including LNnH and LNnO. Notably, the tandem-repeat human Gal-12 showed preferential recognition of 3-fucosylated glycans, a unique feature among members of the galectin family. Finally, Gal-4 presented a distinctive glycan-binding activity characterized by preferential recognition of specific blood group antigens, also validated by saturation transfer difference nuclear magnetic resonance experiments. Particularly, we identified oligosaccharide blood group A antigen tetraose 6 (BGA6) as a biologically relevant Gal-4 ligand, which specifically inhibited interleukin-6 secretion induced by this lectin on human peripheral blood mononuclear cells. These findings highlight unique determinants underlying specific recognition of HMOs and blood group antigens by human galectins, emphasizing the biological relevance of Gal-4-BGA6 interactions, with critical implications in the development and regulation of inflammatory responses.
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The limited availability of efficient treatments for Candida infections and the increased emergence of antifungal-resistant strains stimulates the search for new antifungal agents. We have previously isolated a sunflower mannose-binding lectin (Helja) with antifungal activity against Candida albicans, capable of binding mannose-bearing oligosaccharides exposed on the cell surface. This work aimed to investigate the biological and biophysical basis of Helja's binding to C. albicans cell wall mannans and its influence on the fungicidal activity of the lectin. We evaluated the interaction of Helja with the cell wall mannans extracted from the isogenic parental strain (WT) and a glycosylation-defective C. albicans with altered cell wall phosphomannosylation (mnn4∆ null mutants) and investigated its antifungal effect. Helja exhibited stronger antifungal activity on the mutant strain, showing greater inhibition of fungal growth, loss of cell viability, morphological alteration, and formation of clusters with agglutinated cells. This differential biological activity of Helja was correlated with the biophysical parameters determined by solid phase assays and isothermal titration calorimetry, which demonstrated that the lectin established stronger interactions with the cell wall mannans of the mnn4∆ null mutant than with the WT strain. In conclusion, our results provide new evidence on the nature of the Helja molecular interactions with cell wall components, i.e. phosphomannan, and its impact on the antifungal activity. This study highlights the relevance of plant lectins in the design of effective antifungal therapies.
Assuntos
Antifúngicos , Candida albicans , Parede Celular , Antifúngicos/farmacologia , Antifúngicos/química , Candida albicans/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Helianthus/química , Mananas/química , Mananas/farmacologia , Mananas/metabolismo , Testes de Sensibilidade MicrobianaRESUMO
Colorectal cancer (CRC) represents the third most common malignancy and the second leading cause of cancer-related deaths worldwide. Although immunotherapy has taken center stage in mainstream oncology, it has shown limited clinical efficacy in CRC, generating an urgent need for discovery of new biomarkers and potential therapeutic targets. Galectin-1 (Gal-1), an endogenous glycan-binding protein, induces tolerogenic programs and contributes to tumor cell evasion of immune responses. Here, we investigated the relevance of Gal-1 in CRC and explored its modulatory activity within the CD8+ regulatory T cell (Treg) compartment. Mice lacking Gal-1 (Lgals1-/- ) developed a lower number of tumors and showed a decreased frequency of a particular population of CD8+CD122+PD-1+ Tregs in the azoxymethane-dextran sodium sulfate model of colitis-associated CRC. Moreover, silencing of tumor-derived Gal-1 in the syngeneic CT26 CRC model resulted in reduced number and attenuated immunosuppressive capacity of CD8+CD122+PD-1+ Tregs, leading to slower tumor growth. Moreover, stromal Gal-1 also influenced the fitness of CD8+ Tregs, highlighting the contribution of both tumor and stromal-derived Gal-1 to this immunoregulatory effect. Finally, bioinformatic analysis of a colorectal adenocarcinoma from The Cancer Genome Atlas dataset revealed a particular signature characterized by high CD8+ Treg score and elevated Gal-1 expression, which delineates poor prognosis in human CRC. Our findings identify CD8+CD122+PD-1+ Tregs as a target of the immunoregulatory activity of Gal-1, suggesting a potential immunotherapeutic strategy for the treatment of CRC.
Assuntos
Adenocarcinoma/genética , Linfócitos T CD8-Positivos/imunologia , Colite/genética , Neoplasias Colorretais/genética , Galectina 1/genética , Linfócitos T Reguladores/imunologia , Adenocarcinoma/imunologia , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Animais , Atlas como Assunto , Azoximetano/administração & dosagem , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Colite/induzido quimicamente , Colite/imunologia , Colite/mortalidade , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Biologia Computacional , Sulfato de Dextrana/administração & dosagem , Modelos Animais de Doenças , Galectina 1/deficiência , Galectina 1/imunologia , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade beta de Receptor de Interleucina-2/genética , Subunidade beta de Receptor de Interleucina-2/imunologia , Camundongos , Camundongos Knockout , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Transdução de Sinais , Análise de Sobrevida , Linfócitos T Reguladores/patologia , Carga TumoralRESUMO
Galectins, a family of endogenous glycan-binding proteins, play crucial roles in a broad range of physiological and pathological processes. Galectin-1 (Gal-1), a proto-type member of this family, is overexpressed in several cancers and plays critical roles in tumor-immune escape, angiogenesis and metastasis. Thus, generation of high-affinity Gal-1 inhibitors emerges as an attractive therapeutic approach for a wide range of neoplastic conditions. Small-molecule carbohydrate inhibitors based on lactose (Lac) and N-acetyllactosamine (LacNAc) structures have been tested showing different results. In this study, we evaluated Lac- and LacNAc-based compounds with specific chemical modifications at key positions as Gal-1 ligands by competitive solid-phase assays (SPA) and isothermal titration calorimetry (ITC). Both assays showed excellent correlation, highlighting that lactosides bearing bulky aromatic groups at the anomeric carbon and sulfate groups at the O3' position exhibited the highest binding affinities. To dissect the atomistic determinants for preferential affinity of the different tested Gal-1 ligands, molecular docking simulations were conducted and PRODIGY-LIG structure-based method was employed to predict binding affinity in protein-ligand complexes. Notably, calculated binding free energies derived from the molecular docking were in accordance with experimental values determined by SPA and ITC, showing excellent correlation between theoretical and experimental approaches. Moreover, this analysis showed that 3'-O-sulfate groups interact with residues of the Gal-1 subsite B, mainly with Asn33, while the ester groups of the aromatic anomeric group interact with Gly69 and Thr70 at Gal-1 subsite E, extending deeper into the pocket, which could account for the enhanced binding affinity. This study contributes to the rational design of highly optimized Gal-1 inhibitors to be further studied in cancer models and other pathologic conditions.
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Upon overnutrition, adipocytes activate a homeostatic program to adjust anabolic pressure. An inflammatory response enables adipose tissue (AT) expansion with concomitant enlargement of its capillary network, and reduces energy storage by increasing insulin resistance. Galectin-12 (Gal-12), an endogenous lectin preferentially expressed in AT, plays a key role in adipocyte differentiation, lipolysis, and glucose homeostasis. Here, we reveal biochemical and biophysical determinants of Gal-12 structure, including its preferential recognition of 3-fucosylated structures, a unique feature among members of the galectin family. Furthermore, we identify a previously unanticipated role for this lectin in the regulation of angiogenesis within AT. Gal-12 showed preferential localization within the inner side of lipid droplets, and its expression was upregulated under hypoxic conditions. Through glycosylation-dependent binding to endothelial cells, Gal-12 promoted in vitro angiogenesis. Moreover, analysis of in vivo AT vasculature showed reduced vascular networks in Gal-12-deficient (Lgals12-/-) compared to wild-type mice, supporting a role for this lectin in AT angiogenesis. In conclusion, this study unveils biochemical, topological, and functional features of a hypoxia-regulated galectin in AT, which modulates endothelial cell function through recognition of 3-fucosylated glycans. Thus, glycosylation-dependent programs may control AT homeostasis by modulating endothelial cell biology with critical implications in metabolic disorders and inflammation.
Assuntos
Adipócitos/metabolismo , Células Endoteliais/metabolismo , Galectinas/metabolismo , Neovascularização Patológica/metabolismo , Tecido Adiposo/metabolismo , Animais , Fenômenos Fisiológicos Celulares/fisiologia , Resistência à Insulina/fisiologia , Gotículas Lipídicas/metabolismo , Lipólise/fisiologia , Camundongos Knockout , Polissacarídeos/metabolismoRESUMO
Galectins, soluble lectins widely expressed intra- and extracellularly in different cell types, play major roles in deciphering the cellular glycocode. Galectin-1 (Gal-1), a prototype member of this family, presents a carbohydrate recognition domain (CRD) with specific affinity for ß-galactosides such as N-acetyllactosamine (ß-d-Galp-(1 â 4)-d-GlcpNAc), and mediate numerous physiological and pathological processes. In this work, Gal-1 binding affinity for ß-(1 â 6) galactosides, including ß-d-Galp-(1 â 6)-ß-d-GlcpNAc-(1 â 4)-d-GlcpNAc was evaluated, and their performance was compared to that of ß-(1 â 4) and ß-(1 â 3) galactosides. To this end, the trisaccharide ß-d-Galp-(1 â 6)-ß-d-GlcpNAc-(1 â 4)-d-GlcpNAc was enzymatically synthesized, purified and structurally characterized. To evaluate the affinity of Gal-1 for the galactosides, competitive solid phase assays (SPA) and isothermal titration calorimetry (ITC) studies were carried out. The experimental dissociation constants and binding energies obtained were compared to those calculated by molecular docking. These analyses evidenced the critical role of the glycosidic linkage between the terminal galactopyranoside residue and the adjacent monosaccharide, as galactosides bearing ß-(1 â 6) glycosidic linkages showed dissociation constants six- and seven-fold higher than those involving ß-(1 â 4) and ß-(1 â 3) linkages, respectively. Moreover, docking experiments revealed the presence of hydrogen bond interactions between the N-acetyl group of the glucosaminopyranose moiety of the evaluated galactosides and specific amino acid residues of Gal-1, relevant for galectin-glycan affinity. Noticeably, the binding free energies (ΔGbindcalc) derived from the molecular docking were in good agreement with experimental values determined by ITC measurements (ΔGbindexp), evidencing a good correlation between theoretical and experimental approaches, which validates the in silico simulations and constitutes an important tool for the rational design of future optimized ligands.
Assuntos
Galactosídeos/química , Galectina 1/química , Açúcares/química , Acetilação , Configuração de Carboidratos , Humanos , Simulação de Acoplamento MolecularRESUMO
Chlamydia trachomatis (Ct) constitutes the most prevalent sexually transmitted bacterium worldwide. Chlamydial infections can lead to severe clinical sequelae including pelvic inflammatory disease, ectopic pregnancy, and tubal infertility. As an obligate intracellular pathogen, Ct has evolved multiple strategies to promote adhesion and invasion of host cells, including those involving both bacterial and host glycans. Here, we show that galectin-1 (Gal1), an endogenous lectin widely expressed in female and male genital tracts, promotes Ct infection. Through glycosylation-dependent mechanisms involving recognition of bacterial glycoproteins and N-glycosylated host cell receptors, Gal1 enhanced Ct attachment to cervical epithelial cells. Exposure to Gal1, mainly in its dimeric form, facilitated bacterial entry and increased the number of infected cells by favoring Ct-Ct and Ct-host cell interactions. These effects were substantiated in vivo in mice lacking Gal1 or complex ß1-6-branched N-glycans. Thus, disrupting Gal1-N-glycan interactions may limit the severity of chlamydial infection by inhibiting bacterial invasion of host cells.
Assuntos
Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/metabolismo , Galectina 1/metabolismo , Linfogranuloma Venéreo/metabolismo , Animais , Proteínas de Bactérias/genética , Chlamydia trachomatis/genética , Feminino , Galectina 1/genética , Células HeLa , Humanos , Linfogranuloma Venéreo/genética , Linfogranuloma Venéreo/patologia , Masculino , CamundongosRESUMO
Galectin-8 (Gal-8) is a tandem-repeat type galectin with affinity for ß-galactosides, bearing two carbohydrate recognition domains (CRD) connected by a linker peptide. The N- and C-terminal domains (Gal-8N and Gal-8C) share 35% homology, and their glycan ligand specificity is notably dissimilar: while Gal-8N shows strong affinity for α(2-3)-sialylated oligosaccharides, Gal-8C has higher affinity for non-sialylated oligosaccharides, including poly-N-acetyllactosamine and/ or A and B blood group structures. Particularly relevant for understanding the biological role of this lectin, full-length Gal-8 can bind cell surface glycoconjugates with broader affinity than the isolated Gal-8N and Gal-8C domains, a trait also described for other tandem-repeat galectins. Herein, we aim to discuss the potential use of separate CRDs in modelling tandem-repeat galectin-8 and its biological functions. For this purpose, we will cover several aspects of the structure-function relationship of this protein including crystallographic structures, glycan specificity, cell function and biological roles, with the ultimate goal of understanding the potential role of each CRD in predicting full-length Gal-8 involvement in relevant biological processes.
Assuntos
Metabolismo dos Carboidratos , Galectinas/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Galectinas/química , Humanos , Ligantes , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Herein, we report the synthesis of calix[4]resorcinarene-based multivalent ligands bearing ß-S-GlcNAc and ß-S-AllNAc recognition elements. A clickable ß-S-AllNAc derivative was successfully prepared from a ß-thioalkynyl GlcNAc precursor, making use of a 2,3-oxazoline intermediate, easily formed by intramolecular displacement of a triflate group located at the 3-position by the 2-N-acetate group. By reaction of these alkynyl-functionalized derivatives with an octaazido-calix[4]resorcinarene macrocycle having undecyl chains, two octavalent glycoclusters exposing the epimeric N-acetylhexosamines were obtained. In addition, a related calix[4]resorcinarene-based glycocluster having methyl groups instead of undecyl chains and ß-S-GlcNAc residues was also synthesized. After an initial evaluation of the interaction of the undecyl-functionalized ß-S-GlcNAc octavalent derivative with Wheat Germ Agglutinin (WGA) by a turbidimetry experiment, the interaction of the three synthesized glycoclusters towards WGA was studied by Isothermal Titration Calorimetry. The results showed a favorable effect due to the presence of the undecyl chains in terms of affinity. Surprisingly, the ß-S-AllNAc octavalent compound showed the highest affinity among the evaluated glycoclusters, showing for the first time that WGA interacts with ß-AllNAc-bearing ligands. Molecular docking studies of ß-AllNAc with WGA in comparison with ß-GlcNAc contributed to the understanding of the atomic interactions responsible for this unexpected affinity.
Assuntos
Aglutininas do Germe de TrigoRESUMO
BACKGROUND: We previously demonstrated that the activated leukocyte cell adhesion molecule (ALCAM/CD166) can interact with galectin-8 (Gal-8) in endothelial cells. ALCAM is a member of the immunoglobulin superfamily that promotes homophilic and heterophilic cell-cell interactions. Gal-8 is a "tandem-repeat"-type galectin, known as a matricellular protein involved in cell adhesion. Here, we analyzed the physical interaction between both molecules in breast cancer cells and the functional relevance of this phenomenon. METHODS: We performed binding assays by surface plasmon resonance to study the interaction between Gal-8 and the recombinant glycosylated ALCAM ectodomain or endogenous ALCAM from MDA-MB-231 breast cancer cells. We also analyzed the binding of ALCAM-silenced or control breast cancer cells to immobilized Gal-8 by SPR. In internalization assays, we evaluated the influence of Gal-8 on ALCAM surface localization. RESULTS: We showed that recombinant glycosylated ALCAM and endogenous ALCAM from breast carcinoma cells physically interacted with Gal-8 in a glycosylation-dependent fashion displaying a differential behavior compared to non-glycosylated ALCAM. Moreover, ALCAM-silenced breast cancer cells exhibited reduced binding to Gal-8 relative to control cells. Importantly, exogenously added Gal-8 provoked ALCAM segregation, probably trapping this adhesion molecule at the surface of breast cancer cells. CONCLUSIONS: Our data indicate that Gal-8 interacts with ALCAM at the surface of breast cancer cells through glycosylation-dependent mechanisms. GENERAL SIGNIFICANCE: A novel heterophilic interaction between ALCAM and Gal-8 is demonstrated here, suggesting its physiologic relevance in the biology of breast cancer cells.
Assuntos
Antígenos CD/metabolismo , Neoplasias da Mama/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas Fetais/metabolismo , Galectinas/metabolismo , Mapas de Interação de Proteínas/genética , Antígenos CD/genética , Neoplasias da Mama/patologia , Adesão Celular/genética , Moléculas de Adesão Celular Neuronais/genética , Comunicação Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Células Endoteliais/metabolismo , Feminino , Proteínas Fetais/genética , Galectinas/genética , Glicosilação , Humanos , Ligação Proteica , Propriedades de SuperfícieRESUMO
The synthesis of multivalent sialylated glycoclusters is herein addressed by a chemoenzymatic approach using the trans-sialidase of Trypanosoma cruzi (TcTS). Multivalent ß-thio-galactopyranosides and ß-thio-lactosides were used as acceptor substrates and 3'-sialyllactose as the sialic acid donor. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was shown to be an excellent technique for the analysis of the reaction products. Different eluting conditions were optimized to allow the simultaneous resolution of the sialylated species, as well as their neutral precursors. The TcTS efficiently transferred sialyl residues to di, tri, tetra and octa ß-thiogalactosides. In the case of an octavalent thiolactoside, up to six polysialylated compounds could be resolved. Preparative sialylation reactions were performed using the tetravalent and octavalent acceptor substrates. The main sialylated derivatives could be unequivocally assigned by MALDI mass spectrometry. Inhibition of the transfer to the natural substrate, N-acetyllactosamine, was also studied. The octalactoside caused 82 % inhibition of sialic acid transfer when we used equimolar concentrations of donor, acceptor and inhibitor.
Assuntos
Glicoproteínas/química , Lactose/análogos & derivados , Neuraminidase/química , Proteínas de Protozoários/química , Ácidos Siálicos/química , Tiogalactosídeos/química , Trypanosoma cruzi/enzimologia , Cromatografia Líquida de Alta Pressão , Lactose/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Herein, we describe the design and synthesis of a novel family of hydrolytically stable glycoclusters bearing thiodigalactoside (TDG) analogues as recognition elements of ß-galactoside binding lectins. The TDG analogue was synthesized by thioglycosylation of a 6-S-acetyl-α-D-glucosyl bromide with the isothiouronium salt of 2,3,4,6-tetra-O-acetyl-ß-D-galactose. Further propargylation of the TDG analogue allowed the coupling to azido-functionalized oligosaccharide scaffolds through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) under microwave activation. The final mono-, di-, and tetravalent ligands were resistant to enzymatic hydrolisis by Escherichia coli ß-galactosidase. Binding affinities to peanut agglutinin and human galectin-3 were measured by isothermal titration calorimetry which showed K(a) constants in the micromolar range as well as a multivalent effect. Monovalent ligand exhibited a binding affinity higher than that of thiodigalactoside. Docking studies performed with a model ligand on both ß-galactoside binding lectins showed additional interactions between the triazole ring and lectin amino acid residues, suggesting a positive effect of this aromatic residue on the biological activity.
Assuntos
Arachis/química , Galectina 3/química , Glicoconjugados/síntese química , Lectinas/química , Tiogalactosídeos/química , Proteínas Sanguíneas , Configuração de Carboidratos , Sequência de Carboidratos , Galectinas , Glicoconjugados/química , Humanos , Hidrólise , Ligantes , Modelos Moleculares , Dados de Sequência MolecularRESUMO
In this work we describe the synthesis of mono- and divalent ß-N- and ß-S-galactopyranosides and related lactosides built on sugar scaffolds and their evaluation as substrates and inhibitors of the Trypanosoma cruzi trans-sialidase (TcTS). This enzyme catalyzes the transfer of sialic acid from an oligosaccharidic donor in the host, to parasite ßGalp terminal units and it has been demonstrated that it plays an important role in the infection. Herein, the enzyme was also tested as a tool for the chemoenzymatic synthesis of sialic acid containing glycoclusters. The transfer reaction of sialic acid was performed using a recombinant TcTS and 3'-sialyllactose as sialic acid donor, in the presence of the acceptor having ßGalp non reducing ends. The products were analyzed by high performance anion exchange chromatography with pulse amperometric detection (HPAEC-PAD). The ability of the different S-linked and N-linked glycosides to inhibit the sialic acid transfer reaction from 3'-sialyllactose to the natural substrate N-acetyllactosamine, was also studied. Most of the substrates behaved as good acceptors and moderate competitive inhibitors. A di-N-lactoside showed to be the strongest competitive inhibitor among the compounds tested (70% inhibition at equimolar concentration). The usefulness of the enzymatic trans-sialylation for the preparation of sialylated ligands was assessed by performing a preparative sialylation of a divalent substrate, which afforded the monosialylated compound as main product, together with the disialylated glycocluster.
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Herein, we describe the use of thioglycosides as glycosidase inhibitors by employing novel modifications at the reducing end of these glycomimetics. The inhibitors display a basic galactopyranosyl unit (1â4)-bonded to a 3-deoxy-4-thiopentopyranose moiety. The molecular basis of the observed inhibition has been studied by using a combination of NMR spectroscopy and molecular modeling techniques. It is demonstrated that these molecules are not recognized by Escherichia coli ß-galactosidase in their ground-state conformation, with a conformational selection process taking place. In fact, the observed conformational distortion depends on the chemical nature of the compounds and results from the rotation around the glycosidic linkage (variation of Φ or Ψ) or from the deformation of the six-membered ring of the pentopyranose. The bound conformations of the ligand are adapted in the enzymatic pocket with a variety of hydrogen-bond, van der Waals, and stacking interactions.
Assuntos
Dissacarídeos/farmacologia , Escherichia coli/enzimologia , Modelos Moleculares , Tioglucosídeos/farmacologia , beta-Galactosidase/antagonistas & inibidores , Dissacarídeos/química , Dissacarídeos/farmacocinética , Ligantes , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Tioglucosídeos/química , Tioglucosídeos/farmacocinéticaRESUMO
We report here the synthesis of divalent ligands containing 3-deoxy-4-thiolactose. This thiodisaccharide has been synthesized using the Michael addition of ß-1-thiogalactose to the α,ß-unsaturated system of sugar-derived dihydropyranones, followed by the reduction of the remaining carbonyl group. We were able to control the configuration (S) of the stereocenter linked to sulfur (C-4) of the reducing end by conducting the thioglycosylation at high temperature or by isomerization during the reduction of the 2-ulose thiodisaccharide with NaBH4/THF. The energy profile for this reaction on a model compound was calculated. The anomeric position of the 3-deoxy-4-thiolactose was functionalized with a terminal alkyne, which was coupled to azide-containing sugar scaffolds through CuAAC reaction to afford mono- and divalent ligands. The final products were competitive inhibitors of E. coli ß-galactosidase in the micromolar range. Their binding affinities to peanut agglutinin (PNA) were determined by isothermal calorimetry, which showed a clear decrease in the Ka values for monovalent derivatives compared to lactose. This report contributes to establishing the role of a particular hydroxyl group of lactose in sugar-protein recognition processes.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Lactose/síntese química , Lactose/farmacologia , Ligantes , beta-Galactosidase/metabolismo , Ligação Competitiva , Química Click , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Lactose/análogos & derivados , Estrutura Molecular , TermodinâmicaRESUMO
Galectins are a family of endogenous glycan-binding proteins that have crucial roles in a broad range of physiological and pathological processes. As a group, these proteins use both extracellular and intracellular mechanisms as well as glycan-dependent and independent pathways to reprogramme the fate and function of numerous cell types. Given their multifunctional roles in both tissue fibrosis and cancer, galectins have been identified as potential therapeutic targets for these disorders. Here, we focus on the therapeutic relevance of galectins, particularly galectin 1 (GAL1), GAL3 and GAL9 to tumour progression and fibrotic diseases. We consider an array of galectin-targeted strategies, including small-molecule carbohydrate inhibitors, natural polysaccharides and their derivatives, peptides, peptidomimetics and biological agents (notably, neutralizing monoclonal antibodies and truncated galectins) and discuss their mechanisms of action, selectivity and therapeutic potential in preclinical models of fibrosis and cancer. We also review the results of clinical trials that aim to evaluate the efficacy of galectin inhibitors in patients with idiopathic pulmonary fibrosis, nonalcoholic steatohepatitis and cancer. The rapid pace of glycobiology research, combined with the acute need for drugs to alleviate fibrotic inflammation and overcome resistance to anticancer therapies, will accelerate the translation of anti-galectin therapeutics into clinical practice.
Assuntos
Galectinas , Neoplasias , Humanos , Galectinas/metabolismo , Neoplasias/tratamento farmacológico , Anticorpos Monoclonais , Polissacarídeos/metabolismo , FibroseRESUMO
Development of an aberrant vascular network is a hallmark of the multistep pathological process of tumor growth and metastasis. In response to hypoxia, several pro-angiogenic factors are synthesized to support vascularization programs required for cancer progression. Emerging data indicate the involvement of glycans and glycan-binding proteins as critical regulators of vascular circuits in health and disease. Galectins may be regulated by hypoxic conditions and control angiogenesis in different physiopathological settings. These ß-galactoside-binding proteins may promote sprouting angiogenesis by interacting with different glycosylated receptors and triggering distinct signaling pathways. Understanding the role of galectins in tumor neovascularization will contribute to the design of novel anti-angiogenic therapies aimed at complementing current anti-cancer modalities and overcoming resistance to these treatments. Here we describe selected strategies and methods used to study the role of hypoxia-regulated galectins in the regulation of blood vessel formation.
Assuntos
Galectinas , Hipóxia , Neoplasias , Neovascularização Patológica , Galectinas/metabolismo , Humanos , Hipóxia/fisiopatologia , Neoplasias/irrigação sanguínea , Neovascularização Patológica/fisiopatologia , Transdução de SinaisRESUMO
Combined Antiretroviral therapy (cART) suppresses HIV replication but fails to eradicate the virus, which persists in a small pool of long-lived latently infected cells. Immune activation and residual inflammation during cART are considered to contribute to viral persistence. Galectins, a family of ß-galactoside-binding proteins, play central roles in host-pathogen interactions and inflammatory responses. Depending on their structure, glycan binding specificities and/or formation of distinct multivalent signaling complexes, different members of this family can complement, synergize, or oppose the function of others. Here, we identify a regulatory circuit, mediated by galectin-1 (Gal-1)-glycan interactions, that promotes reversal of HIV-1 latency in infected T cells. We found elevated levels of circulating Gal-1 in plasma from HIV-1-infected individuals, which correlated both with inflammatory markers and the transcriptional activity of the reservoir, as determined by unspliced-RNA (US-RNA) copy number. Proinflammatory extracellular vesicles (EVs) isolated from the plasma of HIV-infected individuals induced Gal-1 secretion by macrophages. Extracellularly, Gal-1 interacted with latently infected resting primary CD4+ T cells and J-LAT cells in a glycan-dependent manner and reversed HIV latency via activation of the nuclear factor κB (NF-κB). Furthermore, CD4+ T cells isolated from HIV-infected individuals showed increased HIV-1 transcriptional activity when exposed to Gal-1. Thus, by modulating reservoir dynamics, EV-driven Gal-1 secretion by macrophages links inflammation with HIV-1 persistence in cART-treated individuals. IMPORTANCE Antiretroviral therapy has led to a dramatic reduction in HIV-related morbidity and mortality. However, cART does not eradicate the virus, which persists in resting CD4+ T cells as the main viral reservoir, consequently requiring lifelong treatment. A major question is how the functional status of the immune system during antiretroviral therapy determines the activity and size of the viral reservoir. In this study, we identified a central role for galectin-1 (Gal-1), a glycan-binding protein released in response to extracellular vesicles (EVs), in modulating the activity of HIV reservoir, thus shaping chronic immune activation in HIV-infected patients. Our work unveils a central role of Gal-1 in linking chronic immune activation and reservoir dynamics, highlighting new therapeutic opportunities in HIV infection.