RESUMO
Access to lipopeptide-based vaccines for immunological studies remains a significant challenge owing to the amphipathic nature of the molecules, which makes them difficult to synthesize and purify to homogeneity. Here, we describe the application of a new peptide ligation technology, the diselenide-selenoester ligation (DSL), to access self-adjuvanting glycolipopeptide vaccines. We show that rapid ligation of glyco- and lipopeptides is possible via DSL in mixed organic solvent-aqueous buffer and, when coupled with deselenization chemistry, affords rapid and efficient access to a vaccine candidate possessing a MUC1 glycopeptide epitope and the lipopeptide adjuvant Pam2Cys. This construct was shown to elicit MUC1-specific antibody and cytotoxic T lymphocyte responses in the absence of any other injected lipids or adjuvants. The inclusion of the helper T cell epitope PADRE both boosted the antibody response and resulted in elevated cytokine production.
Assuntos
Adjuvantes Imunológicos/síntese química , Vacinas Anticâncer/imunologia , Glicopeptídeos/imunologia , Lipopeptídeos/imunologia , Mucina-1/imunologia , Compostos Organosselênicos/química , Sequência de Aminoácidos , Animais , Vacinas Anticâncer/síntese química , Técnicas de Química Sintética/métodos , Feminino , Glicopeptídeos/síntese química , Humanos , Lipopeptídeos/síntese química , Células MCF-7 , Camundongos Endogâmicos C57BL , Repetições Minissatélites , Mucina-1/genética , Compostos Organosselênicos/síntese químicaRESUMO
MUC1 variable number tandem repeats (VNTRs) conjugated to tumor-associated carbohydrate antigens (TACAs) have been shown to break self-tolerance in humanized MUC1 transgenic mice. Therefore, we hypothesize that a MUC1 VNTR TACA-conjugate can be successfully formulated into a liposome-based anticancer vaccine. The immunogenicity of the vaccine should be further augmented by incorporating surface-displayed l-rhamnose (Rha) epitopes onto the liposomes to take advantage of a natural antibody-dependent antigen uptake mechanism. To validate our hypothesis, we synthesized a 20-amino-acid MUC1 glycopeptide containing a GalNAc-O-Thr (Tn) TACA by SPPS and conjugated it to a functionalized Toll-like receptor ligand (TLRL). An l-Rha-cholesterol conjugate was prepared using tetra(ethylene glycol) (TEG) as a linker. The liposome-based anticancer vaccine was formulated by the extrusion method using TLRL-MUC1-Tn conjugate, Rha-TEG-cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a total lipid concentration of 30 mM. The stability, homogeneity, and size characterization of the liposomes was evaluated by SEM and DLS measurements. The formulated liposomes demonstrated positive binding with both anti-Rha and mouse anti-human MUC1 antibodies. Groups of female BALB/c mice were immunized and boosted with a rhamnose-Ficoll (Rha-Ficoll) conjugate formulated with alum as adjuvant to generate the appropriate concentration of anti-Rha antibodies in the mice. Anti-Rha antibody titers were >25-fold higher in the groups of mice immunized with the Rha-Ficoll conjugate than the nonimmunized control groups. The mice were then immunized with the TLRL-MUC1-Tn liposomal vaccine formulated either with or without the surface displaying Rha epitopes. Sera collected from the groups of mice initially immunized with Rha-Ficoll and later vaccinated with the Rha-displaying TLRL-MUC1-Tn liposomes showed a >8-fold increase in both anti-MUC1-Tn and anti-Tn antibody titers in comparison to the groups of mice that did not receive Rha-Ficoll. T-cells from BALB/c mice primed with a MUC1-Tn peptide demonstrated increased proliferation to the Rha-liposomal vaccine in the presence of antibodies isolated from Rha-Ficoll immunized mice compared to nonimmune mice, supporting the proposed effect on antigen presentation. The anti-MUC1-Tn antibodies in the vaccinated mice serum recognized MUC1 on human leukemia U266 cells. Because this vaccine uses separate rhamnose and antigenic epitope components, the vaccine can easily be targeted to different antigens or epitopes by changing the peptide without having to change the other components.
Assuntos
Mucina-1/química , Mucina-1/imunologia , Ramnose/síntese química , Ramnose/imunologia , Animais , Vacinas Anticâncer/síntese química , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Glicopeptídeos/síntese química , Glicopeptídeos/imunologia , Humanos , Imunização/métodos , Lipossomos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The glycopeptide fragment CII259-273 from type II collagen (CII) binds to the murine A(q) and human DR4 class II Major Histocompatibility Complex (MHC II) proteins, which are associated with development of murine collagen-induced arthritis (CIA) and rheumatoid arthritis (RA), respectively. It has been shown that CII259-273 can be used in therapeutic vaccination of CIA. This glycopeptide also elicits responses from T-cells obtained from RA patients, which indicates that it has an important role in RA as well. We now present a methodology for studies of (glyco)peptide-receptor interactions based on a combination of structure-based virtual screening, ligand-based statistical molecular design and biological evaluations. This methodology included the design of a CII259-273 glycopeptide library in which two anchor positions crucial for binding in pockets of A(q) and DR4 were varied. Synthesis and biological evaluation of the designed glycopeptides provided novel structure-activity relationship (SAR) understanding of binding to A(q) and DR4. Glycopeptides that retained high affinities for these MHC II proteins and induced strong responses in panels of T-cell hybridomas were also identified. An analysis of all the responses revealed groups of glycopeptides with different response patterns that are of high interest for vaccination studies in CIA. Moreover, the SAR understanding obtained in this study provides a platform for the design of second-generation glycopeptides with tuned MHC affinities and T-cell responses.
Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Glicopeptídeos/síntese química , Antígenos de Histocompatibilidade Classe II/metabolismo , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Ligação Competitiva , Biotinilação , Avaliação Pré-Clínica de Medicamentos , Glicopeptídeos/análise , Glicopeptídeos/química , Antígeno HLA-DR4/química , Antígeno HLA-DR4/metabolismo , Antígenos de Histocompatibilidade Classe II/química , Humanos , Hibridomas , Análise dos Mínimos Quadrados , Ligantes , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Análise de Componente Principal , Ligação Proteica , Interface Usuário-ComputadorRESUMO
As therapeutic agents of choice in the treatment of complicated infections, glycopeptide antibiotics are often preferentially used in cases of osteomyelitis, an infection located in bone and notoriously difficult to successfully manage. Yet frequent and heavy doses of these systemically administered antibiotics are conventionally prescribed to obtain higher antibiotic levels in the bone and reduce the high recurrence rates. Targeting antibiotics to the bone after systemic administration would present at least three potential advantages: (i) greater efficacy, by concentrating the therapeutic agent in bone; (ii) greater convenience, through a reduction in the frequency of administration; and (iii) greater safety, by reducing the levels of systemic drug exposure. We present here the design, synthesis and in vitro evaluation of eight prodrugs of the glycopeptide antibacterial agents vancomycin and oritavancin taking advantage of the affinity of the bisphosphonate group for bone for delivery to osseous tissues.
Assuntos
Difosfonatos/síntese química , Glicopeptídeos/síntese química , Osteomielite/tratamento farmacológico , Pró-Fármacos/síntese química , Animais , Antibacterianos/farmacologia , Conservadores da Densidade Óssea/síntese química , Conservadores da Densidade Óssea/química , Conservadores da Densidade Óssea/uso terapêutico , Difosfonatos/química , Difosfonatos/uso terapêutico , Glicopeptídeos/química , Glicopeptídeos/uso terapêutico , Humanos , Lipoglicopeptídeos , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pró-Fármacos/química , Pró-Fármacos/uso terapêutico , Ratos , Staphylococcus aureus/efeitos dos fármacos , Vancomicina/uso terapêuticoRESUMO
T cells obtained in a mouse model for rheumatoid arthritis are activated by a glycopeptide fragment from rat type II collagen (CII) bound to the class II major histocompatibility complex Aq molecule. We report a comparative model of Aq in complex with the glycopeptide CII260-267. This model was used in a structure-based design approach where the amide bond between Ala261 and Gly262 in the glycopeptide was selected for replacement with psi[COCH2], psi[CH2NH2+], and psi[(E)-CH=CH] isosteres. Ala-Gly isostere building blocks were then synthesized and introduced in CII260-267 and CII259-273 glycopeptides. The modified glycopeptides were evaluated for binding to the Aq molecule, and the results were interpreted in view of the Aq/glycopeptide model. Moreover, recognition by a panel of T-cell hybridomas revealed high sensitivity for the backbone modifications. These studies contribute to the understanding of the interactions in the ternary Aq/glycopeptide/T-cell receptor complexes that activate T cells in autoimmune arthritis and suggest possibilities for new vaccination approaches.
Assuntos
Artrite Experimental/imunologia , Colágeno Tipo II/química , Glicopeptídeos/química , Antígenos de Histocompatibilidade Classe II/química , Fragmentos de Peptídeos/química , Receptores de Antígenos de Linfócitos T/metabolismo , Alanina/química , Animais , Células Apresentadoras de Antígenos/metabolismo , Artrite Experimental/induzido quimicamente , Ligação Competitiva , Técnicas de Cocultura , Glicina/química , Glicopeptídeos/síntese química , Glicopeptídeos/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Hibridomas , Interleucina-2/metabolismo , Camundongos , Modelos Moleculares , Estrutura Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Ratos , Baço/citologia , Estereoisomerismo , Relação Estrutura-Atividade , Linfócitos T/metabolismoRESUMO
The development of a clinically effective, carbohydrate-based antitumor vaccine is a longstanding ambition in the prevention and treatment of cancer. This review seeks to provide a discussion of some of the unique challenges facing this particular field of immunology. The authors present a historic account of their ongoing research program devoted to the development of fully synthetic, carbohydrate-based anticancer vaccines of clinical value. As will be seen, remarkable advances in carbohydrate and glycopeptide assembly techniques have allowed for the preparation of synthetic constructs of progressively increasing structural complexity. The authors describe the evolution of their synthetic carbohydrate program from first-generation constructs, which were monovalent in nature, to highly complex unimolecular multivalent vaccines, in which multiple carbohydrate antigens are displayed in the context of a single polypeptide backbone. It is the hope that each generation of vaccines represents a move closer to achieving the ultimate objective of developing broadly useful, robust anticancer vaccines.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Neoplasias da Mama/tratamento farmacológico , Vacinas Anticâncer/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Vacinas Conjugadas/uso terapêutico , Animais , Antígenos Glicosídicos Associados a Tumores/química , Vacinas Anticâncer/imunologia , Sequência de Carboidratos , Carboidratos/síntese química , Carboidratos/imunologia , Ensaios Clínicos como Assunto , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Feminino , Glicopeptídeos/síntese química , Glicopeptídeos/imunologia , Humanos , Masculino , Vacinas Conjugadas/imunologia , Vacinas SintéticasRESUMO
New cysteine-containing derivatives of glycyrrhizic acid were synthesized by its coupling with Cys(Bzl) esters or the Cys(Bzl)-Val-OBu(t) dipeptide by the active ester method (DCC/HOSu) or by Woodward's reagent K. The derivatives with Cys(Bzl) and Cys(Bzl)-Val residues attached to the carbohydrate part of the molecule stimulated the primary immune response and the reaction of delayed-type hypersensitivity in mice at a dose of 2 mg/kg. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.
Assuntos
Adjuvantes Imunológicos/síntese química , Cisteína/química , Glicopeptídeos/síntese química , Ácido Glicirrízico/química , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Glicopeptídeos/química , Glicopeptídeos/farmacologia , Ressonância Magnética Nuclear BiomolecularRESUMO
Two different sialic acid containing glycopeptide (sialopeptide) libraries were synthesized using the portion mixing method and ladder synthesis. The libraries were attached via an IMP spacer and a photolabile linker to PEGA(1900) resin in order to facilitate rapid and unambiguous structural analysis of hits by MALDI-TOFMS. One library contained a lactamized sialic acid moiety at the N terminus of a pentapeptide, while a second library displayed a sialic acid residue at the center of a heptapeptide. The sialopeptide libraries were screened against the recombinant binding domain (SnD1) of a sialic acid binding Ig-like protein, sialoadhesin (Siglec-1). No ligands were identified from the lactamized sialic acid library, underscoring the importance of the carboxylic acid moiety for binding. Screening of the second gave few distinct hits (approximately 0.03% of library) with a high consensus. The high-affinity ligands contained, in most cases, a WG motif following the sialylated Thr. The strength of binding of selected ligands was determined by surface plasmon resonance. The best sialopeptide ligand, WLLT(Sa)WGT, exhibited micromolar affinity of SnD1; >10 times the affinity of SnD1 to 3'-sialyl lactose.
Assuntos
Glicopeptídeos/síntese química , Glicoproteínas de Membrana/metabolismo , Oligossacarídeos/fisiologia , Receptores Imunológicos/metabolismo , Sialoglicoproteínas/síntese química , Ligação Competitiva , Técnicas de Química Combinatória , Avaliação Pré-Clínica de Medicamentos/métodos , Glicopeptídeos/metabolismo , Ligantes , Mimetismo Molecular/fisiologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico , Sialoglicoproteínas/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
The preparation of a series of sialylated and fucosylated N,N'-diacetyllactosediamine-type diantennary glycopeptides is reported. By sequential enzymatic action of jack bean beta-galactosidase, snail beta 4-N-acetyl-galactosaminyltransferase, bovine colostrum alpha 6-sialyltransferase and human milk alpha 3-fucosyltransferase, a diantennary glycopeptide obtained from asialo fibrinogen was converted at a 5-mumol scale to a series of structures occurring on the glycoprotein glycodelin A, which potentially inhibit human sperm-egg binding.
Assuntos
Glicopeptídeos/síntese química , Glicoproteínas/química , Proteínas da Gravidez/química , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Bovinos , Fabaceae/enzimologia , Fibrinogênio/química , Glicodelina , Glicosiltransferases/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Plantas Medicinais , Sialiltransferases/metabolismo , Caramujos/enzimologia , beta-Galactosidase/metabolismoRESUMO
A block synthesis of the model compound for the phytoalexin elicitor-active glycoprotein is described. Combination of the C-terminus free compounds, N-(9-fluorenylmethoxycarbonyl)-O-(tert-butyl)-L-seryl-L-proline (1) or N-(9-fluorenylmethoxycarbonyl)-(2,3,4,6-tetra-O-acetyl-beta-D-g luc opyranosyl) -(1-->6)-(2,3,4-tri-O-acetyl-alpha-D-mannopyranosyl)-(1-->6) -(2,3,4-tri-O-acetyl-alpha-D-mannopyranosyl)-L-seryl-L-proline (2) with the N-terminus free compounds, 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl -(1-->6)-(2,3,4-tri-O-acetyl-alpha-D-mannopyranosyl)-(1-->6)-(2,3,4-tri- O -acetyl-alpha-D-mannopyranosyl)-L-seryl-L-prolyl-L-seryl-L-proline methyl ester (4), O-(tert-butyl)-L-seryl-L-prolyl-(2,3,4,6-tetra-O-acetyl-beta-D -glucopyranosyl)-(1-->6)-(2,3,4-tri-O-acetyl-alpha-D-mannopyranosyl) -(1-->6)-(2,3,4-tri-O-acetyl- alpha-D-mannopyranosyl)-L-seryl-L-proline methyl ester (6) or 2,3,4,6-tetra-O-acetyl-beta-D- glucopyranosyl -(1-->6)-(2,3,4-tri-O-acetyl-alpha-D-mannopyranosyl)-(1-->6) -(2,3,4-tri-O-acetyl-alpha-D-mannopyranosyl)-L-seryl-L-prolyl -(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-(1-->6) -(2,3,4-tri-O-acetyl-alpha-D-mannopyranosyl)-(1-->6) -(2,3,4-tri-O-acetyl-alpha-D-mannopyranosyl)-L-seryl-L-proline methyl ester (8), by use of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) gave three hexaglycosyl hexapeptides and a nonaglycosyl hexapeptide derivatives (9, 11, 14, and 17). These N-terminus free compounds were derived from triglycosyl tetrapeptides (3 and 5) or a hexaglycosyl tetrapeptide (7) on selective deblock reaction by morpholine. The hexaglycosyl hexapeptides (10, 13, and 16) and the nonaglycosyl hexapeptide (18) have been prepared by the convergent block synthesis.
Assuntos
Glicopeptídeos/síntese química , Oligopeptídeos/síntese química , Extratos Vegetais/biossíntese , Sequência de Carboidratos , Glicopeptídeos/química , Glicopeptídeos/farmacologia , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Sesquiterpenos , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Terpenos , FitoalexinasRESUMO
A stereo-controlled synthesis of the model compound for the phytoalexin elicitor-active glycoprotein is described. Glycosylation of the trisaccharide, 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl-(1-->6)-2,3,4-tri-O-acetyl- alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidate (12), with N-(9-fluorenylmethoxycarbonyl)-L-seryl-L- proline benzyl ester (3) or N-(carbobenzoxy)-L-seryl-L-proline methyl ester (4) by use of BF3. OEt2 gave the triglycosyl-seryl-proline derivatives. The N- as well as C-terminus of these triglycosyl dipeptides could be deblocked selectively to give compounds 14 and 16, which are versatile intermediates for the completion of model compound synthesis of glycopeptide. Triglycosyl tetrapeptides (18, 21) and hexaglycosyl tetrapeptide (23) have been prepared by the convergent block synthesis.
Assuntos
Glicopeptídeos/síntese química , Oligossacarídeos/síntese química , Extratos Vegetais/química , Sequência de Aminoácidos , Aminoácidos/metabolismo , Configuração de Carboidratos , Sequência de Carboidratos , Fluorenos/metabolismo , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Sesquiterpenos , Terpenos , FitoalexinasRESUMO
Triterpene glycopeptides, derivatives of glycyrrhizic acid, were synthesized with the use of tert-butyl esters of L-amino acids. The condensation of glycoside with esters was carried out with N-hydroxysuccinimide--N,N-dicyclohexylcarbodiimide or N-hydroxybenzotriazol--N,N'-dicyclohexylcarbodiimide in the presence of a base (triethylamine, N-methyl- or N-ethylmorpholine). The protection groups were removed with the use of trifluoroacetic acid.
Assuntos
Adjuvantes Imunológicos/síntese química , Aminoácidos/química , Glicopeptídeos/síntese química , Ácido Glicirretínico/análogos & derivados , Animais , Sequência de Carboidratos , Ésteres , Ácido Glicirretínico/química , Ácido Glicirrízico , Espectroscopia de Ressonância Magnética , Camundongos , Dados de Sequência Molecular , Espectrofotometria UltravioletaRESUMO
A stereocontrolled synthesis of the model compound for the phytoalexin elicitor-active glycoprotein is described. Glycosylation of the disaccharide, 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl-(1-->6)-2,3,4-tri-O-acetyl- alpha- D-mannopyranosyl trichloroacetimidate, with N-(carbobenzoxy)-(2,3,4-tri-O-acetyl-alpha-D-mannopyranosyl)-(1-->3)-L- serine methyl ester or N-(carbobenzoxy)-(2,3,4-tri-O-acetyl-alpha-D-mannopyranosyl)-(1-->3)-L- seryl-L- proline methyl ester by use of AgOTf gave the desired trisaccharide-serine or trisaccharide-seryl-proline derivatives, which were transformed into beta-D-glucopyranosyl-(1-->6)-alpha-D-mannopyranosyl-(1-->6)-alpha-D- mannopyranosyl-(1-->3)-L-serine and triglycosyl-(1-->3)-L-seryl-L-proline via removal of the N-carbobenzoxy group, followed by deacylation.
Assuntos
Glicopeptídeos/síntese química , Extratos Vegetais , Trissacarídeos/síntese química , Configuração de Carboidratos , Sequência de Carboidratos , Dissacarídeos/síntese química , Dissacarídeos/química , Glicopeptídeos/farmacologia , Glicosilação , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Rotação Ocular , Prolina , Serina , Sesquiterpenos , Estereoisomerismo , Terpenos , Trissacarídeos/química , FitoalexinasRESUMO
A series of glycopeptidemimetics based on the hydroxyethylene Phe-Phe isostere have been synthesized and evaluated for their ability to inhibit the enzyme HIV-1 protease. Incorporation of carbohydrate moieties at the P'2-position and elimination of P'3 amino acid in our lead compound 1, provided inhibitors with only nanomolar potencies (400-800 nM). However, incorporation of a carbohydrate moiety at the P'3-position with branched chain amino acid at the P'2-position, resulted in inhibitors with subnanomolar potencies. Within this series, compound 21 was the most potent inhibitor (IC50 value 0.17 nM). This compound has also shown to block the spread of HIV-1 in T-lymphoid cells at an inhibitor concentration of 200 nM.
Assuntos
Glicopeptídeos/síntese química , Inibidores da Protease de HIV/síntese química , HIV-1/enzimologia , Sequência de Aminoácidos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Glicopeptídeos/farmacologia , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Relação Estrutura-Atividade , Linfócitos T/microbiologiaRESUMO
Three triterpene glycopeptides, glycyrrhizic acid monomethly ester derivatives containing peptide fragment of N-acetylmuramoyldipeptide and its analogues, are synthesized. They possess high prednizolone-like antiinflammatory activity and display pronounced stimulative effect on humoral factors of immunity.
Assuntos
Adjuvantes Imunológicos/síntese química , Anti-Inflamatórios não Esteroides/síntese química , Glicopeptídeos/síntese química , Ácido Glicirretínico/análogos & derivados , Aglutininas/análise , Animais , Formação de Anticorpos , Fenômenos Químicos , Química , Glicopeptídeos/farmacologia , Ácido Glicirrízico , Camundongos , Tamanho do Órgão/efeitos dos fármacos , RatosRESUMO
The synthesis of two glycosylated analogs of Substance P is described. The activity of the peptides was assayed on the isolated guinea-pig ileum and their degradation was studied using rat hypothalamus slices. While glycosylation noticeably enhances the solubility of the corresponding compounds, the beta-glucopyranosyl moiety only slightly modifies the biological half-life and the bioactivity of the glycopeptides.