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1.
Biosens Bioelectron ; 24(1): 60-5, 2008 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-18455919

RESUMO

The development of a biosensor based on surface plasmon resonance is described for the detection of carbohydrate-binding proteins in solution on a Biacore 2000 instrument, using immobilized glycopeptides as ligands. Their selection was based on previous screenings of solid-phase glycopeptide libraries with Ricinus communis agglutinin (RCA(120)) and human adhesion/growth-regulatory galectin-1 (h-Gal-1). Glycopeptides were immobilized on Au sensor chips functionalized with mixed self-assembled monolayers of different ratios of 11-mercapto-1-undecanol and 11-mercaptoundecanoic acid, and of 3-mercapto-1-propanol and 11-mercaptoundecanoic acid. The biosensors were optimized for the detection of RCA(120), and a detection limit of 0.13 nM was obtained. Subsequent experiments with h-Gal-1 indicated a detection limit of at least 0.9 nM for this lectin. Additionally, the effect of interfering proteins on the sensitivity of the optimized biosensor was investigated.


Assuntos
Técnicas Biossensoriais/métodos , Glicopeptídeos/química , Ressonância de Plasmônio de Superfície/métodos , Técnicas Biossensoriais/instrumentação , Proteínas de Transporte/análise , Galectina 1/análise , Lectinas de Plantas/análise , Sensibilidade e Especificidade
2.
Anal Biochem ; 378(2): 190-6, 2008 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-18471425

RESUMO

Combinatorial (glyco)peptide libraries offer the possibility to define effective inhibitors of protein (lectin)-glycan interactions. If a (glyco)peptide surpasses the inhibitory potency of the free sugar, then the new peptide-lectin contacts underlying the affinity enhancement may guide further rational drug design. Focusing on the adhesion/growth regulatory human galectins 1 and 3, a screening of three combinatorial solid-phase (glyco)peptide libraries, containing Gal(beta1-O)Thr, Gal(beta1-S)Cys/Gal(beta1-N)Asn, and Lac(beta1-O)Thr, with the fluorescently labeled lectins had led to a series of lead compounds. To define the inhibitory potency of a selection of resynthesized (glyco)peptides systematically, a surface plasmon resonance-based inhibition assay with immobilized asialofetuin was set up. (Glyco)Peptides with up to 66-fold potency relative to free lactose as inhibitor were characterized. The presence of lactose in the most effective glycopeptides indicated the presence of affinity-enhancing peptide-lectin contacts. In addition to drug design, they may be helpful for fine-structural analysis of the binding sites.


Assuntos
Galectina 1/metabolismo , Galectina 3/metabolismo , Glicopeptídeos/metabolismo , Biblioteca de Peptídeos , Ressonância de Plasmônio de Superfície/métodos , Sequência de Aminoácidos , Animais , Assialoglicoproteínas/química , Assialoglicoproteínas/metabolismo , Bovinos , Fetuínas , Galectina 1/química , Galectina 3/química , Glicopeptídeos/química , Humanos , Ligantes , Dados de Sequência Molecular , Ligação Proteica , alfa-Fetoproteínas/química , alfa-Fetoproteínas/metabolismo
3.
Bioorg Med Chem Lett ; 17(3): 793-8, 2007 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17095217

RESUMO

The involvement of human lectins (galectins) in disease progression accounts for the interest to design potent inhibitors. Three fully randomized hexa(glyco)peptide libraries were prepared using the portion mixing method combined with ladder synthesis. On-bead screening with fluorescently labelled galectin-1 and -3 yielded a series of lead structures, whose inhibitory activity on carbohydrate-dependent galectin binding was tested in solution by solid-phase and cell assays. The various data obtained define the library approach as a facile route for the discovery of selective (glyco)peptide-based galectin inhibitors.


Assuntos
Galectinas/química , Glicopeptídeos/síntese química , Linhagem Celular Tumoral , Técnicas de Química Combinatória , Glicopeptídeos/farmacologia , Humanos , Indicadores e Reagentes , Ligantes , Biblioteca de Peptídeos
4.
J Comb Chem ; 8(6): 812-9, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17096569

RESUMO

Two combinatorial glycopeptide libraries were synthesized on solid support via the "split-and-mix" method combined with the ladder synthesis strategy. The O-glycopeptide library contained Gal(beta1-O)Thr, whereas the S-,N-glycopeptide library contained both Gal(beta1-S)Cys and Gal(beta1-N)Asn. In this model study, the two libraries were screened against the fluorescently labeled lectin Ricinus communis agglutinin (RCA120). The screening results showed that both O- and S- or S-,N-glycopeptides were recognized by the lectin with similar amino acid recognition patterns. Surface plasmon resonance interaction studies demonstrated that both the selected S- or S-,N-glycopeptide hits and the O-glycopeptides bound to the lectin with a similar affinity. Structure 19, containing two glycosylated cysteine residues, bound to the receptor with the highest affinity (KA = 3.81 x 10(4) M(-1)), which is comparable to N-acetyllactosamine. Competition assays, in which some selected glycopeptides and methyl beta-d-galactopyranoside competed for the binding site of immobilized RCA120, showed that the glycopeptide-lectin interaction was carbohydrate-specific. Incubation of the O- and S-,N-glycopeptides with beta-galactosidase demonstrated the complete stability of S-,N-glycopeptides toward enzymatic degradation, whereas O-glycopeptides were not completely stable.


Assuntos
Cromatografia de Afinidade/métodos , Técnicas de Química Combinatória/métodos , Glicopeptídeos/química , Lectinas de Plantas/química , Glicopeptídeos/síntese química , Ligantes , Estrutura Molecular , Biblioteca de Peptídeos , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície/métodos , Fatores de Tempo
5.
Org Biomol Chem ; 2(20): 2972-87, 2004 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-15480463

RESUMO

The gut-associated circulating anodic antigen (CAA) is one of the major excretory antigens produced by the parasite Schistosoma mansoni. The immunoreactive part of CAA is a threonine-linked polysaccharide composed of long stretches of the unique repeating disaccharide-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GalpNAc-(1-->. Previously, using surface plasmon resonance and ELISA techniques, it has been shown that some anti-CAA IgM monoclonal antibodies (MAbs) also recognize members of a series of bovine serum albumin (BSA)-coupled synthetic di- to penta-saccharide fragments of the CAA glycan. To generate information on the molecular level about the glycan specificity of the relevant IgM MAbs, two series of oligosaccharides related to the CAA disaccharide epitope were synthesized, and coupled to BSA. The first three analogues, beta-D-GlcpA-(1-->3)-[small beta]-D-GlcpNAc-(1-->O), beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O), and beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O), wherein the native beta-D-GalpNAc moiety was replaced by beta-D-GlcpNAc, were synthesized to investigate the specificity of the selected MAbs to the carbohydrate backbone of CAA. The second series of analogues, beta-D-Glcp6S-(1-->3)-beta-D-GalpNAc-(1-->O), beta-D-GalpNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), and beta-D-Glcp6S-(1-->3)-beta-D-GalpNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), wherein the native beta-D-GlcpA moiety was replaced by beta-D-Glcp6S, was synthesized to evaluate the importance of the type/nature of the charge of CAA for the MAb recognition.


Assuntos
Antígenos de Protozoários/química , Oligossacarídeos/síntese química , Polissacarídeos/química , Schistosoma mansoni/metabolismo , Animais , Antígenos de Protozoários/metabolismo , Configuração de Carboidratos , Estrutura Molecular , Soroalbumina Bovina/química
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