Development of an oxime bond containing Daunorubicin-Gonadotropin-releasing hormone-III conjugate as a potential anticancer drug
2009, Szabó, Ildikó, Manea, Marilena, Orbán, Erika, Csámpai, Antal, Bösze, Szilvia, Szabó, Rita, Tejeda, Miguel, Gaál, Dezsõ, Kapuvári, Bence, Przybylski, Michael, Hudecz, Ferenc, Mezö, Gábor
Here, we report on the synthesis and biological properties of a conjugate in which daunorubicin (Dau) as chemotherapeutic agent was attached through an oxime bond to gonadotropin-releasing hormone-III (GnRH-III) as targeting moiety. In vitro toxicity and the cytostatic effect of the conjugate on MCF-7 human breast and C26 murine colon cancer cell lines were determined, and the results were compared with those obtained for the free daunorubicin, as well as with the doxorubicin containing derivative. In vivo antitumor effect of daunorubicin-GnRH-III was studied on Balb/c female mice transplanted with C26 tumor. Our data indicate that the daunorubicin-GnRH-III conjugate had a lower toxic effect than the free daunorubicin and it was essentially nontoxic up to 15 mg (Dau content)/kg body weight. The treatment of the C26 tumor bearing mice with the conjugate led to tumor growth inhibition and longer survival time in comparison with the controls and with the administration of the free drug. When mice were treated twice with the conjugate (on days 4 and 7 after tumor transplantation), 46% tumor growth inhibition was obtained. In this case, the increase of the median survival time was 38% compared to the controls.
Structure, enzymatic stability and antitumor activity of sea lamprey GnRH-III and its dimer derivatives
2007-04, Mező, Gábor, Czajlik, András, Manea, Marilena, Jakab, Annamaria, Farkas, Viktor, Majer, Zsuzsa, Vass, Elemer, Bodor, Andrea, Kapuvári, Bence, Boldizsar, Mariann, Vincze, Borbála, Csuka, Orsolya, Kovacs, Magdolna, Przybylski, Michael, Perczel, Andras, Hudecz, Ferenc
Direct antitumor activity of sea lamprey (Petromyzon marinus) gonadotropin-releasing hormone III (Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2; lGnRH-III) was described on several tumor cells. To improve the selectivity of antitumor effects without increasing the hormone releasing activity and to enhance the enzymatic stability, IGnRH-III dimers were prepared via disulfide bond formation. Our results demonstrate that the IGnRH-III dimer derivatives exhibited higher antiproliferative effect and enzymatic stability in comparison with the native IGnRH-III, while lower LH-releasing potency was determined. In order to find a correlation between the biological and structural features of these compounds, the conformation of lGnRH-III and its dimer derivatives was determined by ECD, VCD, FT-IR and (1)H NMR.
Synthesis, solution conformation, and antibody recognition of oligotuftsin-based conjugates containing a β-amyloid(4−10) plaque-specific epitope
2005-07, Manea, Marilena, Hudecz, Ferenc, Przybylski, Michael, Mezö, Gábor
One possible therapeutic approach to treat or prevent Alzheimer's disease (AD) is immunotherapy. On the basis of the identification of Aβ(4−10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, conjugates with defined structures containing the epitope peptide attached to a tetratuftsin derivative as an oligopeptide carrier were synthesized and their structure characterized. To produce immunogenic constructs, the Aβ(4−10) epitope alone or flanked by α- or β-alanine residues was attached through an amide bond to the tetratuftsin derivative (Ac-[TKPKG]4-NH2) or to a carrier peptide elongated by a promiscuous T-helper cell epitope (Ac-FFLLTRILTIPQSLD-[TKPKG]4-NH2). The conformational preferences of the carrier and conjugates were examined by CD spectroscopy in water and in 1:1 and 9:1 TFE:water mixtures (v/v). We found that the presence of flanking dimers in the conjugates had no effects on the generally unordered solution conformation of the conjugates. However, conjugates with an elongated peptide backbone exhibited CD spectra indicative for a partially ordered secondary structure in the presence of TFE. Comparative ELISA binding studies, using monoclonal antibody raised against the β-amyloid (1−17) peptide, showed that conjugates with T-helper cell epitope in the carrier backbone exhibited decreased monoclonal antibody recognition. However, we found that this effect was compensated in conjugates comprising the Aβ(4−10) B-cell epitope with the β-alanine dimer flanking regions at both N- and C-termini. Results suggest that modification of the B-cell epitope peptide from Aβ with rational combination of structural elements (e.g. conjugation to carrier, introduction of flanking dimers) can result in synthetic antigen with preserved antibody recognition.
Antibody Recognition and Conformational Flexibility of a Plaque-Specific beta-Amyloid Epitope Modulated by Non-native Peptide Flanking Regions
2008, Manea, Marilena, Kalászi, Adrián, Mezö, Gábor, Horváth, Kata, Bodor, Andrea, Horváth, Anikó, Farkas, Ödön, Perczel, András, Przybylski, Michael, Hudecz, Ferenc
Here we report on the synthesis, antibody binding, and QSAR studies of a series of linear and cyclic peptides containing a β-amyloid plaque-specific epitope (Aβ(4 10); FRHDSGY). In these constructs, two or three α-l-Ala, α-d-Ala, or β-Ala residues were introduced at both N- and C-termini of the epitope as non-native flanking sequences. Cyclization of the linear Aβ(4 10) epitope peptide resulted in reduced antibody binding. However, the antibody binding could be fully compensated by insertion of alanine flanks into the corresponding cyclic peptides. These results indicate that the modification of a β-amyloid plaque-specific epitope by combination of cyclization and flanking sequences could generate highly antigenic peptides compared to the native sequence. A novel 3D QSAR method, which explicitly handles conformational flexibility, was developed for the case of such molecular libraries. This method led to the prediction of the binding conformation for the common FRHDSGY sequence.
Mass spectrometric identification of the trypsin cleavage pathway in lysyl-proline containing oligotuftsin peptides
2007-04, Manea, Marilena, Mező, Gábor, Hudecz, Ferenc, Przybylski, Michael
Trypsin cleaves specifically peptide bonds at the C-terminal side of lysine and arginine residues, except for -Arg-Pro- and -Lys-Pro- bonds which are normally resistant to proteolysis. Here we report evidence for a -Lys-Pro- tryptic cleavage in modified oligotuftsin derivatives, Ac-[TKPKG]4-NH2) (1), using high-resolution mass spectrometry and HPLC as primary methods for analysis of proteolytic reactions. The proteolytic susceptibility of -Lys-Pro- bonds was strongly dependent on flanking residues, and the flexibility of the peptide backbone might be a prerequisite for this unusual cleavage. While -Lys-Gly- bonds in 1 were rapidly cleaved, the modification of these Lys residues by the attachment of a ß-amyloid(4–10) epitope to yield -Lys(X)-Gly derivatives prevented cleavage of this bond, and provided trypsin cleavage of -Lys-Pro- bonds, the pathway of this degradation being independent on the type of Lys-Nε-side chains (acetyl group, amino acid, peptide). Substitution of the Lys residues by Ala at the P′2 positions decreased the tryptic cleavage, while replacement of the bulky side chain of Thr at the P2 positions strongly increased the cleavage of -Lys-Pro- bonds. Circular dichroism (CD) data of the modified oligotuftsin derivatives are in accord with enhanced flexibility of the peptide backbone, as a prerequisite for increased susceptibility to cleavage of -Lys-Pro- bonds. These results obtained of oligotuftsin derivatives might have implications for the proteolytic degradation of target peptides that require specific conformational preconditions.
Synthesis and structural characterization of bioactive peptide conjugates using thioether linkage approaches
2004-12, Mezö, Gábor, Manea, Marilena, Jakab, Annamária, Kapuvári, Bence, Bösze, Szilvia, Schlosser, Gitta, Przybylski, Michael, Hudecz, Ferenc
Applications of cysteine-insertion and thioether linkage approaches to the preparation of a number of bioactive peptide conjugates are reported. Peptides containing epitopes from (i) herpes simplex virus type 1 glycoprotein D, (ii) a specific N-terminal beta-amyloid epitope recognized by therapeutically active antibodies, and (iii) a GnRH-III peptide from sea lamprey with antitumour activity, were elongated with Cys residues and attached to a chloroacetylated tetratuftsin derivative carrier via a thioether linkage either directly, or by insertion of a spacer. The structures and molecular homogeneity of all the peptide conjugates were ascertained by HPLC, MALDI and electrospray mass spectrometry. The use of a spacer such as an oligoglycine or GFLG-tetrapeptide gave an increased yield in the conjugation reaction and enhanced reaction rates. In the formation of cysteinyl-thioether linkages, it was found that the position of flanking Cys residues markedly influenced the conjugation reaction and the formation of intermolecular epitope disulfide-dimers. C-terminal Cys residues gave thioether conjugates with significantly diminished epitope-dimerization, while Cys at the N-terminal caused rapid disulfide-dimerization, thereby preventing efficient conjugation.
Design, structural, and immuno-analytical properties of antigenic bioconjugates comprising a beta-amyloid-plaque specific epitope
2008, Manea, Marilena, Przybylski, Michael, Hudecz, Ferenc, Mezö, Gábor
Immunotherapeutic approaches are investigated for treatment of neurodegenerative diseases of the Alzheimer's dementia (AD) type. The identification of a β-amyloid-plaque specific epitope, Aβ(4-10) (4FRHDSGY10), recognized by therapeutically active antibodies from transgenic AD mice could provide the basis for the development of AD vaccines. Here we report on the synthesis, structural and immuno-analytical characterization of bioconjugates comprising the β-amyloid(4-10) epitope as new vaccine lead structures against Alzheimer's disease. To produce antigenic bioconjugates, potential immunogens, the epitope peptide elongated by a cysteine residue or a cysteinyl-pentaglycine hexapeptide unit either at the N- or C-terminus was attached via a thioether bond to synthetic oligopeptide carriers, such as oligotuftsin derivatives, sequential oligopeptide carrier, or lysine dendrimer. The antigenic properties of these constructs were determined by enzyme-linked immunosorbent assay (ELISA) using an anti-Aβ(1-17) monoclonal antibody. Our results indicate that the major factors which influence the antibody binding of the Aβ(4-10) epitope are (i) the epitope topology and (ii) the presence of a spacer moiety between the carrier and the epitope peptide. Interestingly, the carrier type had no marked effect on the binding of the antibody to the epitope-conjugates. The conformational preferences of the conjugates were examined by circular dichroism spectroscopy in water and in trifluoroethanol. In water, the conjugates adopt random coil conformation independently on their primary structure. However, differences related to the attachment site of the epitope to the carriers were determined in TFE, conjugates in which the epitope was attached to the carrier through the N-terminus exhibiting more ordered secondary structure.
Copper-induced oligomerization of peptides : a model study
2007, Schlosser, Gitta, Stefanescu, Raluca, Przybylski, Michael, Murariu, Manuela, Hudecz, Ferenc, Drochioiu, Gabi
In this work, copper-binding of the tetraglycine peptide (Gly-Gly-Gly-Gly) was studied by electrospray ionization mass spectrometry. Experiments were performed under alkaline conditions, in the presence of ethanolamine (pH 10.95). We observed that the presence of copper(II) ions induces the aggregation of the peptide and the formation of copper-bound complexes with higher molecular mass is favored, such as the oligomer complexes [3M+2Cu-3H](+) and [4M+3Cu-5H](+). At 1:1 peptide-copper(II) ion ratio, the singly charged [3M+2Cu-3H](+) oligomer complex is the base peak in the mass spectrum. Metal ion-induced oligomerization of neurotoxic peptides is well known in the literature; however, there are very few examples in which such oligomerization was directly observed by mass spectrometry. Our results show that application of short peptides can be useful to study the mechanism of metal ion binding and metal ion-induced oligomerization of peptides.
Polypeptide conjugates comprising a β-amyloid plaque-specific epitope as new vaccine structures against Alzheimer's disease
2004, Manea, Marilena, Mezö, Gábor, Hudecz, Ferenc, Przybylski, Michael
Immunotherapeutic approaches designed to induce a humoral immune response have recently been developed for possible vaccination to the treatment of Alzheimer's disease (AD). Based on the identification of Aβ(4–10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, branched polypeptide conjugates with this epitope peptide were synthesized and characterized. In order to produce immunogenic constructs, the Aβ(4–10) epitope alone or together with a promiscuous T-helper cell epitope peptide (FFLLTRILTIPQSLD) were attached via thioether linkage to different branched chain polymeric polypeptides with Ser or Glu in the side chains. A single peptide containing both an Aβ(4–10) and T-helper cell epitope, joined by a dipeptide Cys–Acp spacer, was also attached through the thiol function to chloroacetylated poly[Lys(Seri–DL–Alax)] (SAK). Comparative binding studies of the conjugates with a monoclonal antibody against the β-amyloid(1–17) peptide in mice were performed by direct ELISA. The conformational preferences of carriers and conjugates in water and in a 9:1 triflouroethanol:water mixture (v/v) was analyzed by CD spectroscopy. Experimental data showed that the chemical nature of the carrier macromolecule, and the attachment site of the epitope to the carrier, have significant effects on antibody recognition, but have no marked influence on the solution conformation of the conjugates.