RESUMO
Crude anionic polysaccharides extracted from the Pacific starfish Lethasterias fusca were purified by anion-exchange chromatography. The main fraction LF, having MW 14.5 kDa and dispersity 1.28 (data of gel-permeation chromatography), was solvolytically desulfated and giving rise to preparation LF-deS with a structure of dermatan core [â3)-ß-d-GalNAc-(1â4)-α-l-IdoA-(1â]n, which was identified according to NMR spectroscopy data. Analysis of the NMR spectra of the parent fraction LF led to identification of the main component as dermatan sulfate LF-Derm â3)-ß-d-GalNAc4R-(1â4)-α-l-IdoA2R3S-(1â (where R was SO3 or H), bearing sulfate groups at O-3 or both at O-2 and O-3 of α-l-iduronic acid, as well as at O-4 of some N-acetyl-d-galactosamine residues. The minor signals in NMR spectra of LF were assigned as resonances of heparinoid LF-Hep composed of the fragments â4)-α-d-GlcNS3S6S-(1â4)-α-l-IdoA2S3S-(1â. The 3-O-sulfated and 2,3-di-O-sulfated iduronic acid residues are very unusual for natural glycosaminoglycans, and further studies are needed to elucidate their possible specific influence on the biological activity of the corresponding polysaccharides. To confirm the presence of these units in LF-Derm and LF-Hep, a series of variously sulfated model 3-aminopropyl iduronosides were synthesized and their NMR spectra were compared with those of the polysaccharides. Preparations LF and LF-deS were studied as stimulators of hematopoiesis in vitro. Surprisingly, it was found that both preparations were active in these tests, and hence, the high level of sulfation is not necessary for hematopoiesis stimulation in this particular case.
Assuntos
Dermatan Sulfato , Glicosaminoglicanos , Animais , Glicosaminoglicanos/farmacologia , Dermatan Sulfato/química , Ácido Idurônico , Estrelas-do-Mar , Polissacarídeos , Sulfatos/químicaRESUMO
Preparations of sulfated polysaccharides obtained from brown algae are known as fucoidans. These biopolymers have attracted considerable attention due to many biological activities which may find practical applications. Two Atlantic representatives of Phaeophyceae, namely, Fucus vesiculosus and Ascophyllum nodosum, belonging to the same order Fucales, are popular sources of commercial fucoidans, which often regarded as very similar in chemical composition and biological actions. Nevertheless, these two fucoidan preparations are polysaccharide mixtures which differ considerably in amount and chemical nature of components, and hence, this circumstance should be taken into account in the investigation of their biological properties and structure-activity relationships. In spite of these differences, fractions with carefully characterized structures prepared from both fucoidans may have valuable applications in drug development.
Assuntos
Ascophyllum , Fucus , Phaeophyceae , Ascophyllum/química , Fucus/química , Sulfatos , Phaeophyceae/química , Polissacarídeos/farmacologia , Polissacarídeos/químicaRESUMO
Fucosylated chondroitin sulfates (FCSs) FCS-BA and FCS-HS, as well as fucan sulfates (FSs) FS-BA-AT and FS-HS-AT were isolated from the sea cucumbers Bohadschia argus and Holothuria (Theelothuria) spinifera, respectively. Purification of the polysaccharides was carried out by anion-exchange chromatography on DEAE-Sephacel column. Structural characterization of polysaccharides was performed in terms of monosaccharide and sulfate content, as well as using a series of non-destructive NMR spectroscopic methods. Both FCSs were shown to contain a chondroitin core [â3)-ß-d-GalNAc-(1â4)-ß-d-GlcA-(1â]n bearing sulfated fucosyl branches at O-3 of every GlcA residue in the chain. These fucosyl residues were different in pattern of sulfation: FCS-BA contained Fuc2S4S, Fuc3S4S and Fuc4S at a ratio of 1:8:2, while FCS-HS contained these residues at a ratio of 2:2:1. Polysaccharides differed also in content of GalNAc4S6S and GalNAc4S units, the ratios being 14:1 for FCS-BA and 4:1 for FCS-HS. Both FCSs demonstrated significant anticoagulant activity in clotting time assay and potentiated inhibition of thrombin, but not of factor Xa. FS-BA-AT was shown to be a regular linear polymer of 4-linked α-L-fucopyranose 3-sulfate, the structure being confirmed by NMR spectra of desulfated polysaccharide. In spite of considerable sulfate content, FS-BA-AT was practically devoid of anticoagulant activity. FS-HS-AT cannot be purified completely from contamination of some FCS. Its structure was tentatively represented as a mixture of chains identical with FS-BA-AT and other chains built up of randomly sulfated alternating 4- and 3-linked α-L-fucopyranose residues.
Assuntos
Holothuria , Pepinos-do-Mar , Animais , Anticoagulantes/química , Anticoagulantes/farmacologia , Sulfatos de Condroitina/química , Fucose/química , Holothuria/química , Polissacarídeos/farmacologia , Pepinos-do-Mar/química , Sulfatos/farmacologiaRESUMO
Fucoidans are natural sulfated polysaccharides that have a wide range of biological functions and are regarded as promising antitumor agents. The activity of various fucoidans and their derivatives has been demonstrated in vitro on tumor cells of different histogenesis and in experiments on mice with grafted tumors. However, these experimental models showed low levels of antitumor activity and clinical trials did not prove that this class of compounds could serve as antitumor drugs. Nevertheless, the anti-inflammatory, antiangiogenic, immunostimulating, and anticoagulant properties of fucoidans, as well as their ability to stimulate hematopoiesis during cytostatic-based antitumor therapy, suggest that effective fucoidan-based drugs could be designed for the supportive care and symptomatic therapy of cancer patients. The use of fucoidans in cancer patients after chemotherapy and radiation therapy might promote the rapid improvement of hematopoiesis, while their anti-inflammatory, immunomodulatory, and anticoagulant effects have the potential to improve the quality of life of patients with advanced cancer.
Assuntos
Citostáticos , Neoplasias , Animais , Anti-Inflamatórios , Anticoagulantes/farmacologia , Anticoagulantes/uso terapêutico , Oncologia , Camundongos , Neoplasias/tratamento farmacológico , Polissacarídeos/farmacologia , Polissacarídeos/uso terapêutico , Qualidade de VidaRESUMO
Fucosylated chondroitin sulfates (FCSs) PC and HH were isolated from the sea cucumbers Paracaudina chilensis and Holothuria hilla, respectively. The purification of the polysaccharides was carried out by anion-exchange chromatography on a DEAE-Sephacel column. The structural characterization of the polysaccharides was performed in terms of monosaccharide and sulfate content, as well as using a series of nondestructive NMR spectroscopic methods. Both polysaccharides were shown to contain a chondroitin core [â3)-ß-d-GalNAc (N-acethyl galactosamine)-(1â4)-ß-d-GlcA (glucuronic acid)-(1â]n, bearing sulfated fucosyl branches at O-3 of every GlcA residue in the chain. These fucosyl residues were different in their pattern of sulfation: PC contained Fuc2S4S and Fuc4S in a ratio of 2:1, whereas HH included Fuc2S4S, Fuc3S4S, and Fuc4S in a ratio of 1.5:1:1. Moreover, some GalNAc residues in HH were found to contain an unusual disaccharide branch Fuc4S-(1â2)-Fuc3S4S-(1â at O-6. Sulfated GalNAc4S6S and GalNAc4S units were found in a ratio of 3:2 in PC and 2:1 in HH. Both polysaccharides demonstrated significant anticoagulant activity in a clotting time assay, which is connected with the ability of these FCSs to potentiate the inhibition of thrombin and factor Xa in the presence of anti-thrombin III (ATIII) and with the direct inhibition of thrombin in the absence of any cofactors.
Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Sulfatos de Condroitina/farmacologia , Holothuria/metabolismo , Animais , Anticoagulantes/isolamento & purificação , Antitrombina III/metabolismo , Antitrombinas/isolamento & purificação , Antitrombinas/farmacologia , Sulfatos de Condroitina/isolamento & purificação , Fator Xa/metabolismo , Inibidores do Fator Xa/isolamento & purificação , Inibidores do Fator Xa/farmacologia , Estrutura Molecular , Relação Estrutura-Atividade , Trombina/antagonistas & inibidores , Trombina/metabolismoRESUMO
Immunosuppression derived after cytostatics application in cancer chemotherapy is considered as an adverse side effect that leads to deterioration of quality of life and risk of infectious diseases. A linear sulfated (1â3)-α-l-fucan M-Fuc prepared by chemical modification of a fucoidan isolated from the brown seaweed Chordaria flagelliformis, along with two structurally related synthetic sulfated oligosaccharides, were studied as stimulators of hematopoiesis on a model of cyclophosphamide immunosuppression in mice. Recombinant granulocyte colony-stimulating factor (r G-CSF), which is currently applied in medicine to treat low blood neutrophils, was used as a reference. Polysaccharide M-Fuc and sulfated difucoside DS did not demonstrate significant effect, while sulfated octasaccharide OS showed higher activity than r G-CSF, causing pronounced neutropoiesis stimulation. In addition, production of erythrocytes and platelets was enhanced after the octasaccharide administration. The assessment of populations of cells in blood and bone marrow of mice revealed the difference in mechanisms of action of OS and r G-CSF.
Assuntos
Ciclofosfamida/efeitos adversos , Fármacos Hematológicos/farmacologia , Hematopoese/efeitos dos fármacos , Neutropenia/tratamento farmacológico , Oligossacarídeos/farmacologia , Phaeophyceae/química , Polissacarídeos/farmacologia , Animais , Medula Óssea/efeitos dos fármacos , Modelos Animais de Doenças , Filgrastim/farmacologia , Fármacos Hematológicos/química , Fármacos Hematológicos/isolamento & purificação , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Terapia de Imunossupressão/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neutropenia/sangue , Neutropenia/induzido quimicamente , Neutrófilos/efeitos dos fármacos , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Sulfatos/químicaRESUMO
Fucosylated chondroitin sulfate CD was isolated from the sea cucumber Cucumaria djakonovi collected from the Avachinsky Gulf of the eastern coast of Kamchatka. Structural characterization of CD was performed using a series of non-destructive NMR spectroscopic procedures. The polysaccharide was shown to contain a chondroitin core [â3)-ß-d-GalNAc-(1â4)-ß-d-GlcA-(1â]n where about 60% of GlcA residues were 3-O-fucosylated, while another part of GlcA units did not contain any substituents. The presence of unsubstituted both at O-2 and O-3 glucuronic acid residues in a structure of holothurian chondroitin sulfate is unusual and has not been reported previously. Three different fucosyl branches Fucp2S4S, Fucp3S4S and Fucp4S were found in the ratio of 2:1:1. The GalNAc units were mono- or disulfated at positions 4 and 6. Anti-inflammatory activity of CD was assessed on a model of acute peritoneal inflammation in rats. About 45% inhibition was found for CD, while a structurally related linear chondroitin sulfate SS from cartilage of the fish Salmo salar demonstrated only 31% inhibition, indicating that the presence of sulfated fucosyl branches is essential for anti-inflammatory effect of chondroitin sulfates of marine origin.
Assuntos
Anti-Inflamatórios/farmacologia , Sulfatos de Condroitina/farmacologia , Cucumaria , Peritonite/tratamento farmacológico , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/uso terapêutico , Cartilagem/química , Sulfatos de Condroitina/química , Sulfatos de Condroitina/isolamento & purificação , Sulfatos de Condroitina/uso terapêutico , Dicroísmo Circular/métodos , Modelos Animais de Doenças , Feminino , Humanos , Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular , Peptonas/toxicidade , Peritonite/induzido quimicamente , Ratos , Ratos Wistar , Salmo salar , Relação Estrutura-Atividade , Resultado do TratamentoRESUMO
A gene that encodes fucoidanase ffa2 in the marine bacterium Formosa algae strain KMM 3553T was cloned, and the protein (FFA2) was produced in Escherichia coli. Recombinant fucoidanase FFA2 was purified, and the biochemical properties of this enzyme were studied. The amino acid sequence of FFA2 showed 57% identity with known fucoidanase FcnA from Mariniflexile fucanivorans. The mass of the gene product FFA2 is 101.2 kDa (918 amino acid residues). Sequence analysis has revealed that fucoidanase FFA2 belongs to the GH107 (CAZy) family. Detailed substrate specificity was studied by using fucoidans from brown seaweeds as well as synthetic fucooligosaccharide with distinct structures. Fucoidanase FFA2 catalyzes the cleavage of (1â4)-α-glycosidic bonds in the fucoidan from Fucus evanescens within a structural fragment (â3)-α-l-Fucp2S-(1â4)-α-l-Fucp2S-(1â)n but not in a fragment (â3)-α-l-Fucp2S,4S-(1â4)-α-l-Fucp2S-(1â)n. Using synthetic di-, tetra- and octasaccharides built up of the alternative (1â4)- and (1â3)-linked α-l-Fucp2S units, the difference in substrate specificity and in the rate of enzymatic selectivity was investigated. Nonsulfated and persulfated synthetic oligosaccharides were not transformed by the enzyme. Therefore, FFA2 was specified as poly[(1â4)-α-l-fucoside-2-sulfate] glycanohydrolase. This enzyme could be used for the modification of natural fucoidans to obtain more regular and easier characterized derivatives useful for research and practical applications.
Assuntos
Flavobacteriaceae/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Polissacarídeos/metabolismo , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Glicosídeo Hidrolases/metabolismo , Glicosídeos/química , Glicosídeos/metabolismo , Oligossacarídeos/química , Oligossacarídeos/genética , Polissacarídeos/química , Conformação Proteica , Especificidade por SubstratoRESUMO
A fucosylated chondroitin sulfate (FCS) was isolated from the body wall of Pacific sea cucumber Cucumaria japonicaby extraction in the presence of papain followed by Cetavlon precipitation and anion-exchange chromatography. FCS was shown to contain D-GalNAc, D-GlcA, L-Fuc and sulfate in molar proportions of about 1:1:1:4.5. Structure of FCS was elucidated using NMR spectroscopy and methylation analysis of the native polysaccharide and products of its desulfation and carboxyl reduction. The polysaccharide was shown to contain a typical chondroitin core â 3)-ß-D-GalNAc-(1 â 4)-ß-D-GlcA-(1 â. Sulfate groups in this core occupy O-4 and the majority of O-6 of GalNAc. Fucosyl branches are represented by 3,4- and 2,4-disulfated units in a ratio of 4:1 and are linked to O-3 of GlcA. In addition, â¼ 33% of GlcA are 3-O-sulfated, and hence, the presence of short fucooligosaccharide chains side by side with monofucosyl branches cannot be excluded. FCS was shown to inhibit platelets aggregation in vitro mediated by collagen and ristocetin, but not adenosine diphosphate, and demonstrated significant anticoagulant activity, which is connected with its ability to enhance inhibition of thrombin and factor Xa by antithrombin III, as well as to influence von Willebrand factor activity. The latest property significantly distinguished FCS from low-molecular-weight heparin.
Assuntos
Plaquetas/metabolismo , Sulfatos de Condroitina , Cucumaria/química , Fucose , Agregação Plaquetária/efeitos dos fármacos , Animais , Configuração de Carboidratos , Sulfatos de Condroitina/química , Sulfatos de Condroitina/farmacologia , Fucose/química , Fucose/farmacologia , HumanosRESUMO
Sulfated polysaccharides, fucoidans, from brown algae are built up mainly of α-L-fucopyranosyl units and form a group of natural biopolymers with a wide spectrum of biological activities. Systematic synthesis of oligosaccharides representing fucoidans' fragments gives molecular probes for detecting pharmacophores within fucoidan polysaccharide chains. Recently, it was discovered that the fucoidan from brown seaweed Chordaria flagelliformis contains not only α-L-fucopyranosyl units but also α-L-fucofuranosyl ones. To establish the influence of the unusual α-L-fucofuranose residue on the biological activity and conformational properties of fucoidans, the synthesis of selectively O-sulfated pentasaccharides, which represent the main repeating unit of the fucoidan from C. flagelliformis, was performed. The features of the synthesis were the use of the pyranoside-into-furanoside rearrangement to prepare the fucofuranoside precursor and remote stereocontrolling participation of O-acyl groups to manage stereoselective α-bond formation in glycosylation reactions.
Assuntos
Monossacarídeos/química , Oligossacarídeos/síntese química , Phaeophyceae/química , Polissacarídeos/química , Configuração de Carboidratos , Sequência de Carboidratos , Dados de Sequência Molecular , Oligossacarídeos/químicaRESUMO
Anionic polysaccharides fucosylated chondroitin sulfates (FCS) from holothurian species were shown to affect various biological processes, such as metastasis, angiogenesis, clot formation, thrombosis, inflammation, and some others. To understand the mechanism of FCSs action, knowledge about their spatial arrangement is required. We have started the systematic synthesis, conformational analysis, and study of biological activity of the oligosaccharides related to various fragments of these types of natural polysaccharides. In this communication, five molecules representing distinct structural fragments of chondroitin sulfate have been studied by means of molecular modeling and NMR. These are three disaccharides and two trisaccharides containing fucose and glucuronic acid residues with one sulfate group per each fucose residue or without it. Long-range C-H coupling constants were used for the verification of the theoretical models. The presence of two conformers for both linkage types was revealed. For the Fuc-GlA linkage, the dominant conformer was the same as described previously in a literature as the molecular dynamics (MD) average in a dodechasaccharide FCS fragment representing the backbone chain of the polysaccharide including GalNAc residues. This shows that the studied oligosaccharides, in addition to larger ones, may be considered as reliable models for Quantitative Structure-Activity Relationship (QSAR) studies to reveal pharmacophore fragments of FCS.
Assuntos
Sulfatos de Condroitina/química , Oligossacarídeos/química , Pepinos-do-Mar/química , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Espectroscopia de Ressonância Magnética , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Relação Quantitativa Estrutura-AtividadeRESUMO
Natural anionic polysaccharides fucosylated chondroitin sulfates (FCS) from sea cucumbers attract great attention nowadays due to their ability to influence various biological processes, such as blood coagulation, thrombosis, angiogenesis, inflammation, bacterial and viral adhesion. To determine pharmacophore fragments in FCS we have started systematic synthesis of oligosaccharides with well-defined structure related to various fragments of these polysaccharides. In this communication, the synthesis of non-sulfated and selectively O-sulfated di- and trisaccharides structurally related to branching sites of FCS is described. The target compounds are built up of propyl ß-d-glucuronic acid residue bearing at O-3 α-l-fucosyl or α-l-fucosyl-(1â3)-α-l-fucosyl substituents. O-Sulfation pattern in the fucose units of the synthetic targets was selected according to the known to date holothurian FCS structures. Stereospecific α-glycoside bond formation was achieved using 2-O-benzyl-3,4-di-O-chloroacetyl-α-l-fucosyl trichloroacetimidate as a donor. Stereochemical outcome of the glycosylation was explained by the remote participation of the chloroacetyl groups with the formation of the stabilized glycosyl cations, which could be attacked by the glycosyl acceptor only from the α-side. The experimental results were in good agreement with the SCF/MP2 calculated energies of such participation. The synthesized oligosaccharides are regarded as model compounds for the determination of a structure-activity relationship in FCS.
Assuntos
Sulfatos de Condroitina/química , Fucose/química , Oligossacarídeos/síntese química , Pepinos-do-Mar/química , Animais , Sequência de Carboidratos , Dissacarídeos/síntese química , Indicadores e Reagentes , Modelos Moleculares , Dados de Sequência Molecular , Oligossacarídeos/química , Pepinos-do-Mar/metabolismo , Relação Estrutura-Atividade , Sulfatos/químicaRESUMO
Sulfated polysaccharides of brown algae (fucoidans) attract great attention due to their high and strongly diversified biological activity. This review summarizes recent data on the structural variability of these polysaccharides and reports their anti- and proangiogenic properties. Recent publications have revealed that fucoidans isolated from different algal species may differ considerably in the structures of their backbones and branches, in both monosaccharide composition and sulfate content. It was found that the degree of sulfation significantly influences the biological properties of fucoidans. Additionally, fucoidan action in angiogenesis is highly dependent on molecular weight: antiangiogenic activity is connected with the high-molecular weight of polysaccharide molecules, whereas the low-molecular-weight fractions may act as proangiogenic agents. The influence of other fine structural details of fucoidans on angiogenesis remains to be established.
Assuntos
Inibidores da Angiogênese/metabolismo , Neovascularização Fisiológica , Polissacarídeos/metabolismo , Inibidores da Angiogênese/química , Configuração de Carboidratos , Humanos , Dados de Sequência Molecular , Peso Molecular , Polissacarídeos/químicaRESUMO
Great interest in natural furanoside-containing compounds has challenged the development of preparative methods for their synthesis. Herein a novel reaction in carbohydrate chemistry, namely a pyranoside-into-furanoside (PIF) rearrangement permitting the transformation of selectively O-substituted pyranosides into the corresponding furanosides is reported. The discovered process includes acid-promoted sulfation accompanied by rearrangement of the pyranoside ring into a furanoside ring followed by solvolytic O-desulfation. This process, which has no analogy in organic chemistry, was shown to be a very useful tool for the synthesis of furanoside-containing complex oligosaccharides, which was demonstrated by synthesizing disaccharide derivatives α-D-Galp-(1â3)-ß-D-Galf-OPr, 3-O-s-lactyl-ß-D-Galf-(1â3)-ß-D-Glcp-OPr, and α-L-Fucf-(1â4)-ß-D-GlcpA-OPr related to polysaccharides from the bacteria Klebsiella pneumoniae and Enterococcus faecalis and the brown seaweed Chordaria flagelliformis.
Assuntos
Glicosídeos/química , Oligossacarídeos/síntese química , Sequência de Carboidratos , Enterococcus faecalis/química , Glicosídeos/síntese química , Glicosilação , Klebsiella pneumoniae/química , Modelos Moleculares , Dados de Sequência Molecular , Oligossacarídeos/química , Alga Marinha/químicaRESUMO
Three structurally different fucoidans from the brown seaweeds Saccharina latissima (SL), Fucus vesiculosus (FV), and Cladosiphon okamuranus (CO), two chemically modified fucoidans with a higher degree of sulfation (SL-S, CO-S), and a synthetic totally sulfated octasaccharide (OS), related to fucoidans, were assessed on anticoagulant and antithrombotic activities in different in vitro experiments. The effects were shown to depend on the structural features of the compounds tested. Native fucoidan SL with a degree of sulfation (DS) of 1.3 was found to be the most active sample, fucoidan FV (DS 0.9) demonstrated moderate activity, while the polysaccharide CO (DS 0.4) was inactive in all performed experiments, even at high concentrations. Additional introduction of sulfate groups into fucoidan SL slightly decreased the anticoagulant effect of SL-S, while sulfation of CO, giving rise to the preparation CO-S, increased the activity dramatically. The high level of anticoagulant activity of polysaccharides SL, SL-S, and CO-S was explained by their ability to form ternary complexes with ATIII-Xa and ATIII-IIa, as well as to bind directly to thrombin. Synthetic per-O-sulfated octasaccharide OS showed moderate anticoagulant effect, determined mainly by the interaction of OS with the factor Xa in the presence of ATIII. Comparable tendencies were observed in the antithrombotic properties of the compounds tested.
Assuntos
Plaquetas/efeitos dos fármacos , Hemostáticos/química , Hemostáticos/farmacologia , Polissacarídeos/química , Polissacarídeos/farmacologia , Anticoagulantes/química , Anticoagulantes/farmacologia , Antitrombina III/metabolismo , Plaquetas/metabolismo , Fator Xa/metabolismo , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Fucus/química , Alga Marinha/química , Sulfatos/química , Trombina/metabolismoRESUMO
Human epidermal growth factor receptor 2 (HER2) is overexpressed in numerous cancer cell types. Therapeutic antibodies and chimeric antigen receptors (CARs) against HER2 were developed to treat human tumors. The major limitation of anti-HER2 CAR-T lymphocyte therapy is attributable to the low HER2 expression in a wide range of normal tissues. Thus, side effects are caused by CAR lymphocyte "on-target off-tumor" reactions. We aimed to develop safer HER2-targeting CAR-based therapy. CAR constructs against HER2 tumor-associated antigen (TAA) for transient expression were delivered into target T and natural killer (NK) cells by an effective and safe non-viral transfection method via nucleofection, excluding the risk of mutations associated with viral transduction. Different in vitro end-point and real-time assays of the CAR lymphocyte antitumor cytotoxicity and in vivo human HER2-positive tumor xenograft mice model proved potent cytotoxic activity of the generated CAR-T-NK cells. Our data suggest transient expression of anti-HER2 CARs in plasmid vectors by human lymphocytes as a safer treatment for HER2-positive human cancers. We also conducted preliminary investigations to elucidate if fucosylated chondroitin sulfate may be used as a possible agent to decrease excessive cytokine production without negative impact on the CAR lymphocyte antitumor effect.
RESUMO
Two fucosylated chondroitin sulfates were isolated from the sea cucumbers Psolus peronii and Holothuria nobilis using a conventional extraction procedure in the presence of papain, followed by anion-exchange chromatography on DEAE-Sephacel. Their composition was characterized in terms of quantitative monosaccharide and sulfate content, and structures were mainly elucidated using 1D- and 2D-NMR spectroscopy. As revealed by the data of the NMR spectra, both polysaccharides along with the usual fucosyl branches contained rare disaccharide branches α-D-GalNAc4S6R-(1â2)-α-L-Fuc3S4R â attached to O-3 of the GlcA of the backbone (R = H or SO3-). The polysaccharides were studied as stimulators of hematopoiesis in vitro using mice bone marrow cells as the model. The studied polysaccharides were shown to be able to directly stimulate the proliferation of various progenitors of myelocytes and megakaryocytes as well as lymphocytes and mesenchymal cells in vitro. Therefore, the new fucosylated chondroitin sulfates can be regarded as prototype structures for the further design of GMP-compatible synthetic analogs for the development of new-generation hematopoiesis stimulators.
RESUMO
Hyaluronic acid related di-, tri-, tetra- and hexasaccharide were synthesized as spacer-armed derivatives. 4,6-O-(p-Methoxybenzylidene-2,3-di-O-benzoyl-glucosyl sulfoxide was used as a donor for the formation of ß-D-Glc-(1 â 3)-ß-D-GlcNTCA interunit bond. Selective removal of p-methoxy-benzylidene protecting group, C(6) oxidation by TEMPO-BAIB system followed by methylation led to transformation of Glc unit into GlcA one. Trichloromethyloxazoline donors were used for the formation of ß-D-GlcNTCA-(1 â 4)-ß-D-GlcA linkages. Block-wise [1 + 2], [2 + 2], and [2 + 4] chain assembly afforded to corresponding tri-, tetra-, and hexasaccharide derivatives, respectively. The target compounds were studied as inhibitors of angiogenesis in vitro using endothelial cells and Matrigel as a medium to show the activity of tetra- and hexasaccharide but not for di- and trisaccharide.
Assuntos
Células Endoteliais , Ácido Hialurônico , Sequência de Carboidratos , Oligossacarídeos/química , TrissacarídeosRESUMO
A series of biheterocyclic assemblies comprising of 1,2,5-oxadiazole and azasydnone scaffolds were synthesized and biologically evaluated as novel nitric oxide (NO)-donor and antiplatelet agents. Depending on functional substituents at the biheterocyclic core, all studied compounds demonstrated good NO-donor profiles releasing NO in a wide range of concentrations (19.2%-195.1%) according to a Griess assay. (1,2,5-Oxadiazolyl)azasydnones showed excellent antiplatelet activity in the case of ADP and adrenaline used as inducers completely suppressing the aggregate formation even at the lowest test concentration of 0.0375 µmol/ml, which is a rather unique feature. Moreover, studied biheterocycles possess a selective mechanism of inhibition of platelet aggregation mediated only by ADP and adrenaline, which are considered to be the main inducers causing thrombus formation. In addition, (1,2,5-oxadiazolyl)azasydnones were found to be completely non-toxic to hybrid endothelial cells EaHy 926. Studies of hydrolytic degradation of the synthesized compounds afforded benzoic acid as a sole detectable decomposition product, which is considered advantageous in drug design. Therefore, (1,2,5-oxadiazolyl)azasydnones represent a novel class of promising drug candidates with improved antiplatelet profile and reduced toxicity enabling their huge potential in medicinal chemistry and drug design.
Assuntos
Células Endoteliais , Inibidores da Agregação Plaquetária , Difosfato de Adenosina/farmacologia , Epinefrina/farmacologia , Doadores de Óxido Nítrico/farmacologia , Oxidiazóis , Agregação Plaquetária , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/química , Compostos AzaRESUMO
Fucosylated chondroitin sulfate CJ from the body wall of sea cucumber Cucumaria japonica was depolymerized by the treatment with H2O2 in the presence of Cu(OAc)2. The molecular weight of the polysaccharide was decreased from 32 kDa to 5 kDa. The product CJ-DP was shown to contain l-Fuc, d-GalNAc, d-GlcA, and sulfate in molar proportions of 0.96:0.90:1.00:5.4, which were quite similar to those of the parent polysaccharide CJ. The NMR analysis revealed that CJ-DP, like the parent polysaccharide CJ, consisted of both branched â4)-[3-O-α-l-Fuc]-ß-d-GlcA-(1 â 3)-ß-d-GalNAc-(1â and linear â4)-ß-d-GlcA-(1 â 3)-ß-d-GalNAc-(1â repeating blocks. Sulfate groups occupy O-4 and the majority of O-6 of GalNAc, as well as O-3 of GlcA, in linear blocks and different positions in Fuc branches. This result indicates that depolymerization practically does not diminish the amount of branches and sulfate groups in the product. Both polysaccharides CJ and CJ-DP demonstrated anticoagulant and hematopoiesis-stimulatory activities in vitro.